Medical virology is simply defined as the study of viruses that are of medical importance i.e. those group or families of viruses that cause disease in humans. Virology therefore is the study of viruses inclusive of those ones that are of medical importance and those that are not harmful but are beneficial in nature. Viruses are non-cellular microorganisms that consist of nucleic acids (either DNA or RNA) which are surrounded by a protein coat. They are particles of nucleic acid molecules either DNA or RNA that are usually surrounded by protein molecules and lipid by lipid membranes in some virions. In other words, viruses are acellular complexes or molecules that consist only of a DNA genome or RNA genome and a protein coat that surrounds its nucleic acid molecule.
They are obligate intracellular parasites that only replicate within a living host cell. Viruses are different from the other groups of microorganisms (i.e. bacteria, fungi, protozoa and algae) in several ways. For example, viruses only replicate in particular living cells including cells of other microorganisms, plants, and animals. Viruses are very small; and they are 100 times smaller than bacteria. They rarely exist or reproduce on their own; and thus viruses cannot survive outside a living cell. And viruses do not contain both DNA and RNA in one organism as their genome but rather, each virion either contains DNA or RNA as its nucleic acid and not both in one organism.
A virion (Plural: virions) is a single complete virus particle. Virion, a matured virus is a synonym for virus particle; and it is formed in vivo inside the infected host cell when the invading viral particle takes over the hot cell’s machinery and makes it to produce the components of new viruses that have the same genetic makeup as the parent viral cell. Virion consists mainly of three parts: 1) a nucleic acid genome (DNA or RNA); 2) a capsid or protein coat; and 3) envelop (that surrounds the capsid) which is found in some viruses. The general structure of a virion or viral particle is shown in Figure 1. Another word for capsid is nucleocapsid (a combination of the genome and capsid). Viruses that contain envelops in addition to the usual capsid and genome are generally known as complex (enveloped) viruses while those that contain only the capsid and genome are termed naked (simple) viruses. Scientists or microbiologists that study viruses, and whose area of specialization is on viruses (inclusive of animal, plant and human viruses) are known as virologists.
Capsid (protein coat), a nucleic acid genome (DNA or RNA) and viral envelop are the main components of a viral particle or virion. The capsid or protein coat helps to protect the viral nucleic acid genome from destruction or inactivation by nucleases; and they also facilitate the attachment of viruses to host cells as well as their transfer from one cell to another. The nucleic acid genome contains the genetic information required to produce viral proteins and other molecules necessary for the formation and coupling of new virions. The envelope surrounds the nucleocapsid of some viruses; and they bear specific viral glycoproteins that facilitate viral attachment to host cell(s). As shown in this illustration, the capsid, nucleic acid genome and envelope are the main components of a virus. The nucleocapsid is a combination of the capsid and nucleic acid genome of a virion. While naked viruses lack envelope (an external structure outside the nucleocapsid), naked viruses do not have envelopes.
Figure 1: General structure of a virus. This is an illustration of both naked virus and enveloped virus.
Viruses are ubiquitous in the universe; and they play various roles ranging from causing epidemics and other viral infectious diseases to their utilization in molecular medicine and pharmaceutical industry for the production of effective vaccines for disease prevention and control. Viruses are distinctive from other microbial cells because they infect other forms of life including prokaryotic and eukaryotic cells. Bacteriophages are viruses that infect bacterial cells or prokaryotes. Such group of viruses that infect bacteria can also be known as phages. Bacteriophages are prevalent in nature; and when they attack bacteria especially a bacterial culture, there is an observable appearance of cleared zones known as plaques that shows the points where the bacterial cells growing in the culture plate have been lysed. It is noteworthy that viruses are not actually cells per se; and this is due in part that they solely depend on their host cell for their own cellular and metabolic activities.
However, viruses are not completely regarded as non-living entities because they also exhibit some level of life as other forms of microorganisms. For example, viruses have their own genome (DNA or RNA) like other living organisms. They are capable of autonomous replication with the support of the host cell’s genome which they infect and overpower; and viruses are adaptable to a particular niche or habitat especially those of a living cell that they parasitize in order to assume a living state. Nonetheless, viruses exist as inert cells outside of a living host cells; and they are generally maintained in the laboratory in cell/tissue cultures in vitro and even in egg embryos in vivo. Viruses possess no functional organelles for the synthesis of their own macromolecules as is obtainable in other forms of life including bacteria and fungi. They are metabolically inactive or inert outside their host cell; and in such scenarios viruses only exist as crystallized forms from which they can easily be transmitted to susceptible cells to cause infection.
Since they lack their own independent cellular, metabolic and reproductive prowess, viruses take over the cellular machinery of the cells which they infect and cause same to synthesize products that are beneficial to them (i.e. the invading viral particle). Within the infected host cells, viruses mainly exist as replicating nucleic acid particles (DNA or RNA) coated with protein coat; and they stimulate and overwhelm the host’s metabolic and biosynthesizing machinery to produce the cellular components required for the formation of complete virions. Viruses also exist in the soil, water and air. Viruses like other microbes are ubiquitous and they are found in the air (e.g. influenzae virus), in water and food (e.g. rotavirus); and they can be transmitted to humans through vectors (e.g. yellow fever virus) and through sexual intercourse (e.g. HIV).
The study of virology is significant now than ever before owing to the fact that some viral particles or viral diseases are now emerging and re-emerging. Several outbreaks of viral diseases including but not limited to Ebola haemorrhagic fever and Lassa fever virus have been recently reported in some parts of the world. AIDS, severe acute respiratory syndrome (SARS), influenza virus infection, rabies, respiratory synctial virus (a common cause of pneumonia in infants), human Papilloma virus (causative agent of cervical cancer in women) and hepatitis infection (especially hepatitis B virus, HBV and hepatitis C infection) are some viral infections that still parasitize or harm humanity; and these viral infections amongst others have continued to cause several public health issues across the world, thus putting the public health into jeopardy. These infectious diseases caused by viruses and many other deleterious effects of these unique class of microorganisms (i.e. the viruses) have made the study of viruses more significant than ever before. And it is critical that the microbiologists and other medical and biomedical scientists become well-informed about the basic knowledge of viruses and how these small infectious diseases particles could be properly contained and harnessed for the benefit of all.
Only a proper knowledge of viruses can help health care practitioners and the academia to develop ways of containing viral infections especially now that some recent viral disease outbreak (e.g. Ebola virus disease, EVD) has continued to remind us that pathogenic virus or viral infections is still on the horizon. In this Chapter, we have outlaid the basic concepts of Virology in a more concise manner reliable for teaching, research and public awareness. Though this Chapter on virology is never intended to act as an encyclopedia of viruses as it will take hundreds of volumes of books to do so; it has succinctly explored several fundamental areas of Virology including viral structure, classification, viral cultivation and viral replication amongst others. And it is the optimism of the authors that this Chapter on Virology will help the reader to grasp all the fundamental aspects of Virology.
BRIEF HISTORY OF VIROLOGY
The field of virology (inclusive of medical virology, plant virology and veterinary virology) blossomed following the discovery and development of transmission electron microscopy (TEM). In this section, the historical development of the field of virology shall be succinctly highlighted with particular interest to those areas and scientists that contributed to the foundational advancement of this important discipline of medical/biological sciences. Virology as a field in the biological sciences has spanned over 200 years; and this important discipline has continued to remain relevant to mankind and its environment owing to the varying economic importance of viruses. Mankind has lived with the negative effects of viruses especially in the ability of these small infectious agents to cause devastating diseases (e.g. measles, poliomyelitis and smallpox).
Measles (caused by measles virus) for example is one viral infection that ravaged humanity worldwide causing high mortality rate in children; and the disease is characterized by reddish rash that covers the whole body. Though smallpox have been eradicated worldwide inclusive of measles; poliomyelitis is yet to be fully eradicated in some countries; and there exist vaccines that are used for the prevention of these viral diseases in children. The existence of very small infectious particles or agents too small to be seen by the light microscope and that were able to cause disease in man, plants and animals was long hypothesized by scientists since time immemorial (especially between the 18th to 19th century) even though these disease agents was not actually given a name as at the time. It was discovered as at the time that there exist infectious disease agents (in this case: viruses) that were capable of passing through filters that were very small enough to hold back all infectious agents including the smallest bacteria. These filterable infectious agents were later called “virus” to mean “poisonous liquid” in Latin.
Vaccination (which is the medical process of immunizing a host (man or animal) against an infectious disease by injecting it with substances (i.e. vaccines) that contain antigens to a particular disease so that the host’s body could build up or develop protective antibodies in advance) actually developed from the study of viruses. Today, there are many infectious diseases (including those caused by viruses and bacteria) that humans and animals can be vaccinated against; and this singular process of preventing diseases in advance i.e. before they occur has saved countless number of humans and animals as well from many killer diseases. Vaccination has contributed a lot to humanity in terms of the relief it gives man from infectious disease; and this one aspect gives impetus to the economic importance of virus aside the fact that they cause significant number of diseases in man, plants and animals. Some of the commonly used vaccines that are of viral origin and are used in clinical medicine are shown in Table 1. As at the time that smallpox was ravaging humanity, it was discovered that those who survived the disease outbreak were inoculated with smallpox crusts or pustules; and this singular practice generally known as variolation protected susceptible individuals from contracting smallpox disease. In China precisely (around 1000 BC, smallpox was highly endemic and they used this concept of variolation to protect susceptible persons from smallpox infection.
Variolation is an old or prehistoric practice used by some cultures to protect people from smallpox infection; and it mainly involves the inoculation of susceptible people with smallpox lesions in order to prevent them from contracting the smallpox disease. Variolation is a primitive type of the modern-day vaccination; and the principles of immunization draw its concept or foundation from this obsolete mode of disease prevention. In 1796, a notable scientist known as Edward Jenner (1749-1823) discovered that cow maids infected with the cowpox virus were naturally immune to smallpox infection; and this observation impelled Jenner (a recipient of variolation) into discovering the reason behind this and thus he inoculated humans with cowpox pustules, an act that protected people from smallpox infection as at the time. Jenner inoculated a 13 year old boy with vaccinia virus obtained from a young woman infected with cowpox; and this microbial challenge protected the young lad from smallpox infection.
Dmitri Iwanovski (1864-1920) showed in 1892 that leaf extracts from infected plants could cause disease in healthy plants even after filtration. Dmitri Iwanovski, a Russian bacteriologist attempted to establish the cause of tobacco mosaic disease in 1892 by filtering the sap of diseased tobacco plants through a porcelain filter that was designed to retain bacteria. Scientists in time past had shown that the tobacco mosaic disease could be transmitted from a diseased plant to a healthy plant. To the surprise of Dmitri the infectious agent in the sap of diseases tobacco plants passed through the small pores of the porcelain filter. The healthy tobacco plant came down with tobacco mosaic disease when the infectious sap filtrate was injected into it. Iwanovski called this filterable infectious disease agent a toxin. Dmitri Iwanovski then concluded that the infectious agent responsible for the tobacco mosaic disease was an organism or toxin small enough to pass through the small pores of the porcelain filter.
Table 1: Some commonly used vaccines (of viral origin) in clinical medicine
|Hepatitis B||Hepatitis B infection|
|Yellow fever||Yellow fever infection|
|Hepatitis A||Hepatitis A infection|
This filterable infectious agent that affected plants was later called tobacco mosaic disease virus by Martinus Beijerinck (1851-1931) and his colleagues who in 1898 confirmed the work of Dmitri Iwanovski on plant disease that could be transmitted to healthy plants. Martinus hypothesized that the tobacco mosaic disease in plants was caused by a filterable virus (an infectious particle different from bacteria) since the filtered sap of the diseased plant remained infectious after passing through filters that held back bacteria. They determined that the behavior of the infectious agent causing tobacco mosaic disease in the tobacco plants was different from that of bacteria. And that this infectious particle or agent could only survive inside a living host cell but survive in an inactive state outside living cells.
The tobacco mosaic virus was finally isolated in pure form in 1935 by Wendell M. Stanley. Wendell was the first to establish the chemical nature of viruses; and this was achieved when he showed in the early 1930s through advanced techniques that the tobacco mosaic virus (TMV) is mainly made up of protein molecules. It was later that scientists (particularly Frederick Bawden and Norman Pirie) showed that the TMV does not just contain proteins but nucleic acids. And this was later confirmed in the 1930s following the discovery of the electron microscope that viruses are infectious particles or agents that are made up of proteins and nucleic acids. In 1900, Reed Walter (1851-1902), a U.S army physician showed in 1898 that yellow fever was caused by a virus transmitted via mosquitoes to humans. And this was the first discovery that viruses could be transmitted via insect vectors.
Another milestone in virology was unraveled when in 1915, Frederick William Twort (1877-1950) showed that bacteria could be attacked or infected by virus; and these bacterial viruses were later known as bacteriophages or phages. Viruses that attack Archaea also exist and they are known as archaeal viruses. Archaeal viruses which are mainly DNA-containing virions include those that affect or infect members of the Euryarchaeota and Crenarchaeota families; and no RNA viruses have so far been discovered to infect the Archaea. The DNA genome of the Archaeal viruses is double stranded; and the structure of the virions generally known as the Archaeal viruses is unusual and different from the normal structures of other virions that affect the prokaryotes and eukaryotes.
Notably, most Archaeal viruses assume a spindle-like structure that resembles that of the T4 bacteriophage. The term “bacteriophage” was actually coined by Felix d’Herelle (1873-1949) who used bacteriophage or phage therapy to treat some bacterial related diseases (e.g. cholera, bubonic plague, dysentery, streptococcal and staphylococcal infections) as at the time. This serendipitous discovery of bacteriophages by Felix was noted when he observed that his Shigella cultures were surrounded by plaque-forming agent or filterable virus that encircled the growing bacterial cells on the culture plate. And he later showed that bacteriophages could eat holes in bacterial lawns and that they only reproduce within living cells.
Thus the discovery of bacteriophages was largely accredited to both Twort and d’Herelle who worked independently to discover these bacterial eating viruses known as phages. Many viruses have since been discovered especially the human immunodeficiency virus (HIV) that causes AIDS and have ravaged humanity since its discovery in 1983. Though several studies are underway to find a cure for this world epidemic (AIDS), there is still no permanent cure or vaccine against the dreaded disease. And the reality of AIDS amongst other viral diseases has continued to shed more light on the field of virology which studies viruses. However, several breakthroughs in the field of virology especially in the discovery of potent vaccines and therapeutics as well as the discovery and development of the transmission electron microscope in the early 1930’s has given impetus to the relevance of this subject in our world of today.
WAYS IN WHICH VIRUSES DIFFER FROM OTHER LIVING CELLS
Viruses as aforementioned differ tremendously from other unicellular microorganisms in several ways and these shall be highlighted in this section.
- Viruses reproduce only in living cells. Thus, viruses are obligate intracellular parasites since they only reside and replicate within infected host cells.
- They lack cellular structure; and viruses generally have the ability to infect other forms of life including bacteria, Archaea, animals and plant cells. .
- Viruses lack functional organelles (e.g. ribosomes) for the synthesis of important cellular and metabolic molecules.
- Viruses have the ability to integrate their own genome (i.e. DNA or their RNA transcript) into the genome of their host cell.
- They have simple acellular organization that comprises mainly of a particular nucleic acid (DNA or RNA) and a protein coat.
- Viruses do not carry out binary fission or cell division the same way that the eukaryotic or prokaryotic cells do.
- Viruses lack a metabolic process or system of their own. Instead, they take advantage of the cellular and metabolic processes of their host cells to generate their own energy and thus carry out their metabolic functions.
- They are not inhibited or killed by antibiotics. But viruses can be affected by interferons. Interferons are proteinous substances produced by host cells especially in response to a viral invasion or infection; and they generally help to limit the spread of the virus in the host’s body.
- Viruses are per se the smallest forms of microorganisms and they usually range from 20 – 300 nm or 350 nm in size. And thus viruses cannot be seen with the light microscope (whose resolving power is about 300 nm) but only with the aid of the electron microscope. Due to their relatively small sizes; viruses or virions are measured in nanometers (nm). Parvoviruses are among the smallest viruses (about 20 nm in size) while the largest viral family have a size of about 300-350 nm (e.g. smallpox virus). The largest known virus is mimivirus (Figure 2).
Figure 2: Transmission electron micrograph of mimivirus. Mimivirus is the largest known virus; and its shape is as large as that of some small bacteria (e.g. E. coli). This virion has a large genome and shape. Mimivirus mainly infect amoeba especially the Acanthamoeba species. However, it is also believed to be causative factor in pneumonia in humans.
ECONOMIC IMPORTANCE OF VIRUSES
Though they are known to cause plethora of infectious diseases in man, plants and animals; viruses are very useful tools that can be exploited to the benefit of mankind.
- Viruses are employed in the development of novel vaccines for the prevention of infectious diseases including those caused by viruses.
- Some viruses known as phage or bacteriophage that infect bacteria are used in bacterial taxonomy to classify bacteria. This is known as phage typing of bacteria; and in this technique the bacteria is classified based on the type of bacteriophage that they are susceptible to. And this has helped in the epidemiological containment of diseases especially in disease outbreak.
- Viruses are employed in the production of antiviral drugs and diagnostics used for laboratory diagnosis of some infectious diseases.
- Some viral particles can be used as pesticides to control rodents and pests in the farmlands.
- Reverse transcriptase (RT) enzyme is an enzyme that catalyzes the transcription of RNA into DNA; and this enzyme is applied in recombinant DNA technology or molecular biology for the molecular manipulation of microorganisms. RT which can also be called RNA-dependent DNA polymerase is mainly produced by viruses in the Retroviridae family (e.g. retroviruses).
- Viruses can also serve as vectors for the transmission of genetic materials (i.e. genes or DNA) from one organism to another.
- Some viruses have been employed as anti-cancer agents for the treatment of cancer and other molecular diseases.
- The study of viruses especially at the molecular level using cell/tissue culture techniques and electron microscopy has acquainted biologists with knowledge that led to the development of other fields such as cell and molecular biology. Such studies also led to the discovery of important cellular and metabolic components of cells that allowed scientists to understand the true nature of some molecular and infectious diseases of man.
Prions are sub-viral infectious entities that consist mainly of proteins. They are unique infectious particles that lack nucleic acids inclusive of DNA and RNA. Prions are infectious proteins that generally lack DNA and RNA; and they cause series of diseases in man and animals. They differ from the normal virion in so many ways. Prions are nonimmunogenic while viruses are immunogenic. The immune system of mammals is incapacitated to mount an immune response to the invasion of host cells by pathogenic prion proteins. Viruses possess an RNA or DNA genome that is completely devoid in prions. Prions are composed mainly of an abnormal, pathogenic isoform that is the only identifiable macromolecule in purified preparations of prions. These identifiable macromolecules of prions are generally known as prion protein scrapie (PrPSc), and the PrPSc are the pathogenic forms of the prion protein (PrP). The native cellular isoform of the prion protein which are normally found in mammalian cells are known as prion protein cellular (PrPC).
Mammalian prions reproduce by recruiting the normal, cellular isoform of the prion protein (PrPC) and stimulating its conversion into the disease-causing isoform or pathogenic forms of the prion proteins (PrPSc). Though they lack nucleic acids (which are unique in carrying genetic information for disease initiation in a particular host), the prions cause diseases in animals and mammals as aforementioned. Kuru disease (that occur in cannibalistic mammals), Creutzfeldt- Jakob disease (CJD) that occur in humans, scrapie (that occurs in goat and sheep), bovine spongiform encephalopathy (BSE) that occur in cattle and chronic wasting disease (that occur in deer and elk) amongst others are typical examples of prion diseases in animals and mammals (Table 2).
Transmissible spongiform encephalopathy (TSE) is the fatal prion disease of the nervous system that is characterized by the degeneration of the spongiform of the brain in animals. Animal prion diseases are collectively known as transmissible spongiform encephalopathies (TSEs). Since prions lack nucleic acid molecules, how then do they replicate in vivo to initiate a disease process or produce their own progeny from parental cells? Prion molecules as aforementioned are mainly made up of PrP; and the host cell usually contains a gene that encodes the native form of the prion proteins (i.e. PrPC). This gene in the host cell that encodes for PrPC is known as the human PrP gene (Prnp); and the gene is located in chromosome number 20 in humans and chromosome number 2 in mice.
Table 2: Summary of some prion diseases in man and animals
|Prion disease||Natural host|
|Scrapie||Sheep and goat|
|Creutzfeldt-Jakob disease (CJD)||Humans|
|Chronic wasting disease||Elk and deer|
|Fatal familial insomnia (FFI)||Humans|
|Bovine spongiform encephalopathy (BSE)||Cattles|
|Fatal sporadic insomnia (FSI)||Humans|
|Feline spongiform encephalopathy (FSE)||Cats|
|Gerstmann-Straussler-Scheinker syndrome (GSSS)||Humans|
The pathogenic form of prion proteins (i.e. PrPSc) is identical to the cellular isoform of the prion protein (PrPC), which is non-pathogenic in nature. This genetic similarity between the epitope of PrPC and PrPSc is responsible for the non-immunogenicity of prions, and the reason why host cells fails to mount an immune response to prion invasion the same it mounts an attack against viral agents that enter the body. The invasion of a host cell by PrPSc (pathogenic prion protein) stimulates the genetic conversion of PrPC (the normal and non-infectious prion protein) to PrPSc; and this is particularly applicable in host cells that are expressing PrPC. PrPC and PrPSc are the two known conformations in which prion proteins exist. It is however noteworthy that the replication of prions in host cells is generally facilitated when the sequences of the PrPC and PrPSc are identical.
Thus the presence of PrPC is vital for the development of prion diseases in mammals and animals; and this has been confirmed in knock-out mice (i.e. mice whose Prnp gene have been genetically modified) which are unable to form the PrPC proteins and the prion disease after several challenges with PrPSc, the pathogenic prion proteins. A pathogenic prion protein (PrPSc) replicates itself in host cells be converting healthy prion proteins (PrPC) to the pathogenic isoforms (i.e. PrPSc). PrPC is normally produced by cells of the neurons in mammals and animals; and a misfolding of the PrPC during its synthesis in the neuron can lead to the conversion of PrPC to PrPSc which is insoluble and resistant to proteases (protein degrading enzymes).
The formation of PrPSc leads to the development of aggregates of the pathogenic prion proteins, and this phenomenon causes a damage of the neural tissues, brain tissues and other neurological symptoms that causes prion diseases to ensue. Profound neurological dysfunction in animal and mammalian hosts infected by pathogenic prion proteins is usually fatal in nature. Prion diseases are generally initiated in host cells by a molecular mechanism that causes pathogenic prion proteins to arise spontaneously; and infectious prion proteins are infectious and can be transferred from one infectious host to another susceptible host. Prions have also been discovered to infect fungi especially yeast cells aside animals and mammalian cells that they are mainly known to infect. There is currently no therapeutic option for the treatment of prion diseases even though several compounds have been tested and suggested for use in the management of neurodegenerative diseases caused by pathogenic prion proteins.
Viroids are sub-viral infectious entities that infect plants. They are acellular infectious agents like the prions that lack the essential features of a virus; and which are capable of directing their own replication since they have small naked RNA as their genome. Unlike the prions which lack nucleic acids (inclusive of DNA and RNA), viroids contain single-stranded RNA molecules. Viroids are generally plant-pathogenic infectious particles that comprises of small, naked and circular ssRNA. They lack protein coat since they are naked ssRNA molecules. The ssRNA depends entirely on the infected host cell for its replication since they lack genes that encode protein biosynthesis; and they rarely code for any protein molecule.
Viroids can exist outside of their plant host cell unlike viruses. Viroids cause significant damage to crop plants. And this lead to considerable commercial damage to agricultural products, a factor that can impact negatively on food security. Viroids are transmissible from plants to plants, and they are the smallest known pathogens that contain a nucleic acid molecule (particularly ssRNA). Some of the plant diseases caused by viroids include: tomato apical stunt viroid (TASV), coconut cadang-cadang viroid (CCCV), potato spindle tuber viroid (PSTV), tomato bunchy top viroid (TBTV), cucumber pale fruit viroid (CPFV), citrus exocortis viroid (CEV) and hop stunt viroid (HSV) amongst others. Cocaviroid, Popsiviroid, Coleviroid, Hostuviroid and Pelamoviroid are some examples of viroid families or genera that cause disease in plants.
The symptoms of plant infection by viroids is usually seen as plant discolouration, leave malfunction, fruit malfunction and stunted growth of particular crop plants. The transmission of viroids amongst crop plants usually occur during crop propagation such as in grafting of plants and via other mechanical damage to the plant; and viroids can also be transmitted via pollen grains and seeds amongst plants. The mechanical damage that causes viroids to penetrate food crops or plants can be caused by farming implements such as cutlasses, hoes and diggers. Some insects especially aphids are notorious in transmitting plant viruses (i.e. viroids) via insect bites; and this occurs when the insects feed on plant saps. Viroids are not known to cause disease in animals; and none have also been identified to infect bacterial cells like the bacteriophages.
Viruses are infectious agents that have a simple acellular structure that is mainly made up of a protein coat or capsid and a nucleic acid genome which can either be DNA or RNA. Some viruses also have envelopes (which are lipid-containing outer membranous layer that surround the nucleocapsid in some viruses) while others lack them, and are thus generally known as naked viruses. Viruses with envelopes are known as enveloped viruses. Viruses are unique group of microorganisms that are composed of several chemical molecules or structures that are vital to their development and replication within a particular host cell. These chemical compositions of viruses include the viral proteins, viral envelopes, viral glycoproteins and viral genomes; and they form the integral parts of mature viruses.
- Viral proteins: Viral proteins are important components of a virus because they serve as the main antigenic determinants or epitopes of an infecting virus. The viral proteins bind specifically to the receptor molecules on host cells, and this facilitates their penetration or entry into the cell. Viral proteins form parts of the viral capsid or protein coat which serves to protect the nucleic acid genome of the infecting virus. This protective function prevents any external damage to the viral nucleic acid either by enzymes that readily hydrolyze or destroy the viral genome. Generally, the viral proteins provide and maintain the structural integrity or symmetry of viruses.
- Viral enzymes: Viruses like other organism contain enzymes (which are proteinous in nature) that facilitate their development and replicative processes within their host cell. These enzymes which include reverse transcriptase (RT) and RNA polymerase enzymes play critical role in the replication process of viruses that harbour them, even though the enzymes may have little or no function in the protein coat of the virus.
- Viral genomes: The nucleic acid genome of a virus is either DNA or RNA. No virus contains both DNA and RNA as its nucleic acid genome. Viral nucleic acid genome is important because it is what contains the genetic information necessary for the replication of an infecting virus within a host cell. The genome of a virus can assume several orientations. The genome of a virus can either be circular or linear in structure. Some are double-stranded (ds) while others are single-stranded (ss). The viral genome can also be segmented or non-segmented; and while some RNA-containing viruses have genomes with negative-sense strands (-) others have genomes with positive-sense (+) strands. Some RNA-containing viruses have genomes that are ambisense – because they have a partial genetic coding sequence in the positive-sense as well as in the negative-sense. Arenaviruses are examples of viruses with ambisense genomes. There is a great variation in the nucleic acid genome of a virus depending on whether the virus is a DNA-containing virus or an RNA-containing virus. Also, the genome size of DNA viruses (which is usually in the range of 3.0-370 kbp) is different from the genome size of RNA viruses (which is usually in the range of 7-30 kb). All animal viruses including those that infect humans have a dsDNA genome. The only exception is parvoviruses (in Parvoviridae family) which have ssDNA genome. All RNA viruses that infect animals and humans have ssRNA genome with the exception of reoviruses (in Reoviridae family) which have dsRNA genome.
- Viral glycoproteins: Viral glycoproteins are viral molecules that comprises of proteins and carbohydrates which are attached together. They are mainly found on enveloped viruses; and viral glycoproteins are virus-encoded in that they are generated by the viruses themselves. Viral glycoproteins form the surface glycoproteins of enveloped viruses and they aid in the adsorption of enveloped viruses to specific molecules on the surface of the host cell. Naked viruses lack viral glycoproteins.
- Viral envelopes: Some viruses are known as enveloped viruses because they contain envelope while other are referred to as naked viruses because they lack envelopes. Enveloped viruses acquire their envelopes (which are lipid-like) through the cytoplasmic membrane of their host cell by a budding process. And this occurs during the maturation and release of the virus from the host cell. Since they contain lipids, enveloped viruses are sensitive to lipid-solvents, ether and other organic solvents unlike naked viruses that lack lipid-envelopes and are resistant to treatment with organic solvents.
MORPHOLOGY OF VIRUSES
As shown in Figure 1 above, a virion is composed mainly of three parts viz: the nucleic acid genome (DNA or RNA); the capsid or nucleocapsid; and the envelopes which surround the capsid or protein coat. The nucleic acid genome of a virus or virion can assume any of the following genetic composition:
- Double-stranded DNA (dsDNA)
- Single-stranded DNA (ssDNA)
- Double-stranded RNA (dsRNA)
- Single-stranded RNA (ssRNA)
Viruses assume different shapes and sizes. Morphologically, viruses have four main types of structures based on their capsid structure viz: icosahedral or cubic viruses, complex viruses and helical viruses. Some viruses however have envelops and are thus known as enveloped viruses. Icosahedral or cubic viruses have a symmetry that is in a closed shell; and typical example of viruses in this morphological group is the adenoviruses (Figure 3). The icosahedral symmetry is exhibited by both DNA and RNA viruses. Some viruses have a spherical symmetry (Figure 4). The helical symmetry is most common amongst viruses in the orthomyxoviridae family and the tobacco mosaic viruses that infect plants also have a helical structure. Helical structure is formed when the protein subunits of the virion are bound in a periodic pattern to the viral nucleic acid in such a way that it is wound into a helix (Figure 5). Viruses with a spherical shape usually assume a circular or globular structure. Influenzavirus in the Orthomyxoviridae family is an example of a virus with a spherical structure.
Icosahedral symmetry is an example of cubic symmetry assumed by most viruses. And the icosahedral symmetry is exhibited by both DNA-containing viruses and RNA-containing viruses. Typical examples of viruses with the icosahedral symmetry are those in the viral families: Caliciviridae, Astroviridae, Picornaviridae, Birnaviridae, Reoviridae, Parvoviridae, Polyomaviridae, Papillomaviridae, Adenoviridae, Hepadnaviridae and Bornaviridae. Viral spikes are external polypeptide projections found on the surface of viruses and which aid in the attachment or adsorption of viruses to the surface of their host cell(s). They form part of the polypeptide molecules that make up a virion. Other viral polypeptides aside viral spikes include: membrane proteins, haemagglutinins, membranes and nucleocapsid. Helical symmetry is another typical viral shape assumed by most viruses especially the RNA-containing viruses. Most of the animal viruses with helical symmetry that infect animals including humans have RNA-containing genomes. The tobacco mosaic virus (TMV) – which infects plants, is a typical example of virus with a helical symmetry.
Figure 3: Illustration of viral icosahedral symmetry.
Viruses that do not show either the icosahedral or helical symmetry form a complex structure; and such viruses have an architectural plan that resembles a brick-shaped configuration with ridges on the outside and lateral bodies and core on the inside (Figure 5). Poxvirus and bacteriophages are examples of viruses that form complex symmetry. Complex viruses as shown in Figure 6 have a tail or tube-like structure through which its nucleic acid genome is inserted into the host cell they infect. The viral nucleic acid genome (DNA or RNA) contains the genetic information of the virion and it is also responsible for directing the infectiousness of the virus. The capsid or protein coat (which can be icosahedral, helical or complex) provides a protective function for the virion in that it protects the nucleic acid genome from enzymatic action. It is antigenic in nature, and the capsid also acts as specific binding sites that mediate binding or attachment of the virus to their host cell. The envelope consists mainly of lipid bilayers and glycoproteins which facilitate the entry of the virion into the infected host cell.
Figure 4: Illustration of spherical symmetry.
Figure 5: Illustration viral helical symmetry.
Figure 6: Structure of T4 bacteriophage, a complex virus. The different components of the phage are: baseplate, tail fibers, sheath, core, collar and head.
TAXONOMY OF VIRUSES
The taxonomy of viruses unlike the classification of other microorganisms such as bacteria is not actually straight forward and it is quite different from the normal classification of bacteria as aforementioned in Chapter 3 of this textbook. Due to the nature of these infectious particles or agents (i.e. viruses) especially due to the fact that they only contain a particular nucleic acid genome (DNA or RNA) that is enclosed in a protein coat known as the capsid or nucleocapsid; viral classification is done with considerations to some features about the infectious agent. Viruses are mainly classified on the basis of: 1) their nucleic acid genome; 2) the morphology of the virion; and 3) the mode of replication of the virus under consideration. The presence or absence of envelops in a particular virus as well as its size or shapes are other factors considered in viral classification.
Viruses are generally placed in different families based on these aforementioned criteria. And within each viral family are subdivisions known as genera or genus which usually carries the suffix “virus”. The taxonomy of viruses is governed by a Universal System of Virus Taxonomy generally called the International Committee on Taxonomy of Viruses (ICTV) as established in the early 1960’s. It is now on the shoulders of the ICTV formally known as the “International Committee on the Nomenclature of Viruses, ICNV” that the responsibility of classifying and naming viruses as they are being discovered rest upon. So far, the ICTV have classified viruses into different genera’s and families; and this includes viruses that infect or cause disease in humans, plants and animals.
Thus, in the current classification of viruses according to the ICTV, viruses are classified into different taxa that include viral families, orders, genus and species. Since the nucleic acid genome of viruses is either a DNA or RNA, viruses are classified into different families based on this; and thus there are DNA viruses and RNA viruses. The DNA genome can be single-stranded (ss) or double-stranded (ds) and this also applies to the RNA genome. However, the ssRNA viruses can be further divided into two groups depending on whether their ssRNA is a negative strand (-RNA) or a positive strand (+RNA). It is worthy of note that all DNA viruses (excluding those that belong to the Parvoviridae family) are double-stranded and all RNA viruses (excluding those that belong to the Retroviridae family) are single-stranded.
REPLICATION IN VIRUSES
Replication is defined as the process in which a cell divides to make copies of its genome or itself. Cell division or replication in viruses is different from what is obtainable in other microbes such as bacteria that mainly replicates by binary fission. Replication in viruses only occurs inside a suitable host, and in such cases the virus redirects the metabolic and cellular machinery of its host cell to support the replication of the virus in vivo. Viral genomes (DNA or RNA) are usually introduced into the host cell through infection, and once they have gained entrance the virus ensures that the host cell is overwhelmed to produce the different viral components (e.g. capsid and nucleic acid) required to generating new virions that will be released to continue the infection process. Viral replication usually involves several phases from the point of attachment of the virus to its host cell and the release of new virions from the host cell. Attachment, penetration, uncoating, expression of viral nucleic acid, biosynthesis of viral components, assembly and the release of mature and complete virions are the main stages that characterize the replication of viruses in a host cell (Figure 7). These important factors of the viral replication cycle are highlighted in this section.
1. ATTACHMENT STAGE OF VIRAL REPLICATION
As aforementioned, viruses usually gain entry into their host cell through infection. Attachment or adsorption is the first step in viral replication; and this stage is critical because without it the infecting virus cannot gain entry into its target host cell. In adsorption (attachment), the infecting virus attaches to specific receptors on the cell membrane of its target host cell through its capsid or surface proteins and this interaction between the infecting virus and the target host cell is vital for viral entry. Absence of a particular receptor site on the host cell membrane that the infecting virus can recognize means that the infecting virus will not attach and this ultimately prevent infection because the infecting virus cannot attach.
One of the major aims of an infecting virus is to replicate its genome especially in a host cell, and in order to achieve this the virus must find a way to first of all enter the target host cell and then takeover its metabolic and cellular machinery to manufacture its own viral components so that new virions can be generated and released. But this cannot be possible if the infecting virus fails to attach itself first to specific protein molecules (inclusive of other lipoproteins, carbohydrates and glycoproteins) found on the cell or plasma membrane of the target host cell. The first step involved in the replication of a virus is the attachment or adsorption of the infecting virus to the surface proteins found on the surface of its host cell. The protein molecules found on the capsid of the virus interact specifically with the surface receptors on the host cell; and this facilitates the entry or penetration of the infecting virus into the cell.
After entry, the infecting virus uncoats and releases its nucleic acid genome (DNA or RNA) from its nucleocapsid or capsid. The released nucleic acid genome takes over the cellular machinery of the host cell and starts expressing its own genetic makeup. The expression of viral nucleic acid genome within the host cell leads to the biosynthesis of specific viral proteins and viral nucleic acids required for the assembling of new virions. Newly synthesized virions must be packaged into a complete virion or viral particle before it can be released from the host cell to infect new cells; and this is prerequisite for the infection of new cells within the organism. After proper assembling or packaging, the newly synthesized virions are released from the host cell through cell lysis (for naked viruses) or through budding (for enveloped viruses).
Figure 7: Overview of viral replication.
These receptors are unique and specific in nature, and viruses usually have several multiple protein sites that can bind to host cell receptors in a specific fashion in order to facilitate their entry into the host cell. Viral pathogenesis (i.e. the ability of a virus to cause infection in its host organism or cell) is largely dependent on the ability of the infecting virus to attach to receptors on the target host cell membrane; otherwise there will be no initiation of infection because viral entry will not be facilitated if the infecting virus fails to adsorb or attach to a specific receptor expressed the surface of a susceptible host cell. It is noteworthy that susceptible host cells that fail to express specific receptors for viral attachment cannot be infected by the infecting virus; and the host cell can also become resistant to the infecting virus in cases where the target receptor on the surface of the host cell becomes mutated and altered in such a way that it cannot be recognized. The host cell is said to be resistant in such scenarios. However, some mutated viruses can still come up and attach to the mutated cell surface receptors. The cell surface receptors on the host cell generally determine whether the host cell will be infected or not by a particular infecting virus.
2. PENETRATION (ENTRY) STAGE OF VIRAL REPLICATION
Penetration is the next stage that follows a successful viral attachment in the viral replication cycle. Viruses usually penetrate or enter their target host cell after attachment through a biological process known as endocytosis. Endocytosis is simply defined as the process in which a virus is taken intact into a host cell. Viral entry into their host cell is critical because viral replication can only occur within a particular host cell and not outside it. The mode of penetration of the infecting virus is usually different depending on whether the virus is a naked virus or an enveloped virus. However, the main aim of this stage is to ensure that the infecting viral genome penetrates the host cell so that it can be replicated. In naked or non-enveloped viruses, the plasma membrane of the susceptible host forms an endosome or endosomal cavities that enclose the infecting virus and this facilitate its entry through endocytosis into the host cell. Enveloped viruses usually uncoat at the plasma membrane and this facilitates the entry of the virion content into the host cell. The fusion of the lipid envelop of enveloped viruses with the plasma membrane of the susceptible host cell facilitates the entry of the enveloped viruses into the host cell. Nonetheless, endocytosis is the main process via which the naked viruses and enveloped viruses enter their host cells.
3. UNCOATING STAGE OF VIRAL REPLICATION
Uncoating is another key step in the viral replication cycle. This is because it is at this stage that the viral nucleic acid genome (DNA or RNA) is released from the capsid or nucleocapsid of the infecting virion. This important stage of viral replication cycle can be inhibited by the host’s defense mechanisms such as the release of enzymes (e.g. lysozymes) that inactivate the pathogenic potential of the infecting virus prior to uncoating. During uncoating, the viral nucleocapsid or capsid and other associated protein molecules is physically separated from the genome or removed in order to expose the viral genome which takes over the cellular and metabolic machinery of the host cell for the commencement of viral replication and the production of new viral components. At this stage of uncoating, the infecting parent virus losses its infectivity and thus it can longer be infectious since its protein coat or capsid has been physically separated from its genome.
4. EXPRESSION OF VIRAL NUCLEIC ACID AND BIOSYNTHESIS OF VIRAL
After uncoating as aforementioned, the viral nucleic acid genome (DNA or RNA) becomes expressed within the host cell and this stage usually follows the central dogma of molecular biology in which DNA is transcribed to mRNA that is later translated to specific proteins. Specific mRNA must be transcribed from the viral genome (e.g. DNA) for the successful expression and duplication of the genetic information of the infecting virus. DNA replication mainly occurs in the nucleus of the infected host cell for DNA viruses; while for RNA viruses (whose genome is mainly mRNA in form), the viral RNA is mainly replicated in the cytoplasm of the cell. However, some DNA viruses (e.g. poxviruses) can still replicate their genome in the cytoplasm; and this variation is observable in some RNA viruses (e.g. retroviruses) that replicate their genome in the nucleus of the infected host cell. Following infection, it is critical to produce new copies of the viral genome inside the host cell and this is usually accompanied by the synthesis of viral proteins as shall be highlighted later.
An understanding of the replication mechanisms of infecting viruses (DNA and RNA viruses inclusive) is vital to the development of novel drugs that can interfere with and inhibit the pathogenic or virulent capabilities of pathogenic viruses especially in viral disease conditions. The synthesis of virus nucleic acid and proteins is vital to viral replication, and this is carried out by the metabolism of the host cell as directed by the infecting virus. New copies of viral genome and specific viral proteins must be synthesized in the infected host cell prior to the formal replication of the said virus. The replication of a particular virus is usually directed by the orientation or structure of its genome. Generally, the messenger ribonucleic acid (mRNA) of a virus is defined as “positive sense” (designated with the symbol ‘+’) while its complementary strand is defined as “negative sense” (designated with the symbol ‘—’). This implies that a nucleic acid strand that has the same sequence as mRNA is designated (+) strand while the one with a complementary strand is designated (—) strand.
And since all viruses (both DNA and RNA viruses) must express their genes or genetic makeup as functional mRNA molecules early enough in the course of their infection or disease development in vivo (i.e. within a living host cell), the mRNA (which is unique in carrying and decoding the genetic information for protein synthesis from the gene) of the infecting virus must always be in preference to the host’s mRNAs potential viral proteins must be synthesized for the coupling of new virions. Viruses as aforementioned are microbes that are composed mainly of nucleic acids and protein molecules and envelopes in the case of enveloped virus. The mRNA is responsible for the translation of genetic information it encodes (especially for the synthesis of specific viral proteins). It converts the genetic information it is carrying into a sequence of polypeptide in order to form a particular protein molecule; and this phenomenon is of utmost importance to virus because if the viral capsid (which is proteinous in nature) is not well synthesized, the viral genome will not be well packaged or assembled; and this generally exposes the virus to destruction by the host immune mechanisms and by other antiviral agents.
Thus, in order to direct the host cell’s translational machinery to make viral proteins; it is critical that the correct viral mRNAs which are designated as positive sense or negative sense depending on the genomic orientation of the virion are always available and well aligned to carry out this important genetic transformation. The phrases ‘sense’ and ‘strand’ are used synonymously in describing the genomic orientation of viruses in this section. It is noteworthy that some viruses can have both the negative and positive strand, and for such viruses; their genomic orientation is said to be ambisense in nature. Different viral families inclusive of DNA and RNA viruses have different genomic orientation that can either be single-stranded(ss) or double stranded (ds); and the polarity of their mRNA can either be positive sense, negative sense or ambisense as the case may be. These variations or features are critical in understanding the replication pattern of each of these viruses that make up the different viral families; and because of the differences in the transcription of the genomes of DNA viruses and RNA viruses during viral replication, it is important to use these designations as aforementioned in the discussions of viral replication for clarity.
The replication pattern of a particular virus is usually determined by the structural orientation and makeup of its genome. For example, viruses with single-stranded positive sense RNA (ss+RNA) genome make use of their positive RNA (+RNA) as mRNA during transcription. These viruses uses the ribosomes and enzymes of the host cell to decode the information encoded in the +RNA genome of the virus during translation to produce protein molecules (e.g. RNA-dependent RNA polymerase). RNA-dependent RNA polymerase is responsible for transcribing the RNA genome of RNA viruses into RNA genomes so that such viruses can be further replicated. Eukaryotic cells do not possess enzymes for the replication of RNA genomes; and thus the infecting virus must provide replicase enzymes such as the RNA-dependent RNA polymerase which initiate the replication of RNA genome of viruses in the host cell(s).
For viruses containing single-stranded negative sense RNA (ss-RNA) genome, their ss-RNA genome must first be converted to a +RNA strand. The +RNA strand is used as an mRNA template for transcription to the genomic –RNA genome. And this is enzymatically catalyzed by RNA-dependent RNA polymerase proteins. In the replication of double stranded RNA (dsRNA) genome of dsRNA viruses, the –RNA strand of the dsRNA genome must first be converted to a complementary +RNA strand that will serve as a template for the replication of the viral genome. RNA-dependent RNA polymerase also plays a role in the replication of the viral genome. Retroviruses (e.g. human immunodeficiency virus, HIV) are RNA viruses that differ markedly from other RNA viruses in that the retroviruses synthesize mRNA and replicate their genome by means of an RNA-dependent DNA polymerase (also known as reverse transcriptase, RT). Hepadnaviruses are another group of animal viruses that utilizes RT in the replication of their genome apart from the viruses in the Retroviridae family (e.g. Retroviruses).
The Retroviruses have an ss+RNA. They do not use their ss+RNA genome as an mRNA template in the replication of their genome because it is not used as a messenger RNA or message. Rather, the RNA genome of Retroviruses is first transcribed into complementary DNA strands by RT in a genetic process known as reverse transcription (which is quite different from the central dogma of molecular biology – in which DNA is transcribed to RNA and then translated to protein). Double stranded DNA (dsDNA) viruses depend solely on the cellular DNA replication of their host cell since the mRNA of dsDNA viruses is transcribed in the same manner akin to DNA replication of host cells. For single stranded DNA (ssDNA) viruses, their ssDNA is first converted to a dsDNA molecule that is transcribed to form mRNA. The host RNA polymerase plays a role in the transcription of the genome of DNA viruses into mRNA; and the viral mRNAs are later translated to specific proteins in the ribosomes located within the cytoplasm of the host cell.
The newly synthesized viral proteins are transported back to the nucleus of the host cell where the progeny viral particles are assembled and released from the infected host cell(s). It is however, noteworthy that new viral proteins are required for the replication of a viral genome (whether DNA or RNA genome) within an infected host cell; and the synthesis of these viral proteins are encoded by mRNAs transcribed from the genome of the infecting virus. And as aforementioned, the viral genomic RNA can also serve as the mRNA (especially in RNA viruses), and in others, the viral genome can serve as a template for the synthesis of the mRNA – which is to be translated for viral protein synthesis. And some viruses such as the Retroviruses contain reverse transcriptase or RNA-dependent DNA polymerase that helps to carryout the transcriptional process in which the RNA genome of the virus is converted to ds DNA that act as the template for the synthesis of mRNA by normal cellular enzymes of the infected cells.
With the exception of Poxviruses and Hepadnaviruses, all animal DNA viruses replicate in the nucleus of their host cells; and the replication of viral genome varies from one virus to another depending on the genome the virus possess and its configuration i.e. whether it is double-stranded (ds) or single-stranded (ss) and whether it is positive sense (+) or negative sense (—). All RNA viruses replicate in the cytoplasm of the infected host cells with the exception of Orthomyxoviruses and Retroviruses (which can replicate in both the nucleus and cytoplasm).
5. ASSEMBLY STAGE OF VIRAL REPLICATION
The assembly of synthesized viral proteins and other associated molecules or particles is critical for viral pathogenesis in living cells. The viral proteins and viral genome must be packaged into a complete virion or viral particle before it can be released from the host cell. Virions carry out a self-assembling mechanism of the viral proteins and viral genomes within the infected host cell. The release of naked viruses form the host cell after packaging is quite different from the release of enveloped viral particles. Viral assembly is the last stage in the life cycle of viral infection; and it is accompanied by the release of the complete viral particles from the infected host cell. Viral replication can occur in any of two ways: lytic cycle of viral replication and lysogenic cycle of viral replication (Figure 8).
The lytic cycle is the normal process of viral reproduction involving penetration of the cell membrane, nucleic acid synthesis, and lysis of the host cell (Figure 8). It involves the reproduction of viruses using a host cell to manufacture more viruses; the viruses then burst out of the cell. Viruses overtake a living host cell and use the cellular machinery of the host cell to reproduce and form its own molecules – since viruses are not capable of independent or self replication. And one of the ways the infecting virus may choose to leave the affected host cell is by destroying the host cell. Viruses usually leave their host cell by cutting (lysing) their way out of the host cell. This is called the lytic cycle of a virus replication. In a lytic cycle, the virus reproduces thousands to millions of times in just a few hours and they produce many viral progeny during this time or process.
This result to the weakening of the host cell wall enough that the cell will lyse, or burst open, setting the army of new viruses free. The lytic cycle results in the destruction of the infected cell and its membrane unlike the lysogenic cycle which does not lead to the destruction of the host cell and its membrane. The lysogenic cycle involves the incorporation of the viral genome into the host cell genome, this infecting the host cell from within (Figure 8). It is a form of viral reproduction involving the fusion of the nucleic acid of a bacteriophage with that of a host, followed by proliferation of the resulting prophage. In the lysogenic cycle, the phage DNA first integrates into the bacterial chromosome to produce the prophage. When the bacterium reproduces, the prophage is also copied and is present in each of the daughter cells. The daughter cells can continue to replicate with the prophage present or the prophage can exit the bacterial chromosome to initiate the lytic cycle. Prophage is the latent form of the virus genome that remains within the host cell without destroying it.
Figure 8: Illustration of the lytic and lysogenic cycles of viral replication.
The difference between the lytic and lysogenic cycles of viral replication is that in the lytic phage, the viral DNA exists as a separate molecule within the bacterial (host) cell, and replicates separately from the host (bacterial) DNA while the location of viral DNA in the lysogenic cycle is within the host (bacterial) DNA. And while the lytic cycle of viral replication leads to the destruction of the host cell and membrane as aforementioned, the lysogenic cycle of viral replication does not lead to the destruction or lysis of the host cell and its membrane. Lytic cycle is a type of viral life cycle that ends with host cell lysis and the release of numerous and newly synthesized viral progenies. This type of viral replication (i.e. lytic cycle) is usually carried out by virulent viruses that naturally lyse their host cells during the reproductive cycle. However, in lysogenic cycles, viruses engage in a different type of relationship with their host. And in this type of viral replication (i.e. lysogenic cycle), the viral genome does not take total control of the host and destroy it while it is still synthesizing new virions or phages as the case may be.
In lysogenic cycle, the viral genome remains within the host cell and replicates its own genome alongside the host (bacterial) genome to produce new cells that continue to grow and divide over a long period of time. The infected host cell looks normal; and each of the infected host cells can go on to produce new virions or phages which will lyse under normal conditions. The infecting virus establishes a type of relationship known as lysogeny – in which both the virus and the host cell coexist without destroying each other. Lysogeny is defined as a relationship in which a virus or phage genome remains within its host cell (e.g. a bacterial cell) after infection and reproduces alongside the host genome instead of taking total control of the host cell and destroying it in the process of replication. The viruses that enter into this type of relationship with their host cell are known as temperate phages while the bacterial host cell that are able to produce new viral or phage particles under these conditions are known as lysogens; and they are said to be lysogenic.
6. RELEASE OF MATURE AND COMPLETE VIRIONS
Completely assembled and packaged viral genome and viral proteins are released from infected host cell as the self-assembly of the individual particles are complete. Naked viruses are usually released from the infected host cell via the lysis of host cells while enveloped viruses are released via the plasma or cell membrane of the host cell in a budding process in which the virion acquires its envelope in the process. The morphogenesis of viral particles usually occurs simultaneously with viral release even though the process may vary in both naked viruses and enveloped viruses. While enveloped viruses mature by a budding process before their release from host cells as aforementioned, matured naked viruses do not undergo budding before they are released. Instead, mature naked viruses are immediately released from their host cells via apoptosis or cell lysis once the complete virion have been well assembled and packaged. Upon the infection of new host cells, the virion disassembles spontaneously and penetration of the virus is initiated in the process of the viral infection. Virus-host interaction is critical to the initiation of a particular disease process in a host; and disease development is dependent on many factors including the amount of released viral particles; the host immune state; the type or number of organs infected by the virus; the virulence factors of the infecting virus; environmental factors and the persistence of the infecting virus within the host amongst other host and viral factors.
HOST IMMUNITY TO VIRAL INFECTION
Most viral infections do not result to disease development with the exception of AIDS and some haemorrhagic viral infections such as Ebola and Lassa fever that leads to serious disease in the affected host and even death. The effectiveness of the immune system of the viral-infected host is one singular factor there is that can significantly affect the outcome of a particular viral infection in an individual. Even though the immune system may not eliminate totally the infecting virus, a strong immune system helps to contain the pathogenicity or virulence of a given pathogenic virus. The administration of potent vaccines and other antiviral agents to affected hosts and other susceptible host can help to treat the disease or infection and even prevent the contamination of a particular viral infection respectively.
Vaccine development (which is mainly based on the isolation or cultivation, attenuation or inactivation and the direct or indirect injection of a susceptible host with killed, live or purified subunits of causative pathogenic microorganism) has saved mankind from the onslaught of some life-threatening infectious diseases such as measles and smallpox through the control and the complete eradication of these diseases in some cases. The timely vaccination of a large population of susceptible host against a particular viral infection helped to minimize the spread of the pathogen in the immunized populace via herd immunity. And vaccination/immunization has helped to increase the life expectancy of mankind through the protection of the population from life-threatening diseases especially those caused by pathogenic viruses.
Viral infection unlike other microbial infections stimulate an immune response in the host; and this serves to protect the host from immediate attack and even from futuristic viral infection through specific immune responses. Pathogenic viruses as obligate intracellular microorganisms engage in uniquely intimate host-parasite relationships with the living organisms (plant, animal or man) that they infect, and this is due in part to the fact that viruses only exist or replicate in living cells. In the course of their pathogenicity or virulence in a particular host, pathogenic viruses express gene products that act to circumvent one or more of the several antiviral defense mechanisms (e.g. production of interferons) developed by the host organisms.
Nevertheless, the host resistance to viral infections involves both the humoral immunity and cell-mediated immunity. While some viral infections (e.g. influenza and common colds) can be contained by the host’s immune system; some others such as the causative agent of AIDS (i.e. HIV) overpowers the host’s immune system and makes it incapable to fight against the invading viral agent. Infections with some pathogenic viruses may lead to apoptosis; and this programmed cell death is a host defense mechanism that can be inhibited by some viruses. Antibodies produced by humoral immunity can neutralize pathogenic viruses by interfering with their attachment to host cells; and the production of antibodies also enhances the destruction of viral particles via phagocytosis. However, antibodies cannot completely eliminate the infecting virus once the virion has incorporated its genome into that of the host.
The cell-mediated immunity is one of the major important arms of the immune system that interfere with viral replication in vivo. Activated lymphocytes including cytotoxic T lymphocytes (CTL), helper T cells (CD+4) and cytotoxic T cells (CD+8) can recognize and destroy viral infected cells especially when these organisms bud off from their host cells. The production of interferons (which are protein substances produced by cells during viral infection) helps to reduce the spread of virus especially in some benign viral infections such as influenzae and cold. They stimulate the production of natural killer (NK) cells and T cells; and interferons also accelerate the immune response of a host to viral infection. And by acting on other effector cells of the immune system, interferons (which are antiviral cytokines) generally reduce the susceptibility of other uninfected cells of the host to the invading virus, and they do so by localizing the pathogenic virus so that they do not easily spread in the host’s body.
DNA viruses have only the deoxyribonucleic acid (DNA) molecules as their nucleic acid; and the DNA can either be double-stranded or single-stranded as the case may be. The replication site of all DNA viruses is the nucleus of their host cell. Nevertheless, Poxviruses (which are also DNA viruses) replicate outside the nucleus of their host cell. Poxviruses replicate in the cytoplasm of their host cell. All DNA viruses are double-stranded with the exception of Parvoviruses that are single-stranded. Double stranded DNA (dsDNA) viruses infect man, animals, mycoplasmas, algae, fungi and protozoa. However, viruses in this category rarely infect plants. Some DNA viruses have double stranded DNA genome while others have single stranded DNA (ssDNA) genome; and both category of DNA viruses cause infections in humans (Table 4). A handful of viruses in this category (inclusive of dsDNA and ssRNA viruses) cause a variety of infections and diseases in man and other vertebrates. The replication of the DNA viruses is usually not as complex as is the case for RNA viruses as aforementioned.
During the replication process of most DNA-containing viruses, the genome of the host cell (i.e. the DNA) is stimulated by the viral genome which overpowers it and causes it to drive their own DNA replication. Some of the viral families that make up the DNA containing viruses and that parasitize humans and other vertebrates include: Parvoviridae, Adenoviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Polyomaviridae, Papillomaviridae, Iridoviridae, and Asfarviridae. These viral families have viruses that contain dsDNA genome. Those viral families with dsDNA genome that parasitize non-vertebrate hosts such as bacteria, Archaea, algae and mycoplasmas include: Myoviridae (bacteria), Siphoviridae (bacteria), Podoviridae (bacteria), Tectioviridae (bacteria), Corticoviridae (bacteria), Plasmaviridae (mycoplasma), Lipothrixviridae (Archaea), Rudiviridae (Archaea), Fuselloviridae (Archaea), Guttaviridae (Archaea), and Phycodnaviridae (algae). The ssDNA viruses (with their host in parenthesis) include: Microviridae (bacteria), Inoviridae (Mycoplasma and bacteria), Circoviridae (vertebrates), and Densovirinae (invertebrates, mosquitoes and silkworm). The characteristics of some DNA-containing viruses especially those that are of clinical or medical importance to man shall be highlighted in this section.
Parvoviridae family has six (6) genera of virus which include Parvovirus, Contravirus, Erythrovirus, Dependovirus, Densovirus and Iteravirus. Parvovirus or the human parvovirus B19 is the major viral agent in this family of virus because it causes infections in humans. The family Parvoviridae also contains viruses that cause viral infection in animals. The human parvovirus B19 is small, non-enveloped virus with a linear, single-stranded DNA (ssDNA) genome. It is an erythrovirus in the Parvoviridae family. Parvovirus measures between 18-26 nm in diameter; and they have a single-stranded DNA (ssDNA) genome. Their replication is within the nucleus of their host cell (specifically the erythroid precursor cells responsible for erythrocyte production in the bone marrow); and viruses in the Parvoviridae family are resistant to ether but sensitive to chlorine compounds, formalin and ultraviolet (UV) light. Viruses in the Parvoviridae family lack envelope (i.e. they are naked viruses), and they have an icosahedral shape or structure.
They are relatively stable at high temperature; and viruses in this family are released through the lysis of infected host cell(s) since they lack envelope. Human parvovirus B19 causes erythema infectiosum in children and aplastic crises in sickle cell and anaemic patients. Erythema infectiosum is also called the “fifth disease”; and it is usually characterized clinically as the onset of an erythematous rash (known as slapped cheek syndrome) on the face of the infected child. It is called “slapped cheek syndrome” because the both cheeks of infected children appear as though they have been slapped on both sides. There is rapid destruction of the red blood cells (RBCs) of anaemic or sickle cell individuals infected with the virus due to its replication in the blood cells or bone marrow of these persons.
Parvovirus B19 viral infection has also been associated with spontaneous abortion in humans. The incubation period of human parvovirus B19 infection is usually between 12-18 days; and the disease onset is usually characterized by the appearance of a maculopapular rash on the body of infected children. Upper respiratory symptoms, fever and malaise are other associated signs of the disease. Its transmission route is via infected blood and the respiratory route. Parvovirus infects mostly children, and the disease has a worldwide prevalence. However, immunocompromised individuals, pregnant women and people with haemolytic anaemia are also at risk of contamination. No specific prophylaxis or vaccine exists for human parvovirus infection; and there’s also no specific antiviral treatment for the disease.
Poxviridae family is a distinct family of viral genera that contain viruses that replicate in the cytoplasm of their host cells (inclusive of vertebrate and invertebrate cells). Cowpox, smallpox or variola virus, vaccinia virus and monkey pox viruses are typical examples of viruses in this family. The Poxviridae family contains viruses that infect humans, other vertebrates, invertebrates, birds and insects. Viruses in this family especially the poxviruses are the largest in size of all known viruses; and they generally have a double-stranded (dsDNA) genome.
Entomopoxvirinae (which infects invertebrates) and chordopoxvirinae (which infect vertebrates) are the two subfamilies of the Poxviridae family; and the Chordopoxvirinae subfamily contain eight genera of viruses (inclusive of poxviruses which causes infection in humans) while the Entomopoxvirinae subfamily contain only three (3) genera of viruses that parasitize invertebrates. Orthopoxvirus (that comprises of the human poxviruses, vaccinia, variola, and monkey pox viruses), Parapoxvirus (that parasitize humans and animals), Avipoxvirus (that infect birds), Yatapoxvirus (that infects primates), Capripoxvirus (that infect sheep and cattle), Molluscipoxvirus (that parasitize humans), Suipoxvirus (that infect pigs) and Leporipoxvirus (that infect rabbits) are the eight genera that make up the Chordopoxvirinae subfamily of Poxviridae family.
Human poxviruses are unique and different from other DNA-containing viruses because they replicate in the cytoplasm of their host cell instead of in the nucleus (as is applicable with DNA-containing viruses). Poxviruses have a brick-shaped structure; and their size is 220-450 nm long, 140-260 nm wide and 120-240 nm thick. Poxviruses have an enveloped genome and they are released from their host cell by budding – which allows them to acquire envelope. Infections caused by poxviruses are highly contagious and can be spread by body contact in a human population. However, treatment and vaccination exist for the disease, and some of the diseases caused by the human poxviruses have been eradicated. For example, smallpox (caused by variola virus) was eradicated in 1977; and there has not been any reported case of the disease since then. Patients become infectious once the maculopapular rash start appearing, and the incubation period of the disease is usually between 10-12 days after exposure. Sudden onset of fever, back pain and headache are some of the early signs of the disease. Human to human infection of smallpox virus occurs via the respiratory route after contact with an infected person.
The viral family Papillomaviridae comprises of papillomaviruses (abbreviated as PVs); and they were previously classified together with polyomavirus in the Papovaviridae family which is no longer in use in viral taxonomy for DNA containing viruses. Papillomavirus is the only viral genome in the Papillomaviridae family; and the papillomaviruses are oncogenic i.e. they are cancer-causing viruses. Human papillomavirus (HPV) causes papillomas on the lips as well as on the skin and mucous membranes of infected individuals. Papillomavirus also causes infection in non-human hosts such as cattle, rabbit, horse, elephant, dog, and birds. Viruses in this family have a circular dsDNA genome that is devoid of envelope. They are icosahedral in shape, and measure about 55 nm in diameter. They are also resistant to ether but sensitive to UV light and formalin. The replication site of viruses in this family is the nucleus of their infected host cell and they are released from the host cells they infect via lysis.
Papillomaviruses induce benign lesions of the skin and mucous membranes in individuals infected by the virus. The lesions produced on the skin are known as warts while lesions produced on the mucous membranes are known as condylomas. Genital warts (with occur on the external genitalia of men and women) and cervical cancer are the two most significant infections caused by the papillomavirus in humans. The papillomaviruses are of immense clinical importance because of their ability to initiate the development of cancer in infected individuals. Some of the cancers associated with the papillomaviruses include cancer of the cervix, mouth, skin, urogenital tract, eye, gastrointestinal tract, and cancer of the eyes. HPV is implicated clinically as the causative agent of cervical cancer in women, and the virus has a worldwide distribution. Studies have shown that genital HPV infection is one of the world’s most common sexually transmitted viral infections; and the disease occurs in both men and women with varying prevalence’s.
Since the contraction of one sexually transmitted disease (STD) in a person can place an individual at a higher risk of acquiring another STD or STD agent, the risk of infection with HPV is also high especially in HIV-infected persons whose immune system have been suppressed by viral infection. The sexual route is the main route of transmission of the disease but vertical transmission from mother to child especially during delivery and transmission via direct contact with infected materials such as sharp objects can also occur. There is no specific antiviral agent for the treatment of the disease; and the skin lesions are usually self-limiting. Surgery is often considered in some cases of papillomavirus infection especially those infections involving the genitalia.
Polyomaviridae family contains DNA tumour viruses like the Papillomaviridae family. Polyomavirus is the only viral genera or genus in the Polyomaviridae family. They are so named because the polyomaviruses causes tumour in various organs of the body including the kidneys, ureters, bladder, brain and bones. However, there are various genotypes of viruses in this family that infect humans and other vertebrates/mammals such as monkey, cattle, mice, rabbits and birds. The polyomavirus SV40 (which infects rhesus monkeys) and the BK polyomavirus and JC polyomavirus which are both human polyomaviruses are the typical examples of viruses in this family. SV stands for “simian vacuolating” virus.
The initials BK and JC represent the initials of the names of the individuals from whom the virus was initially isolated from the early 1970s. Viruses in this family have a circular dsDNA genome that is devoid of envelope. They are icosahedral in shape, and measure about 45 nm in diameter. They are also resistant to ether but sensitive to UV light and formalin. The replication site of viruses in this family is the nucleus of their infected host cell and they are released from the host cells they infect via lysis. BK polyomavirus causes cystitis and nephropathy while JC polyomavirus causes a fetal brain disease (known as progressive multifocal leukoencephalopathy) in immunocompromised individuals respectively. Progressive multifocal leukoencephalopathy (PML) is a rare but fatal viral disease that occurs mainly in AIDS patients. Humans are the natural host of the BK and JC polyomavirus while rhesus monkeys and macaques are the natural host of SV40 virus.
BK and JC polyomaviruses can both cause disease in children and adults especially in the immunocompromised adult hosts. SV40 which is known to infect only rhesus monkeys (their natural host) but it was discovered that the virus occur in individuals who received early jabs of killed and live poliovirus vaccine that had been cultivated in monkey cells which were unknowingly infected with SV40. This contamination occurred during the production of the vaccine. And thus, the SV40 is currently being considered as a pathogenic viral agent in human population due to its detection in human samples including blood and urine. Whether the presence of SV40 in human population was due to the use of contaminated poliovirus vaccine or from other sources is still a subject of discussion in the medical community, but the truth remains that the virus occur in human population. Highly active antiretroviral therapy (HAART) and other antiviral therapy are available for the treatment of infections caused by polyomaviruses.
Hepadnaviridae family consists of two viral genera which are Orthohepadnavirus (which contain viruses that infect humans and other mammals) and Avihepadnavirus (which contain viruses that infect birds); and these viruses are generally called hepadnaviruses. The viruses in this family include human hepatitis B virus, animal hepatitis viruses and duck hepatitis virus. Hepadnaviruses are spherical in shape, and they measure between 40-48 nm in diameter. They are sensitive to ether, heat, acid, organic solvents and detergents; and they have no envelope. However, some viruses also possess envelope. They generally replicate in the nucleus and are thus released from their host cell via lysis. The human hepatitis B virus is the main viral representative of the Hepadnaviridae family; and the virus has a worldwide distribution. Hepatitis B virus (HBV) is the only causative agent of viral hepatitis that has a dsDNA genome (Table 3).
The other causative agents of hepatitis (liver inflammation) in humans are caused by viral agents whose genome type is ssRNA. HBV is a unique DNA virus because it shares an encoding reverse transcriptase (RT) with the retroviruses (e.g. HIV); and their replication is through an RNA-DNA route because of the RT they encode. HBV has a high affinity for liver cells (i.e. hepatocytes); and they have two major proteins hepatitis B surface antigen (HBs Ag) and hepatitis B core antigen (HBc Ag) that both aid the virulence of the pathogens in humans. While HBc Ag antigens are basically expressed within hepatocytes, the HBs Ag is expressed outside the liver cells when the virus replicates in the liver. The main route of transmission of human HBV is via sexual contact, congenitally (i.e. from an infected mother to unborn child) and parenterally especially via exposure to contaminated blood.
The incubation period of HBV infection in humans is between 6 weeks to 6 months; and the disease can be acute or chronic in nature depending on the persistence of the virus in the infected host. The clinical outcome of the disease is usually characterized by liver dysfunction and jaundice. Prolonged and untreated disease can result to liver cancer and cirrhosis. Intravenous drug users, healthcare workers, people who receive tattoos and acupuncture, blood recipients, homosexuals, people who keep multiple sex partners, those who share sharp objects and sex workers are people who are at risk of infection with HBV. HBV infection can be prevented via proper vaccination of susceptible population with hepatitis B vaccine, and treatment is done using antiviral drugs. However, HBV infection is self-limiting in some individuals, and some patients can spontaneously clear the virus from their system.
Table 3: Summary of the causative agents of liver inflammation (hepatitis) in humans
|Heptitis A||Picornaviridae||ssRNA||Feacal-oral route|
|Hepadnaviridae||dsDNA||Sexual contact, blood and from mother to child|
|Flaviviridae||ssRNA||Sexual contact, blood and from mother to child|
|Hepatitis D||Deltavirus||ssRNA||Parenteral or via blood contact|
|Hepatitis E||Hepeviridae||ssRNA||Feacal-oral route|
HBV infection is a common nosocomial viral infection that can spread within a particular hospital environment or healthcare setting owing to the ease with which the viruses can be contracted via blood and other blood-contaminated body fluids or instruments such as syringes, scissors, and other hospital equipments. It is therefore critical that healthcare workers and laboratory personnel’s wear the correct protective coverings such as gloves and hospital/laboratory gowns when attending to patients or processing patient’s specimens. And all blood samples or blood-containing body fluids should be treated as “HIGHLY INFECTIOUS” in order to avoid contamination.
Adenoviridae family is made up of two viral genera which are Mastadenoviridae (which contain viruses that infect humans and other mammals) and Aviadenovirus (which contain viruses that infect birds). Adenoviruses are one of the major viruses in this family; and they have a worldwide distribution. Adenoviruses have an icosahedral shape, and they have no envelope. They measure between 80-110 nm. Adenoviruses are resistant to ether but sensitive to formalin and lipid solvents. They generally replicate in the nucleus and are released via the lysis of infected host cell. There are several serotypes of adenoviruses that cause infection in humans and other mammals; and adenoviruses cause several febrile illnesses in man and animals. Adenoviruses cause respiratory diseases, infantile gastroenteritis and conjunctivitis. And they also cause disease in the urinary tract. Most adenovirus infection is asymptomatic in infected individuals.
The major routes of entry of adenovirus into the body of host include the mouth, the nasopharynx, and the ocular conjunctiva. The virus replicates in the epithelial cells of the eye conjunctiva, respiratory tract and the gastrointestinal tract; and as aforementioned most clinical cases of adenovirus infection are covert in nature (i.e. they do not produce clinical symptoms). The incubation period of adenovirus infection is between 2-5 days; and patients become infectious when they are symptomatic. Adenovirus infections occur worldwide in humans as well as in a variety of animals. People at risk of infection with adenoviruses include young children, infants, the immunocompromised host and people in overcrowded military camps. The major routes of transmission of adenoviruses in human population include the feacal-oral route and the respiratory route.
Human infection with adenoviruses induces a lifelong immunity in infected hosts; and the likelihood of a re-infection with the virus is unlikely in individuals who have been previously exposed or infected. However, there is no specific treatment for adenovirus infection since most adenovirus infections in humans are asymptomatic in nature. And preventive measures depend on the maintaining proper personal hygiene especially hand washing. The adenoviruses are of immense medical importance because they could serve as vectors for vaccination purposes and they have also been employed in gene therapy techniques. The use of adenovirus for these purposes is because adenoviruses hardly integrate their genome into the chromosome of their host cells; and the virus is one of the most studied viruses. They can be easily grown in cell culture in vitro; and adenoviruses have a higher stability than other DNA-containing viruses.
Herpesviridae family is a distinct viral family that contains a wide variety of viruses that are distributed worldwide and which infect humans, mammals and other vertebrates. There are three subfamilies that make up the Herpesviridae family, and they include Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. Simplexvirus (e.g. herpes simplex virus) and Varicellovirus (e.g. Varicella-zoster virus) are the two genera of Alphaherpesvirinae that contain viruses that infect humans. In the Betaherpesvirinae subfamily, there are only two genera of viruses that infect humans; and these include Cytomegalovirus (that contain cytomegalovirus) and Roseolovirus (that contain human herpes virus types 6 and 7). Gammaherpesvirinae family has two genera viz: Lymphocryptovirus (that contain Epstein-Barr virus) and Rhadinovirus (that contain human herpesvirus 8).
Viruses in this family include herpes simplex virus (HSV) type 1 and 2 and Varicella-zoster virus (VZV) in the Alphaherpesvirinae subfamily; cytomegalovirus (CMV) in the Betaherpesvirinae family; and Epstein-Barr virus (EBV) which is in the Gammaherpesvirinae subfamily. Morphologically, the herpes viruses have a dsDNA genome, and they measure about 150 nm or 100-300 nm in diameter. Structurally, herpes viruses have an icosahedral shape. They are enveloped viruses. Herpes viruses replicate in the nucleus and they are released from their host cell via budding through the cytoplasmic membrane of the infected cell. Chickenpox (caused by VZV), Burkitt’s lymphoma (a tumour caused by EBV), fever blisters or cold sores (caused by HSV-1) and genital herpes (caused by HSV-2) which are both caused by the human herpes simplex viruses type 1 and 2 respectively are typical examples of clinical diseases caused by viruses in the Herpesviridae family (Table 6).
Some viruses in the Herpesviridae family such as CMV are common amongst individuals with weakened immune system especially AIDS patients Lesions or blisters on the penis (in males), vagina or cervix (in females) and blisters appearing on the lips and mouth regions of infected individuals are some of the clinical symptoms of the disease. Some of the route of transmission of the disease includes direct sexual contact with an infected individual, transmission via aerosols, and other body fluids such as saliva. Herpes simplex virus can also be transmitted from an infected mother to the unborn child. It is noteworthy that herpes viruses have the ability to establish a latent infection in their natural host; and thus the virus is capable of re-emerging in its virulence in the future in an asymptomatic host. Prophylaxis and antiviral therapy exist for the treatment of some human herpes virus infections.
Viruses as aforementioned are basically classified based on their genome content as DNA-containing viruses and RNA-containing viruses. RNA viruses are also different from the DNA viruses in terms of their site of replication aside their genomic difference from DNA viruses. All RNA viruses replicate in the cytoplasm of their host cell. The only exception in this case is Retroviruses and Orthomyxoviruses found in the Retroviridae family and Orthomyxoviridae family respectively which can replicate in the nucleus of their host cell. In addition, Retroviruses and some Hepadnaviruses (that have RNA genomes) can replicate in both compartments of the nucleus and the cytoplasm of their host cell. All RNA viruses have a single-stranded RNA (ssRNA) genome. The only exceptions in this case are Rotavirus, Reoviruses or Orthoreoviruses (found in the Reoviridae family) that have a double-stranded RNA (dsRNA) genome. RNA-containing viruses must provide a replicase enzyme (i.e. an RNA-dependent RNA polymerase) for the replication of its genome since the host cell (especially those of eukaryotic origin) do not innately possess enzymes for the replication of RNA genomes.
Structurally, RNA viruses have icosahedral shape or helical shape; and they can either be enveloped or naked viruses. The major families of RNA viruses include Retroviridae, Orthomyxoviridae, Paramyxoviridae, Filoviridae, Togaviridae, Bunyaviridae, Picornaviridae, Caliciviridae, Reoviridae, and Arenaviridae. These varieties of RNA viruses have unique replication strategies; and typical amongst these are the retroviruses in the Retroviridae family that have a reverse transcriptase (RT) enzyme. Retroviruses (such as the human immunodeficiency virus, HIV) use RT, an RNA-dependent DNA polymerase to produce a DNA copy of its own RNA genome. And in this case the RNA genome of the virus serves as a replication template for the biosynthesis of a DNA copy of the RNA genome of the virus.
The normal operation of the flow of genetic information in living systems according to the central dogma of molecular biology is from DNA — RNA — protein. But in the case of replication in retroviruses, genetic information now flows from the RNA to the DNA because the virus has an enzyme (reverse transcriptase) that carries out this function in a reverse fashion; and this process occurs in the cytoplasm of the cell. The dsDNA synthesized is then transported to the nucleus of the cell where it is inserted into the genome of the host cell as a provirus (a viral DNA). The provirus is later transcribed by the host cell into an RNA molecule (i.e. mRNA) that enters the cytoplasm or ribosome where viral proteins are finally synthesized and assembled for the formation of new virions.
Retroviridae family is a unique family of viruses that contain reverse transcriptase (RT) which allows all viruses in this family to carryout reverse transcription of their genome. Viruses in this family are usually referred to as retroviruses because of their possession of RT. Retroviruses are distributed worldwide. Reverse transcription is the genetic process of copying the genetic information found in the RNA genome of an organism into DNA. Viruses in this family cause various types of tumours, lymphomas or sarcomas in animals. And this implies that some retroviruses are oncogenic or cancer-causing in nature. Retroviruses also infect invertebrates as well as vertebrates. Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) which causes AIDS and simian AIDS in humans and monkeys respectively are the most important viruses in the Retroviridae family.
Viruses in the Retroviridae family have an icosahedral shape; and they measure between 80-110 nm in diameter. They possess a ss(+)RNA, and they replicate in the nucleus of their host cell. Retroviruses are enveloped viruses, and their envelope is rich in lipids and proteins. Retroviruses are released via the process of budding from the cytoplasmic membrane of their host cell. There are seven (7) genera of viruses in the Retroviridae family. These viral genera are Lentivirus, Spumavirus, Epsilonretrovirus, Betaretrovirus, Alpharetrovirus, Deltaretrovirus and Gammaretrovirus. HIV and SIV, which are one of the most significant viruses in this family, are found in the genus Lentivirus. The human T-cell leukemia virus-1 (HTLV-1), a Deltaretrovirus is another important member of the Retroviridae family which causes lymphoma in humans. With the help of reverse transcriptase, the ssRNA genome of retroviruses is transcribed by RT into a dsDNA molecule otherwise known as provirus or viral DNA.
The provirus is finally integrated into the chromosomal DNA of the host cell; and the viral DNA surmounts or takes over the cellular machinery of their host cell to produce viral RNAs and protein molecules required for the formation of new virions. It is noteworthy that the provirus generally serves as the template for the biosynthesis of viral RNA and protein molecules that are required for the assembling of new viral progenies (i.e. new retroviruses). Infections or diseases caused by retroviruses especially AIDS still have no permanent cure but the disease can be clinically managed or treated using some antiviral drugs such as zidovudine or azidothymidine (AZT) which are nucleoside analogs that inhibit the activities of RT during the replication of retroviruses.
HUMAN IMMUNODEFICIENCY VIRUS (HIV)
Human immunodeficiency virus is the causative agent of acquired immunodeficiency syndrome (AIDS) in humans. There are two variants of HIV that causes AIDS in humans viz: HIV-1 and HIV-2. While HIV-1 is mainly transmitted from mother to child and is thus a neonatal infection acquired congenitally, HIV-2 is transmitted via blood and sexual contact or having unprotected sex with an already infected person. HIV was first discovered as a human viral pathogen in the early 1980’s; and the disease (i.e. AIDS) has since caused innumerable number of morbidity and mortality in the human population. The actual origin of AIDS in the human population is as a result of cross-species infection of humans by a chimpanzee Lentivirus particularly the simian immunodeficiency virus (SIV), which infects only monkeys, chimpanzees and like primates in the west central African region. The scourge of the AIDS disease has impacted negatively on the economies of the world due to its ability to deteriorate the immune system of HIV-infected individuals.
PATHOGENESIS OF HIV
HIV is mainly transmitted via sexual intercourse especially unprotected sexual intercourse with an infected person. Transmission via contact with blood and other sharp objects contaminated with blood of an HIV-positive individual is also possible. HIV can also be transmitted congenitally from an infected mother to an unborn child. Intravenous drug users, heterosexuals and homosexuals are also at high risk of infection with HIV. HIV is entirely an infection of the immune system of humans, and the CD4 T helper cells (TH) of the cellular immune system are the main receptor of the virus. HIV binds to the CD4 cells and also on other cellular cells of the human host that bears the CD4 marker on their surfaces. This binding leads to the suppression of the individual’s cellular immune response due to the loss of CD4 T helper cells.
The CD4 T helper cells are unique in the cellular immune response of humans in that they play a central role in both cellular and humoral immune response when foreign bodies (i.e. antigens inclusive of pathogenic viruses like HIV) invades the body. CD4 cells secrete numerous cytokines that activates other specific components of the immune system such as the macrophages; delayed-type hypersensitivity T cells (TDTH) and the cytotoxic T cells (CD8) which carries out the killing of antigens in the body. HIV infects and kills immune system cells (especially the CD4 lymphocytes) that are vital for effective immune response against pathogens that invades the body. Once the HIV has attached to the CD4 cells, it facilitates its entry into the cell through several cell-entry techniques. And once inside the cell, the virus uncoats and its RNA genome is transcribed with the help of its reverse transcriptase (RT) to a viral DNA or provirus that is integrated into the chromosomal DNA of the host cell. Since the CD 4 cells are primarily responsible for the mediation or activation of T cell immunity during antigen invasion of the body, acquired immunodeficiency syndrome (AIDS) finally results following a marked decrease or suppression of the CD4 cells of the affected individual by HIV.
HIV infection can be acute (during which the infection is rapid in progression) or chronic (which the infection is slow in progression) in occurrence but the actual clinical outcome of the disease is dependent on several factors which include the genetics of the infecting virus, the genetics of the affected host, the virulence of the infected virus and the immune status of the host. HIV-infected people remain infectious throughout the period of their lifetime especially in the acute stage of the disease, and such individuals are potential routes via which the disease can be transmitted to susceptible populations. At the acute stage of HIV infection, the viral load in infected individuals is very high and such patients show a high level of viraemia at this phase when viral count of their blood is taken. During the chronic stage of the disease, some of the dead CD4 cells are rapidly replaced and the patients usually show low viral load count and normal CD4 cell count too.
But AIDS definitely sets in; and it is generally characterized by the emergence of several opportunistic infections caused by bacterial, fungal and protozoal pathogens and even some viruses such as cytomegalovirus (CMV). Development of tumours (especially cancers of the skin) and some neurological disorders such as wasting and aseptic meningitis are other accompanying (opportunistic) infections that characterize AIDS in HIV-infected individuals. These opportunistic infections exemplify the main clinical features of AIDS in HIV infected individuals and they appear during the progressive loss of CD4 cells in infected persons. Unexplained weight loss, fever, pharyngitis, headache, arthralgia, myalgias, and malaise are some of the main non-specific symptoms or clinical features that characterize the primary stage of HIV infection (i.e. the stage at which the individual is newly infected).
The incubation period of the disease is usually 2-6 weeks after exposure but this can also last up to 3 months or 6 months. It is noteworthy that the course of HIV infection in humans varies from one individual to another. While some individuals can show clinical signs of AIDS within 10 years of infections, others can live beyond this limit without showing any clinical signs of AIDS. However, the AIDS stage of HIV infection is usually defined by a marked decrease in the CD4 level of the infected host. A CD4 cell count of less than 200 cells/µL is clinically indicative of AIDS stage. The appearance of some opportunistic infections, cancers or tumours can also define the AIDS-stage of the disease. AIDS usually appear after about 10 years of infection with HIV; and due to the marked disintegration of the host immune system especially that of the cellular immunity, the individual is exposed to plethora of opportunistic infections caused by other microbes including bacteria, viruses, protozoa and fungi as aforementioned. The severity of HIV infection according to studies have been linked to co-infection with other non-retroviral agents such as hepatitis B virus (HBV), hepatitis C virus (HCV) and CMV amongst others; and this has affected or increased the rate of progression of the disease in HIV-positive patients who have these co-infections.
LABORATORY DIAGNOSIS OF HIV
Clinically, the presence of opportunistic infections such as cryptococcal meningitis and recurrent vulvovaginal candidiasis amongst others mentioned above without a known cause of immunodeficiency in a person should raise suspicion of HIV infection. Nevertheless, the laboratory diagnosis of HIV infection is largely dependent on the detection of HIV-1 or HIV-2 antibodies in the serum or blood samples of patients using serological or molecular techniques that includes enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and other rapid diagnostic laboratory techniques. In addition, specific antigens of the virus especially the capsid proteins of HIV such as p24 can be detected in serum following infection using HIV antigen detection kits. The detection of HIV capsid proteins some days after infection aids in the early diagnosis of infection since these proteins appear in the serum or blood of infected individuals few days after infection and before HIV-1 or 2 antibodies starts to appear. The AIDS defining illness of the disease can also be confirmed in the laboratory by determining the patients CD4 count. And if the CD4 cell count is less than 200 cells/µL, then the individual is in the AIDS phase of the disease.
TREATMENT OF HIV
One of the major impediments to the effective treatment of HIV infection is the development of resistance to some readily available antiviral drugs especially when such drugs are used singly for treatment. Aside this, the development and preservation of latent HIV-1 in some cellular and anatomical sites of HIV-1 infected individuals has contributed a great deal to the inability of current antiretroviral therapy (ART) to eradicate the virus from the body of people living with the disease. These immunological and pharmacological privileged sites of the body such as lymph nodes are known as reservoir sites; and they are important sanctuaries where transcriptionally silent but replication competent latent HIV-1 are hiding in the body of people living with HIV/AIDS (PLHA).
More so, upon the discontinuation of ART, the reservoir sites of latent HIV-1 in PLHA continue to serve as repertoire and source via which the general circulation is replenished with the infectious virus. This has made curing and eradication of HIV-1 difficult. Which is why researchers are actively looking out for chemicals known as latency reversing agents (LRAs) that can purge the reservoir sites, and thus flush out the latent HIV-1 from their hiding places in the body of PLHA. In lieu of this, antiviral drugs are now administered in combinations of three antivirals i.e. as a triplet; and this has helped to contain the possibility of development of resistance to antiretroviral drugs when they are used singly. To this end, highly active antiretroviral therapy (HAART) which is the type of HIV treatment in which antiviral drugs are used in combination as triple therapy is now being used for HIV treatment. In HAART, one nucleoside analog and one protease inhibitor are included in the antiretroviral therapy, and the main aim of this triple therapy is to slow the development of resistance to any of the agent and also to ensure effective treatment.
Most antiretroviral drugs target key steps in the replication cycle of retroviruses in order to inhibit or slow their replication. Also, antiretroviral drugs help to reduce the number of viruses in the individual as quick as possible so that the nefarious activity of the virus (especially in suppressing the cellular immunity) can be contained and assuaged. The major classes of antiretroviral drugs available for the treatment of HIV-infection are nucleoside inhibitors and protease synthesis inhibitors. The nucleoside inhibitors include zidovudine or azidothymidine (AZT), lamivudine (3TC) and didanosine (ddl). These antiviral drugs are nucleoside analogs and they basically inhibit the activities of reverse transcriptase (RT) during HIV replication. They bind to the active site of RT, and thus becomes incorporated into the growing DNA strands (i.e. the viral DNA).
By interfering with the synthesis of viral DNA, AZT acts as chain termination to stop the elongation of the DNA strand so that an incomplete genome of the virus will be produced. Protease inhibitors basically act by inhibiting the production of protease which is vital for the formation of viral proteins. Examples of antiviral agents that are protease inhibitors include ritonavir (RTV), indinavir (IDV) and saquinavir (SQV). Protease inhibitors generally inhibit viral maturation since proteases play critical roles in the synthesis of viral proteins required for the coupling of new virions.
No effective vaccine currently exist for the vaccination of susceptible human population against HIV infection but vaccine development for HIV prevention is still underway and putative. The AIDS virus (HIV) has a rapid rate of maturation or replication, and its genetics is inconsistent due to the ease with which the virus mutates into different and new forms. And the human body can still not produce protective immunoglobulins against HIV. These factors have greatly affected the development of novel and potent vaccine for the effective vaccination of the human race against the scourge. Having safe sex and sticking to only one partner when married, screening of blood and organs before transfusion or transplantation and avoiding the sharing of sharp objects such as needles are some of the measures that can be taken for the prevention of HIV infection.
OVERVIEW OF HAART, ART AND HIV-1 RESERVOIR
HAART is the acronym for highly active antiretroviral therapy. The term combination antiretroviral therapy (cART) can also be used synonymously with cART. Thus, HAART can also be referred to as cART. For the purposes of this review, the term HAART will be used. HIV is a highly mutating viral pathogen; and thus single therapies or monotherapies directed at the virus in vivo might end up not producing the desired clinical outcome or prognosis – since the targeted virus might change and not respond to the therapy. In addition, monotherapy in HIV treatment and management processes might also contribute and lead to the development and resistant strains of the virus. Despite this, the bioavailability of cART/HAART in PLHA is critical to measure the prognosis of the individual especially with the notable mutation rate of HIV. But how is HAART measured in PLHA? The effect of highly active antiretroviral therapy (HAART) in the treatment of HIV infection is usually measured by survival, CD4 lymphocyte counts, HIV-1 RNA viral load testing, and the occurrence of opportunistic infections.
Having these parameters in mind, and as a way of determining the in vivo efficacy of HAART in PLHA, it is also important to determine and evaluate the bioavailability of these drugs in several different anatomical sites especially those tissues that have been previously characterized or are currently emerging anatomical reservoir sites for HIV-1 such as adipose tissues. To effectively treat HIV and suppress the viral load of the infection to undetectable limits (<50 copies/ml), a combination of antiretrovirals that target different and specific stages of the HIV replication stages or processes are often used clinically in combination; and these agents are administered to PLHA as a regimen to achieve HIV suppression. However, HIV in its ingenuity and high mutational rate still finds a way to come out once in a while from its hiding place and cause one or two infections even in people on cART/HAART. The drugs used in HAART target four (4) different steps in the HIV replication cycle including the viral entry step, reverse transcription step, integration step and maturation step. The different antiretroviral agents used for HIV treatment are usually divided into four (4) classes of agents that are used in HAART; and these are briefly summarized.
- Nucleotide/nucleoside reverse transcriptase inhibitors (NRTIs): Reverse transcriptase (RT) enzyme is an important enzyme in HIV replication. RT helps to reverse transcribe the HIV RNA into HIV proviral DNA (in a process known as reverse transcription), which can carry out the normal process of the central dogma of molecular biology (i.e. DNA-RNA-Protein). Without RT, it will be impossible for HIV to replicate in vivo; and thus the virus may not be a serious threat to humanity as it is now. Reverse transcription is usually the first thing the virus must do once it enters a host cell in order to maintain its continued perpetuation or existence in the infected host cell and elsewhere. NRTIs are antiretrovirals that target the reverse transcription step that converts the viral genomic RNA into linear double stranded DNA. Typical examples (generic name shown) include zidovudine, emtricitabine, tenofovir alafenamide, tenofovir, lamivudine and abacavir to mention a few.
- Non-nucleotide reverse transcriptase inhibitors (NNRTIs): NNRTIs like NTRIs target the reverse transcription step that converts the viral genomic RNA into linear double stranded DNA. Examples include (generic name shown) efavirenz, nevirapine, etravirine and rilpivirine.
- Protease inhibitors (PIs): Protease are other important enzymes that help to breakdown or cleave the polyproteins produced during HIV replication into their respective constituent and smaller proteins required for the formation of a new virion. Protease is critical for the maturation of viral particles that bud out from infected host cells Typical examples include (generic name shown) indinavir, ritonavir, saquinavir, tipranavir and atazanavir.
- Integrase inhibitors: Integrase enzyme helps the HIV genome to be successfully integrated into the genome of the infected host cell. Without this enzyme, HIV replication may not proceed further. Integrase inhibitors can also be called integrase strand transfer inhibitors (INSTIs), and they block integrase strand transfer activity that is required to insert viral DNA into a host cell chromosome. Examples include (generic name shown) raltegravir, dolutegravir and elvitegravir.
It is no doubt that the persistence of HIV infection in PLHA, and the reason for the perpetuated viral rebound experienced in these individuals is mostly attributable to the lurking, preservation and protection of latent strains of HIV-1 and/or SIV (in non-human primates) in certain cellular and anatomical sites of the body as has been already highlighted in this review. These cellular and anatomical reservoir sites including lymph nodes, CD4+ T cells, spleen and macrophages to mention a few continue to serve as repertoire from which the peripheral circulation is replenished with infectious virus that causes the viral rebound (or new infections) experienced in PLHA even in the face of HAART or cART. Thus, the current HAART/cART should be re-engineered to target and possible enter both cellular and anatomical reservoir sites of HIV in order to ensure adequate antiviral activity in vivo.
More so, they should be able to maintain appropriate intercellular concentration and reach the bioavailability level required to possibly purge the reservoir sites of the virus. It is therefore important that the current ongoing reserve on finding a functional cure for HIV takes into account the different pharmacological differences that exist amongst these cellular and anatomical sites since certain antiretroviral drugs penetrate some tissues or cells more efficiently than they do in other sites where a pharmacological barrier or wall has been constituted, maybe due to differences in drug concentrations relative to the cells membranes and other physiological and metabolic processes. Though HAART has contributed a great deal towards viral suppression and HIV-1 control in PLHA, it is still important for new drug targets to be rediscovered for novel drug targets; and also, further drug developments in the search for HIV cure should take into consideration the other uncharacterized reservoir sites of HIV-1 with a view to finding out how they respond to these novel agents.
Togaviridae family comprises of viruses that are usually transmitted through arthropods such as insects. But some viruses in this family lack arthropod transmission. There are basically two genera of viruses in the Togaviridae family; and they are Alphavirus and Rubivirus. The viruses in the genera Alphavirus are basically transmitted to humans via arthropods especially blood-sucking mosquitoes while those in the genera Rubivirus lack arthropod transmission. Chikungunya virus (found in Asia and Africa), Mayaro virus (found in South America), Ross River virus (found in Australia) and Sindbis virus (found in Australia, Asia and Africa) are some examples of viruses that make up the genera Alphavirus. Rubella virus is the only viral species in the genera Rubivirus. Pestiviruses and Flaviviruses are other members of the Togaviridae family. Rubella virus which causes German measles in children is one of the most important members of this family, and this is because of the disease they cause in human population. Rubella virus is found in the genera Rubivirus.
Viruses in the Togaviridae family have an icosahedral nucleocapsid and they are generally enveloped viruses. The viruses in this family exit their host cell through the plasma or cytoplasmic membrane by budding. They have a ss(+)RNA and their replication is in the cytoplasm. They measure between 50-70 nm in diameter. The viruses in the Togaviridae family have a worldwide distribution. Chikungunya virus, and Alphavirus and rubella virus, a Rubivirus are the two viruses in the Togaviridae family that causes infection in humans. It is noteworthy that viruses in the genera Alphavirus are usually geographically limited to some regions of the world. German measles or rubella is a benign form of measles in children and young adults, and the disease is usually characterized by the appearance of maculopapular rash on the skin. Rubella used to be a worldwide epidemic but since the advent of vaccination, the disease has been significantly contained in countries where it is usually endemic.
Pregnant women especially those in their first trimester of pregnancy who are infected with rubella virus can induce serious congenital defects due to rubella virus infection in their unborn child. Rubella is a highly in factious disease and its transmission route is via the respiratory tract of humans (in respiratory droplets). Arthralgia (joint pains), fever, and maculopapular rash are some of the clinical symptoms of rubella (German measles); and there is no active treatment for the disease since most cases are self-limiting in affected individuals. Active rubella immunization still remains the basis for the effective control of rubella infection in human population. Vaccines are also available for the prevention of infection caused by Alphaviruses.
Bunyaviridae family comprises of viruses that infect humans, animals and even plants. The main viral genera that make up the Bunyaviridae family are Orthobunyavirus, Phlebovirus, Nairovirus, and Hantavirus genera (which all infect animals) and the genera Tospovirus (which infect plants). Majority of viruses in the Bunyaviridae family are arthropod-borne viruses because they are transmitted by arthropods especially blood-sucking insects such as mosquitoes and ticks. Viruses that are transmitted via arthropods or insects to humans are generally known as arboviruses (Table 8). Viruses in this family have a helical nucleocapsid and they are enveloped viruses. They replicate in the cytoplasm and they are released from their host cell via cytoplasmic membrane by a budding process. The viruses in Bunyaviridae family measure between 80 to 120 nm in diameter.
Bunyaviruses have a ss(-)RNA genome. The viruses that cause infection in humans are found in the genera Orthobunyavirus (e.g. La Crosse virus), Nairovirus (e.g. Crimean-Congo haemorrhagic fever virus) and Phlebovirus (e.g. Sandfly Fever Sicilian virus). Generally, the viruses in the Bunyaviridae family that infect humans cause haemorrhagic fever; and they can also be referred to as “haemorrhagic fever viruses” as is applicable to Ebola and Lassa fever viruses. The infections caused by Bunyaviruses are usually benign, and they are characterized by febrile infections that include haemorrhagic fevers. In severe cases of the disease, the central nervous system (CNS) may also be affected. Orthobunyaviruses causes benign forms of encephalitis; Nairoviruses causes haemorrhagic fevers; Phleboviruses causes sandfly fever or phlebotomous fever; and Hantaviruses causes Hantavirus pulmonary syndrome (HPS) in humans. All the genera of Bunyaviridae family that cause infections in humans with the exception of Hantavirus genera are transmitted to humans via arthropods.
Hantaviruses are transmitted to humans through rodents such as mice and rats. And a human infection with Hantaviruses (which is notorious in causing haemorrhagic fevers in humans) is through the inhalation of aerosols from the excreta or body secretions (e.g. urine, saliva and feaces) of these animals that harbour the virus. Such transmission of viruses from rodents to humans is generally known as an aerogenical transmission. Rodents are the natural reservoirs of Hantavirus, and they remain apathogenic in these animals (i.e. they do not cause infections in them). However, they cause serious infections in humans when they are aerogenically transmitted to humans. Prevention of infections caused by Bunyaviruses is through avoidance of contact with rodents and insect bites.
Orthomyxoviridae family comprises of unique classes of viruses (i.e. Orthomyxoviruses) that causes highly contagious respiratory disease known as influenza or viral flu in humans. Orthomyxoviruses viruses are zoonotic disease agents transmissible from animals to humans. The Orthomyxoviruses are distributed worldwide and they cause respiratory infections in both humans and animals. Orthomyxoviruses have a ss(-)RNA genome, and they have a helical nucleocapsid. They measure between 80-120 nm in diameter. They replicate in the nucleus. Orthomyxoviruses are enveloped viruses. Though the synthesis of the RNA genomes of RNA viruses occurs in the cytoplasm; that of the Orthomyxoviruses are known to occur in the nucleus of the cell, and this is possible because they contain or harbour an RNA-dependent RNA polymerase (as aforementioned) which convert their ss(-)RNA genome to a ss(+)RNA genome.
Orthomyxoviruses are released from the host cell via the cytoplasmic membrane by the process of budding. There are five genera of viruses that make up the Orthomyxoviridae family; and three of these viral genera cause significant human infections. Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus and Isavirus are the five genera that make up the orthomyxoviridae family. Influenzavirus A (which comprise of influenza A), Influenzavirus B (which comprise influenza B) and Influenzavirus C (which comprise influenza C) are the genera of Orthomyxoviridae family that cause infections in humans. Thogoto virus (found in the Thogotovirus genera) and infectious salmon anemia virus (found in Isavirus genera) cause infections in mammals and fish respectively. The influenza viruses (especially influenza A and B) are important human pathogens that are primarily transmitted via the respiratory tract or route in aerosols of infected persons. Influenza viruses have continued to cause epidemics amongst human population; and this is due in part to the reassortment of the genetic makeup of the organism. And this mutation or gene reassortment in influenza viruses has impacted negatively on the development of long-lasting immunity against the pathogen due to the continuous change in the structural and genetic makeup of the organism.
When the RNA genome of two genetically distinct strains of influenza virus that infect the same cell or organism are re-assorted, a biological process known as antigenic shift occurs; and this leads to the production of a new virus that is different from that of the two original virions from which it was initially developed from. Influenza viruses also undergo antigenic drift in their quest to mutate into new virions. Antigenic drift occurs when the protein structure of the virus (especially the neuraminidase and haemagglutinin) undergoes mutation to produce newer virions that will not be recognized by protective antibodies produced by the host’s immune system. Antigenic drift and antigenic shift are mainly responsible for the continuous emergence of new epidemics of influenza virus because these processes lead to the formation of novel strains of the virus. Neuraminidase (NA or N) and haemagglutinin (HA or H) are the two important proteins found on the surface of influenza virus; and they are used in the typing of the organism especially in the face of outbreak of disease caused by influenza virus. HA and NA are the two major antigens of influenza viruses.
While HA is mainly involved in the attachment and fusion of influenza virus to the host cell, NA basically aids in the release of the virion from the cell. There are several serotypes or subtypes of influenza viruses, and these have been classified based on their HA and NA variations. Influenza viruses are mainly reserved in animals inclusive of birds from which human infections have also occurred. The H5N1 influenza virus that caused influenza outbreaks in parts of Asia, Europe and Africa is mainly transmitted from birds to humans; and human to human transmission of this particular strain has not been recorded but likely. (The first outbreak of the H5N1 influenza virus infection known as “Avian Flu” occurred in poultry farms in Hong Kong in 1997; and human outbreaks of avian flu have been recorded). The virus replicate in the mucosa of the nasopharynx where they cause pharyngitis. The incubation period of the disease is between 2-3 days; and the infection can reach the lungs where pulmonary infections also occur.
The clinical symptoms of infections caused by the influenza viruses include fever, headache, cough, sore throat and nasal congestion. Secondary bacterial infections can occur in influenza infections; and these infections which occur mainly in the lungs are caused by Streptococcus and Haemophilus species. However, secondary bacterial infections due to influenza are common amongst the immunocompromised host and people who are seriously ill. Flu is usually the general name used to describe viral infections caused by influenza virus. But the infection is sometimes confused with common cold and other bacterial respiratory diseases which present with similar symptoms. Proper clinical and laboratory diagnosis are critical for the effective treatment of flu caused by influenza viruses. Antiviral therapy such as the use of amantadine (a synthetic amine and a viral uncoating blocker) is available for the treatment of flu caused by influenza virus. And since the organism is a genetically variable viral pathogen (as exemplified by their antigenic drift and antigenic shift), effective vaccination and prophylaxis should be administered to vulnerable groups of the human population.
Bornaviridae family contains only one genus of virus known as Bornavirus. Bornavirus contain only one species of virus, Borna disease virus – the causative agent of Borna disease (a slow virus infection). Borna disease virus (BDV) is the only viral species in the genus Bornavirus, and they have a ss(-)RNA genome. The ss(-)RNA genome of BDV is converted to a ss(+)RNA genome with the virus’ RNA-dependent RNA Polymerase or replicase that carries out this function. They measure about 90 nm in diameter and BDV are generally enveloped viruses. BDV like the Orthomyxoviruses replicate in the nucleus of their host cell, and they are released via a budding process through the cytoplasmic membrane of the cell. The nucleocapsid of BDV is spherical; and the virus is sensitive to UV light, organic solvents and detergents. The natural hosts of BDV are horses and sheep.
Aside sheep and horses, BDV can also cause infection in a wide variety of other mammals including deer’s, donkeys, shrews, pigs, mules, rats, cattle’s and rabbits and birds. BDV causes lassitude or lethargy, spasm and paralysis in animals especially in horses. And BDV causes a neuropsychiatric disorder in humans. Characteristically, BDV causes infected horses to walk in a staggering manner. Abnormalities in the movement and behavioural patterns of animals infected with BDV are typical characteristics of BD. Infections by BDV in animals and humans are generally asymptomatic in nature. Borna disease (BD) occurs mostly in Europe where the first case of the infection was reported (precisely in a town in Saxony, Germany), but the disease now occur worldwide and have been reported in Africa and Asia.
BDV is highly neurotropic in nature; and this implies that the virus has a high affinity for the nerve cells of the CNS. The affinity of BDV for the nerve cells of its host explains the neuropsychiatric disorder of BD in its host. BDV has the ability to downregulate its replication in vivo or in its host, and this leads to a decrease in the amount of virions that will be produced during an infection. Though the immune system mounts a putative attack against the virus, the low production of infectious virions by BDV allows the pathogen to establish a persistent or chronic infection in its host. Amantadine, an antiviral drug that inhibits the uncoating of viruses is usually used for the treatment of BD in horses, other mammals and humans. The transmission route of BDV still remains largely obscure, and vaccination against BDV infection is available in some economies where the disease is endemic particularly in horses.
Caliciviridae family is a family of non-enveloped viruses that possess a ss(+)RNA genome. Their site of replication is in the cytoplasm, and viruses in this family have an icosahedral nucleocapsid. They measure between 27-40 nm in diameter. Viruses in the Caliciviridae family are generally called caliciviruses. Members of the calciviruses cause viral gastroenteritis in humans. Caliciviridae family comprises of four genera of viruses; and these are Vesivirus, Lagovirus, Norovirus and Sapovirus genera. Only Norovirus and Sapovirus genera contain viruses that cause infections in humans. Vesivirus and Lagovirus contain viruses that cause infections in other mammals including pigs, rabbits and cats. Norwalk virus (NV) and Sapporo virus (SV) are the two important members of the genera Norovirus and Sapovirus respectively; and they both cause epidemic gastroenteritis in humans. NV and SV have a worldwide distribution, and they are transmitted in human population via the feacal-oral route.
Together with adenoviruses and rotaviruses, caliciviruses are the most frequently reported viral agents that cause gastroenteritis in human populations. Gastroenteritis caused by NV and SV occurs in both children and adults. And infections with NV are mostly common in overcrowded settings such as in military camps and nursing homes. Human infections due to Noroviruses are usually associated with the consumption of fresh or raw foods such as salads and sandwiches during outdoor activities or camping. Noroviruses enter the human body predominantly via the oral route. Transmission of NV from person-to-person and via contaminated water or food is also possible. The clinical symptoms of the disease include vomiting, anorexia, abdominal cramp, fever, myalgias, chills, diarrhea, sore throat, headache and nausea.
Noroviruses causes prolonged gastroenteritis in immunocompromised patients as opposed to individuals with active or strong immunity that can contain the infection to some extent. Generally, viral gastroenteritis due to NV can be benign and self-limiting especially at the covert stage of the disease; and thus most cases of the infection are not treated with antivirals due to non-complications of the disease. However, NV infection can be very debilitating when the disease is at its symptomatic stage. Oral administration of bismuth subsalicylate and other oral rehydration therapy (O.R.T) is vital to contain the disease at its symptomatic stage. Regular hand washing and proper disposal of sewage (so that they do not contaminate the water ways) as well as good environmental and personal hygiene are important for the control and prevention of gastroenteritis due to caliciviruses. No vaccine exists for the vaccination of human population against NV infection.
Picornaviridae family represents a large family of virus that causes infections in humans, primates and other mammals. Enteroviruses, polio virus, foot-and-mouth disease virus (the first animal virus to be discovered), hepatitis A virus (HAV) and rhinoviruses are some examples of viruses in the Picornaviridae family. With the exclusion of polio infection or poliomyelitis (caused by polio virus) which has been eradicated in some parts of the world (e.g. USA), Picornaviruses have a worldwide distribution. There are nine genera of viruses in the Picornaviridae family; and these include Aphthovirus, Cardiovirus, Enterovirus, Erbovirus, Hepatovirus, Kobuvirus, Parechovirus, Rhinovirus, And Teschovirus. Only five genera of Picornaviridae family including Enterovirus, Hepatovirus, Kobuvirus, Parechovirus and Rhinovirus contain Picornaviruses that cause infections in humans.
Enteroviruses and polio virus are in the genus Enterovirus; foot-and-mouth disease virus (which causes foot-and-mouth disease in animals) is in the Aphthovirus genera; HAV is found in the Hepatovirus genera and rhinoviruses are found in the genera Rhinovirus. The human rhinovirus is the causative agent of the majority of common cold in humans; and the disease is characterized by inflammation of the nasal cavity. Rhinoviruses are transmitted via the respiratory route or tract of infected persons. HAV found in the Hepatovirus genera is the causative agent of infectious hepatitis in humans; and HAV is transmitted via the feacal-oral route. Kobuvirus species which is transmitted via the feacal-oral route causes gastroenteritis in humans; and infection with the virus is common amongst people who eat raw sea foods such as oysters.
Viruses in the Picornaviridae family are non-enveloped viruses that posses a ss(+)RNA genome. They measure between 28-30 nm in diameter and this makes members of viruses in this family one of the smallest viruses in terms of the size of their virion. Picornaviruses are resistant to ether. Enteroviruses which include polio virus, Coxsackie A, Coxsackie B and echoviruses are the different serotypes that make up the Enterovirus genera. Enteroviruses are transient inhabitants of the GIT of humans and they cause mild gastrointestinal disease in infected individuals. Poliomyelitis or polio caused by polio virus is one of the most serious diseases caused by Enteroviruses. But the disease which is characterized by the deformity of the limbs of infected children has been contained in most parts of the world due to massive immunization programmes against the disease. Polio virus infects motor neurons of the spinal cord and other parts of the CNS, and this leads to flaccid paralysis. Enteroviruses are transmitted via the feacal-oral route.
Astroviridae family contains only two genera of viruses; and these include Mamastrovirus (which contain the human Astroviruses and animal Astroviruses) and Avastrovirus (which contain viruses that infect birds such as ducks and turkeys). The human Astroviruses cause infection in humans while the animal Astroviruses infect other mammals including pigs, cattle, sheep and cats. Astroviruses are non-enveloped viruses that posses a ss(+)RNA genome. They have an icosahedral nucleocapsid and viruses in this family measure between 28-30 nm in diameter. Astroviruses are resistant to ether, chloroform and lipid solvents. Just like the Picornaviruses, Astroviruses are also amongst the smallest known viruses owing to the size of the virion. Astroviruses replicate in the cytoplasm and they are released from their host cell via cell lysis.
The Astroviruses cause disease in both humans and animals. Astroviruses cause an acute gastrointestinal disease or diarrhea in humans; and this intestinal viral infection is usually self-limiting in nature. They cause gastrointestinal infections in people of all age groups but infections with Astroviruses are most common in immunocompromised people, young children and older people. Astroviruses like the sapoviruses, Picornaviruses, reoviruses, caliciviruses and adenoviruses can be generally referred to as enteric viruses because they cause mild diarrhea and other forms of enteritis in their human host. They have a worldwide distribution. Astroviruses are transmitted in human populations through the feacal-oral route. And the consumption of food or water contaminated with the virus is a usual means of transmission of the pathogen.
Astroviruses are significant food borne pathogens, and they are responsible for a number of foodborne illnesses across the globe. Person-to-person transmission of Astroviruses via direct body contact and transmission through fomites or contaminated surfaces is also possible. Astroviruses are shed in large amounts in the feaces of infected people; and thus such persons could serve as active means of transmitting the disease agent in defined human population. Frequent and proper hand washing, proper sewage disposal, and good environmental sanitation or decontamination is vital to the effective control and prevention of Astrovirus infection inclusive of other gastroenteritis caused by other enteric viruses as aforementioned.
Reoviridae family is a unique family of RNA-containing viruses in that it is the only family of RNA viruses that contain viruses which posses a double-stranded DNA (dsRNA) genome; and one which cause infection in humans and other mammals. Other families of RNA viruses with dsRNA genome include Birnaviridae, Chrysoviridae, Cystoviridae, Hypoviridae, Partitiviridae, and Totiviridae. Only the Birnaviridae and Reoviridae families contain viruses that infect vertebrates. The other five families (including Chrysoviridae, Cystoviridae, Hypoviridae, Partitiviridae, and Totiviridae) contain viruses that infect other forms of life including fungi, birds, plants, protozoa, insects and bacteria. All RNA viruses with the exclusion of reoviruses have single-stranded RNA (ssRNA) genome. Reoviruses are non-enveloped viruses with dsRNA genome and they measure between 60-80 nm in diameter.
They have an icosahedral nucleocapsid, and reoviruses are resistant to ether. However, reoviruses are sensitive to UV light, formalin and chlorine compounds. Reoviruses replicate in the cytoplasm and they exit their host cell via cell lysis. During their replication in the cytoplasm of their host cell, the RNA-dependent RNA polymerase which is contained in the virion is used for the transcription of the organism’s dsRNA into an mRNA since the dsRNA genome is entirely inactive to act as mRNA template in the synthesis of viral proteins. There are twelve (12) genera of viruses in the Reoviridae family; and these include Orthoreovirus, Orbivirus, Coltivirus, Rotavirus, Seadornavirus, Aquareovirus, Cypovirus, Idnoreovirus, Fijivirus, Phytoreovirus, Oryzavirus and Mycoreovirus. Only members of the genera Orthoreovirus, Rotavirus, Orbivirus, Coltivirus and Seadornavirus infect humans and other vertebrates. The other genera of Reoviridae family contain viruses that cause infection in non-vertebrates, some vertebrates and microorganisms such as fungi, protozoa and bacteria.
Rotaviruses in the genus Rotavirus are the most important human pathogen of the Reoviridae family; and this is because they cause diarrhea in young children including infants. The elderly, transplant patients and the immunocompromised host are also not left out as rotaviruses cause diarrhea in these individuals. Rotaviruses are distributed worldwide; and they are the most frequent cause of severe diarrheal disease or gastroenteritis in newborns/infants and young children. Different serotypes of rotaviruses exist including rotavirus A, B, C, D, and E; and rotaviruses are usually transmitted via the feacal-oral route and respiratory route. Rotaviruses invade the microvilli or villi of the small intestines where they attach to the mucosa of the intestine to cause several diarrheal illnesses. The incubation period of the disease is between 1-2 days; and clinical symptoms of diarrhea due to rotaviruses include vomiting, diarrhea (characterized by profuse passing of watery stool) and abdominal cramp. No antiviral treatment exists for infections caused by rotaviruses.
And the management of the diseases is usually based on the administration of O.R.T to affected patients on time. The disease can be fatal if care is not administered on time to individuals especially infants and young children who ingests the virus via contaminated hands, water or food. Severe diarrheal disease caused by rotaviruses is responsible for a significant amount of infant and child morality across the globe. And the mortality rate of the disease is high in developing countries where environmental sanitation and personal hygiene in young children is averagely poor. Children who have been previously exposed to rotavirus infection usually develop passive immunity against the infectious agent. Due to the worldwide morbidity and mortality caused by rotaviruses in infants and young children, there have been steps to develop vaccines against the infection and some of such potent vaccines (usually administered orally) are currently in use in some developed economies. Frequent and proper hand washing is vital to the prevention of rotavirus infection especially in hospitals and other healthcare facilities where outbreaks due to rotaviruses are possible.
Arenaviridae family is comprised of viruses that are transmitted from animals particularly rodents such as mice, rabbits and rats to humans when they come in direct contact with the aerosolized excreta including urine, saliva and feaces of these animals. These rodents serve as the natural host or reservoirs of the viruses in the Arenaviridae family; and human infection occurs via contact with the excreta of these animals. Generally, the viruses in this family cause serious disease in humans; and these infections are collectively known as haemorrhagic fevers because of the invariable bleeding from different body sites of infected humans. Filoviridae family, Bunyaviridae family and Flaviviridae family are other viral families aside the Arenaviridae family that contain viruses which cause haemorrhagic fevers in humans.
Viruses in the Arenaviridae family have an ambisense genome; and this implies that their genetic makeup is partially coded as positive (+) sense and as negative sense (-) at the same time. The phrase “ambisense” is generally used to describe viruses with a single-stranded RNA genome, and whose genetic coding sequences is expressed in the positive sense (+) as well as in the negative sense (-). Arenaviruses (i.e. viruses found in the Arenaviridae family) are typical examples of viruses whose genomes are expressed in the ambisense configuration as ‘ss(+/-)’. Arenaviruses are enveloped viruses, and they possess an ssRNA genome that is ambisense in nature as aforementioned. They have a helical nucleocapsid; and Arenaviruses replicate in the cytoplasm of their host cell. Arenaviruses are released by a budding process through the cytoplasmic membrane of their host cell since they are enveloped viruses. They measure between 110-300 nm in diameter. Arenaviruses are sensitive to UV light, chlorine compounds, high temperatures and to other organic compounds.
Viruses in the Arenaviridae family are distributed worldwide, and they cause haemorrhagic fevers that are chronic in nature. Arenaviruses cause asymptomatic infections in rodents such as rats, mice and rabbits (which are the natural hosts of these viruses). However, humans show clinical signs of the disease and become symptomatic when they come in contact with the body fluids or excreta of the rodents containing the pathogen. Arenaviruses are basically divided into two genera: Old World Arenaviruses and New World Arenaviruses. Viruses in the Old World Arenavirus genera which cause infections in humans include Lassa virus (LASV) and Lymphocytic choriomeningitis virus (LCMV). Machupo virus (MACV), Junin virus (JUNV), Sabia virus (SABV) and Guanarito virus (GTOV) are examples of viruses in the New World Arenavirus genera that cause disease in humans; and these viruses are geographically limited because they are mostly found in South American countries including Venezuela (GTOV), Argentina (JUNV), Bolivia (MACV) and Brazil (SABV) where they cause haemorrhagic fevers that are peculiar to each region.
LASV cause haemorrhagic fever in countries in the African continent especially in West Africa including Nigeria; and the causative agent of this disease is naturally found in the rodent species known as Mastomys natalensis, a multimammate mouse that readily enter people’s houses especially in the rural areas where these rodents are also eaten as a delicacy. LCMV, the causative agent of aseptic meningitis is distributed worldwide; and the pathogen causes febrile illness and central nervous system disease occasionally. Lymphocytic choriomeningitis virus (LCMV) is naturally found in the house mouse species known as Mus musculus, from which human infections occurs especially when people come in contact with the excreta or body fluids of the pathogen’s natural host. Lassa virus (LASV), the causative agent of Lassa fever (a type of haemorrhagic fever) is endemically found in Africa particularly in West Africa where it was first discovered in a town known as Lassa in the Northern part of Nigeria in 1969 after it killed an American missionary nurse stationed at that location as at the time.
The virus has caused a number of outbreaks in recent times in many regions of West Africa including Nigeria where periodic outbreak of Lassa fever normally occurs even in healthcare facilities. The mortality rate of Lassa fever is high. Arenaviruses including LASV are aerogenically introduced into the human body via contact with excreta of rodents (in this case: Mastomys natalensis rats for LASV) that naturally harbour the virus. After invasion, affected individuals show symptomatic viraemia; and this is immediately followed by invasion and attack of various internal organs of the body. Clinical symptoms of haemorrhagic fever due to LASV infection include myalgia, fever, severe prostration, body pain or weakness and other haemorrhagic or neurological signs.
This is also followed by severe gastrointestinal upset such as abdominal pain, nausea and vomiting, and diarrhea. Profuse bleeding from the nose, eyes, mouth and ears (which is usually rare and occurs in some cases of the disease) occurs in infected individuals; and at this stage of bleeding the disease becomes fatal and may claim the life of the patient. The incubation period of LASV infection is between 10-21 days; and infected patients becomes symptomatic immediately after infection. The clinical management of patients infected with LASV is usually based on a supportive therapy that is geared toward stopping the bleeding and maintaining the normal functioning of affected vital organs of the body. However, the antiviral drug ribavirin has been used for treatment as well as for prophylaxis purposes especially in those who are exposed to the virus.
LASV can be transmitted from an infected person to a susceptible or unexposed host; and human contact with excreta of rodents harbouring the virus is the most common route of contamination or contraction of the pathogen. Consumption of food infected or contaminated with the excreta of rodents that harbour the pathogen is also another route via which human infection can occur. LASV and other Arenaviruses form stable and infectious aerosols when shed via feaces, saliva or urine of the rodents; and this account for the ease with which human infections occurs especially when humans come in contact with excreta from the multimammate mouse or other materials including food stuffs contaminated with the excreta. The prevention and control of haemorrhagic fever caused by Arenaviruses especially LASV that ravages some parts of West Africa is mainly based on effective rodent control measures since these pathogens are naturally harboured in these animals.
In most rural communities in West Africa, the rodents (which are the natural hosts or reservoirs of these viruses) are eaten as delicacies; and this could serve as route via which the disease can spread in a defined human population. No vaccine currently exists for the prevention of haemorrhagic fever caused by LASV. The primary transmission of LASV is from its natural host i.e. multimammate mouse (M. natalensis) to humans who eat the animals and those who come in contact with the excreta or aerosols contaminated with the excreta of the rodents. Thus people who live in regions where the disease is endemic or where these natural hosts of Arenaviruses are common should avoid contact with them as much as possible. Raw food stuffs should be properly stored and covered in containers so that the rats do not excrete on them. Healthcare practitioners and laboratory personnel’s should always wear the correct protective wears including laboratory/hospital gowns, eye masks or goggles and gloves when clerking patients and handling samples from potentially infected individuals because the Arenaviruses are highly infectious.
Filoviridae family consists of viruses generally known as Filoviruses. Filoviruses have a ss(-)RNA genome. They are enveloped viruses because they have envelope. Viruses in this family are pleomorphic in nature, and thus have varying shapes. They actually appear in very long and thread-like structure. The length of Filoviruses can be above 10,000 nm in size due to the variability of the pathogen; and they measure about 80 nm in diameter. But the normal length of Filoviruses is between 800-1000 nm. The Filoviridae family is comprised of only two genera of viruses viz: Marburgvirus and Ebolavirus, both of which cause haemorrhagic fevers in humans. The viruses in each of these genera can generally be called Marburg virus and Ebola virus as the case may be; and they are usually named according to the geographic region where they cause a disease outbreak. Marburg virus is the causative agent of Marburg disease, a type of haemorrhagic fever generally known as Marburg haemorrhagic fever (MHF). And Marburg virus is only a single-type virus i.e. it is the only virus in the genus Marburgvirus.
MHF has an incubation period of 5-10 days; and the disease only occurs in Africa. Maculopapular rash, nausea, vomiting, chest pain, diarrhea, abdominal pain and massive haemorrhagic and multiple organ dysfunction or failure are some of the clinical symptoms of the disease. Marburg virus infects humans and other primates; and human-to-human transmission of the disease is possible. Ebola virus is the causative agent of ebola disease, a severe haemorrhagic fever that occurs in parts of Africa and the Philippines. There are four subtypes of ebola virus that have been recognized to infect humans; and these are: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Cote d’Ivoire ebolavirus (CIEBOV) and Reston ebola virus (which occurs only in the Philippines). ZEBOV, SEBOV and CIEBOV infect humans while CIEBOV infect monkeys in the Philippines.
The incubation period of ebola disease is 2-21 days; and the disease is characterized clinically by high fever, joint pains, muscle aches, sore throat, diarrhea, headache and vomiting. A maculopapular rash which appears all over the body; and severe internal and external bleeding is also associated with ebolavirus infection. Filoviruses (i.e. viruses in the Filoviridae family) are primate-borne viruses that are usually transmitted to humans who come in contact with excreta or body secretions of the animals or primates that serve as natural host for these viruses. Filoviruses are of biosafety and biosecurity concern because of the bioterrorism threat they pose; and thus viruses in the Filoviridae family are classified as biosafety level 4 (BSL-4) pathogens.
Coronaviridae family is comprised of viruses known as coronaviruses. There are only two genera of viruses in the Coronaviridae family. Coronavirus (which contain Coronaviruses) and Torovirus (which contain Toroviruses) are the two main genera of Coronaviridae family. Both coronaviruses and toroviruses have a worldwide distribution. Viruses in this family include those that infect humans and other mammals such as pigs, mice, cattle, birds and bats. Coronaviruses are enveloped viruses that replicate in the cytoplasm of their host cell. Coronavirus are released from their host cell by a budding process via the cell membrane after completing the cycle of their replication in affected host cell. They have a ss(+)RNA genome and coronaviruses measure between 120-160 nm in diameter. Coronaviruses have a helical nucleocapsid. Viruses in this family cause respiratory infections in humans and other animals that they infect.
The commonest viral member of the Coronaviridae family is severe acute respiratory syndrome (SARS) coronavirus – that causes a severe respiratory infection or lung disease that is characterized by pneumonia of the lower respiratory tract of humans. In 2003, SARS coronavirus caused several outbreaks of SARS in most parts of the world; and the epidemic was marked by several morbidity and mortality especially in hospital environments and amongst healthcare practitioners who had direct contact with cases of SARS. The first outbreak of SARS actually occurred in the Chinese city of Guangzhou province in 2002 from where the world outbreak of the disease started from. Aside the lower respiratory tract of humans which SARS coronavirus is known to colonize, the virus also attack the gastrointestinal tract and neurological tissues of the body. The main routes via which coronaviruses are transmitted are via aerosols, body contact and through the oral-feacal route (especially for some toroviruses that infect animals).
But the main major route via which SARS coronaviruses are spread in human population is via the respiratory tract of infected individuals especially when they cough and sneeze. SARS coronavirus infection in humans has an incubation period of 2-7 days and infected individuals are highly infectious especially when they present with respiratory symptoms that characterize the disease. Common cold, malaise, and fever are some of the asymptomatic symptoms of SARS; and when individuals become very symptomatic, infected individuals have myalgia, dry throat and even shortness of breath. No specific antiviral therapy exists for the treatment of SARS in humans. However, the management of the disease is mainly based on supportive therapy.
Paramyxoviridae family is a large family of viruses that infect both humans and animals. Viruses in this family are generally called paramyxoviruses; and they include the most important causative agents of respiratory infections in young children and infants (i.e. respiratory synctial virus and parainfluenza viruses) as well as the causative agent of measles – a highly infectious viral disease of global health importance that have been eradicated. Paramyxoviruses have a ss(-)RNA genome; and they are enveloped viruses. They have a helical nucleocapsid and paramyxoviruses measure between 150-350 nm in diameter. Paramyxoviruses replicate in the cytoplasm of their host cell and they are released by a budding process through the cytoplasmic membrane.
The Paramyxoviridae family is divided into two subfamilies viz: Paramyxovirinae and Pneumovirinae. Paramyxovirinae subfamily is comprised of five genera: Respirovirus (which contain the human parainfluenza viruses – types 1 and 3), Rubulavirus (which contain mumps virus and the human parainfluenza viruses – types 2 and 4), Avulavirus (which contain Newcastle disease and avian parainfluenza viruses), Morbillivirus (which contain measles virus) and Henipavirus (which contain Hendra and Nipah viruses). The Pneumovirinae subfamily contains only two genera: Pneumovirus (which contain the human respiratory synctial virus) and Metapneumovirus (which contain viruses that infect birds such as turkey). Paramyxoviruses have a worldwide distribution with the exception of viruses in the genera Henipavirus – which are geographically limited and are found in parts of Asia including Malaysia, Singapore and Australia.
Majority of the diseases caused by paramyxoviruses including mumps, parainfluenza and measles are known as notifiable diseases that must be reported to public health authorities for proper action to be taken to contain their outbreak and spread. Paramyxoviruses are mainly spread via aerosols and the respiratory tract as droplet infections; and they majorly affect children and infants inclusive of adults. Attenuated live vaccines exist for the prevention of mumps and measles in children and other susceptible human population. And antiviral agents are also available for the treatment of infections caused by paramyxoviruses. Due to the high morbidity and mortality associated with paramyxoviruses infection especially measles (a childhood disease characterized by the appearance of maculopapular rash all over the body); infections caused by paramyxoviruses still remain amongst the preventable diseases that newborns and susceptible populations are vaccinated against all over the world.
Rhabdoviridae family is comprised of viruses generally known as rhabdoviruses. Rhabdoviruses have a ss(-)RNA genome and they measure between 45-100 nm in diameter. They are enveloped viruses and rhabdoviruses replicate in the cytoplasm of their host cell. They are released from their host cell by a budding process through the cytoplasmic membrane. Rhabdoviruses have a helical nucleocapsid. Viruses in the Rhabdoviridae family infect humans, animals, fish, insects and even plants. There are six genera of viruses that make up the Rhabdoviridae family, and these are: Vesiculovirus, Lyssavirus, Ephemerovirus, Novirhabdovirus, Cytorhabdovirus and Nucleorhabdovirus. Only the Vesiculovirus and Lyssavirus contain important human viruses. Vesicular stomatitis virus (VSV) and rabies virus which are found in the Vesiculovirus and Lyssavirus genera respectively are of importance to man because they cause significant human disease.
VSV causes an asymptomatic herpes-like disease in humans especially those in close contact with animals such as horses, pigs and cattle that are naturally infected by the agent. And VSV is mostly endemic in South American countries. Rabies is an acute and fatal, zoonotic viral infection of the CNS of humans. And the disease which is prevalent worldwide occurs in both animals and humans. Human infection with rabies virus occurs following the bite of a rabid dog or other animals such as wolves, foxes, jackals, bats and monkeys. The virus is present in the saliva of rabid animals and it is passed on or transmitted to humans by the bite of the animal and even when they lick an open wound on the body. Rabies virus is destroyed by UV radiation, heat, ether treatment and trypsin treatment.
Rabies virus multiplies in the connective tissues or muscle cells at the site of bite; and the virus is transported to peripheral nerves of the CNS from where neurological dysfunction occurs. Sore throat, headache, lack of appetite, malaise and fever are other mild symptoms of rabies. The incubation period of the disease is 1-3 days after exposure but it can last for weeks and even months and years depending on the intensity and site of bite. Localized paralysis, jerky movements, increased muscle tone and hydrophobia (the fear of water) are the most significant clinical syndromes of the rabies disease; and infected patients may due within weeks after infection especially when appropriate medical intervention is delayed. Human-to-human transmission of rabies virus is not possible. No treatment exists for rabies virus infection once the virus has invaded the body and symptoms starts to appear. However, supportive therapy is administered to infected individuals in order to save their lives. And rabies vaccine exists as a prophylactic measure in susceptible human population.
VIRAL CULTIVATION IN THE LABORATORY
Viral cultivation unlike the cultivation or culture of other microbial forms including bacteria and fungi is quite unique and different from the usual routine cultivation techniques undertaken in the conventional microbiology laboratory (both in the hospital and in an academic institution). One of the major reasons for this is because viruses unlike other microbes exist as a living thing inside a living host cell. Viruses only replicate inside a living cell including those of humans, plants, animals, other mammals and microbes as well. They are obligate intracellular parasites that engage in a close relationship with their host; and this allows viruses to take over the cellular and replication machinery of their host cell to their own advantage.
Virtually all the materials required for viral replication including cellular energy are provided by the viral-infected host cell. They rarely replicate independent of a living host cell. Thus, the culture of viruses requires the culture of living host cells as hosts for the unperturbed growth and replication of the virus being propagated. Viruses cannot grow and replicate in non-living culture media or agar plates as is applicable to bacteria and fungi that can readily be propagated in such growth medium. These are some of the factors that distinguish viral replication from the propagation or cultivation of prokaryotic and eukaryotic cells in the laboratory. Several reasons exist for the cultivation of viruses in the laboratory. Some of the major reasons for viral cultivation are highlighted in this section.
- Viruses are cultivated in order to prepare viruses used for the production of vaccines – that are used as prophylaxis and preventive measures for some infectious diseases.
- To isolate pathogenic viruses from clinical samples as an aid in the diagnosis of viral infections.
- To identify and classify viruses from clinical samples and other viral-laden samples.
- Viruses are cultivated in order to study their host-parasite relationship.
- To study the genetics of viruses inclusive of their structures and replication patterns.
- They are cultivated for other research purposes especially in studying the efficacy of novel antiviral drugs.
Viruses can be cultivated in vitro and in vivo – depending on the discretion of the researcher and the type of viral cultivation to be undertaken. The in vivo viral cultivation techniques include: the use of a natural viral host, experimental animals, transgenic animals and embryonated eggs. In vivo viral cultivation techniques are generally undertaken in living host cells which include embryonated eggs, experimental animals and transgenic animals as aforementioned. However, this is not the case for the in vitro viral cultivation technique – which is usually carried out in cell culture or tissue culture plates that contain living cells of animals that support the growth of the virus(s) being propagated. Organ culture (performed for viruses that attack specialized organs of the body) and explant culture (which are rarely performed) are two other methods or techniques of cell culture that can be used to cultivate viruses in the laboratory. Generally, viral cultivation can be divided into three (3) main groups.
ANIMAL INOCULATION SYSTEMS OF VIRAL CULTIVATION
The animal models used for viral inoculation include experimental animals such as transgenic animals, monkeys, primates, hamsters, guinea pig, and laboratory mice or rabbits. Animal inoculation is generally used for the cultivation of viruses that cannot be cultivated in embryonated eggs or cell culture systems. The newborns of these animals are usually preferred in the cultivation of viruses when viral cultivation using animal models is anticipated. Animal inoculation systems however, where mostly used before the advent of the cell/tissue culture systems and embryonated egg systems. Even though animals still play an essential role in studying viral pathogenesis or virulence; the use of animals for viral cultivation is gradually being replaced by other means of viral cultivation such as the cell/tissue culture systems and the use of embryonated eggs as well. The viruses are usually inoculated subcutaneously or intraperitoneally; and the inoculated experimental animal(s) is observed for the development of the disease caused by the said pathogenic virus. Death usually occurs in most cases. And the virus is then isolated from the tissues or organs of the dead animals and purified for further studies. Nevertheless, animal inoculation systems for viral cultivation has some disadvantages and advantages. The extinction of these animals aside other factors is one of the reasons limiting the use of animals for viral cultivation.
CELL/TISSUE CULTURE SYSTEMS OF VIRAL CULTIVATION
Cell/tissue culture is the in vitro technique by which cells or tissues obtained from an organism are maintained under controlled laboratory conditions outside its natural host. It provides the most widely used and most powerful hosts for cultivation and assay of viruses since the animal inoculation systems are not widely accepted due to some ethical issues surrounding the use of animals in research. Animal viruses can be grown and/or cultivated efficiently in vitro in cell/tissue culture systems due to the availability of animal cells that can be propagated outside their host organisms in an efficient manner over certain period of time. Table 4 shows the advantages and disadvantages of animal inoculation systems used for viral cultivation.
Table 4: Advantages and disadvantages of animal inoculation systems for viral cultivation
|It is the primary method used for the isolation of some
|Animal systems for viral cultivation are very expensive.|
|Animal inoculation systems helps the researcher to
determine the pathogenesis and clinical symptoms of
the virus being propagated.
|They are difficult to maintain; and choosing a unique animal to cultivate a given virus is usually intricate.|
|They provide a reliable method of studying viral
replication patterns. Animal inoculation systems helps in the identification of antibodies produced against the virus.
|Not all human viruses are cultivated in animals.
Ethical issues limit the use of animals for viral cultivation since animal welfare is given top-most priority in some regions.
|It is the perfect medium for studying immune responses
to a particular virus.
|Some animals such as mice do not provide the ideal environment for the development of human vaccines.|
Tissue culture has enhanced the process of viral cultivation; and the practice is now widely used for the isolation and identification of viruses. The development of growth media that support the thriving of animal cells outside their normal environment as wells as the development of antibiotics and drugs that inhibit the growth of bacteria and fungi in cell/tissue culture plates during cell culture has also enhanced the use of this technique to cultivate viruses in vitro. The growth or replication of virus in cell culture plates can be deciphered in several ways including the development of cytopathic effects, hemadsorption and the formation of plaques. Cytopathic effects are observable morphological changes that occur in cells because of viral replication. Death of the cells in culture, ballooning and the clustering or binding together of the cells are some examples of cytopathic effects observable in cell/tissue culture plates during viral cultivation.
Hemadsorption is the phenomenon that occur when red blood cells (RBCs) added to a cell culture plate during the incubation of the plate gets attached to the plasma membrane of the infected cultured cells which have been altered by the cultivated virus. Plaque is the area of lysis or hole formed in a lawn of cells in cell/tissue culture due to the infection or replication of a virus. It is the localized areas of cellular destruction and lysis of cells in cell culture due to viral infection. Plaque formation is not usually used to measure or detect viral replication in cell cultures because they do not always form when viruses are cultivated in vitro in tissue culture vessels. Nevertheless, plaque assay is an important viral assay used to quantify the amount of infectious virus in a sample. Plaque assay is a technique that is used to determine the concentration of infective particles in a virus solution or sample; and it is usually expressed as plaque-forming units per ml (pfu/ml).
In plaque assay, the number of plaques induced on a lawn of bacteria or eukaryotic cells in cell culture plates is generally used to evaluate the concentration of infective particles of viruses present in a given sample. PFU is defined as the number of plaques formed per unit of volume or weight of a virus suspension or sample. Though the number of plaques formed in the cell culture plates does not necessarily reflect or show the actual number of virus particles present in the samples, it gives a proportionate ratio of the infecting agent present. It is noteworthy that the number of plaques formed is generally expressed as pfu/ml because of the uncertainty that a single plaque arose from a single infectious viral particle. To avoid contamination of the process, viral cultivation is undertaken inside a biosafety laminar flow cabinet or hood. Both cytopathic effects and hemadsorption can be detected microscopically using inverted microscopes meant for this purpose. Cell/tissue culture techniques for viral cultivation are divided into three (3) types.
PRIMARY CELL CULTURES OF VIRAL CULTIVATION
Primary cells are cells obtained directly from freshly killed animals; and they can only be passaged or subcultured once or twice. They support the cultivation of many viruses. The cells used here can also be obtained from humans. Primary cells are heterogeneous at the stage of collection. But they are homogenized with trypsin, a proteolytic enzyme prior to their usage. Chelating agents like ethylene diamine tetra-acetic acid (EDTA) can also be used for the dispersal of cells in tissues prior to cell culture. The homogenization of these tissues helps in the release of single cells and small aggregates of cells capable of initiating optimal growth of the cultivated virus(s). Kidney cells and lung tissues from animals are typical examples of sources of cells for primary cell culture. Primary cell culture though best, is expensive; and it is usually difficult to obtain a reliable supply of normal cells from freshly killed animals to undertake primary cell culture during viral cultivation. Primary cell lines are generally used for viral isolation and for vaccine preparation.
- Continuous cell cultures: Continuous cells are cell lines that are capable of more prolonged and indefinite growth. They are immortalized cell lines that can be passaged or subcultured several times unlike the primary cell lines that can only be passage once or twice. HeLa cells (human carcinoma of cervix cell line) are typical examples of continuous cell lines capable of indefinite growth. Continuous cell lines are usually derived from tumour or cancerous cells. These cells are not used for vaccine preparation since they are derived from malignant tumours or cancer cells. Continuous cell culture can only handle a limited number of viruses unlike the primary cell culture that handles a wide variety of viruses even though they are the easiest type of cell culture technique to undertake for viral cultivation.
- Semi-continuous cell culture: Semi-continuous cell lines which can otherwise be known as diploid cell lines are cell lines that contain the same number of chromosomes as the parent cells from which they are derived. Though they can be passaged several times, semi-continuous cell lines cannot be subcultured indefinitely like the continuous cell lines used in continuous cell culture. However, semi-continuous cell lines can be passaged up to 50 times unlike the primary cell lines that can only be passaged once or twice. Examples of semi-continuous cell lines include cells from the rhesus monkeys and human embryonic lungs. They can be used for vaccine preparation and for the isolation of fastidious viruses.
One of the disadvantages of using cell culture techniques in carrying out viral cultivation is that cell culture requires specialized and trained personnel with experience to do it. It is not a routine practice in some hospital laboratories – since they rarely isolate and identify pathogenic viruses from clinical samples. Most clinical samples collected for viral examinations are sent to reference laboratories where trained personnel’s and equipments are available for their processing. Cell/tissue culture technique does not support the cultivation of some animal viruses. Nevertheless, cell culture technique has a high sensitivity is identifying a virus and the technique is relatively cheap and easy to perform.
EMBRYONATED EGG INOCULATION OF VIRAL CULTIVATION
Embryonated egg inoculation technique is one of the common techniques employed for virus isolation and identification. Embryonated chicken eggs provide an ideal environment for the cultivation of pathogenic viruses and other forms of virus for various pharmaceutical, medical and research purposes. Since its development and use in the early 1930’s, embryonated egg inoculation technique for viral cultivation has played tremendous role in the study of virology especially in their application for the production of vaccines (Figure 9).
Figure 9: Scientists working on vaccine production for the dreaded H1N1 influenza virus strain at a pharmaceutical company in Hangzhou in East China’s Zhejiang province.
Developing chick embryo eggs that are about 10-14 days old are often the most preferred for embryonated egg inoculation technique. This is because such category of embryonated eggs provides a variety of differentiated tissues or cells such as the chorioallantoic membrane, amniotic fluids, allantoic cavity and the yolk sac region – which alls serve as suitable substrates for the growth of a wide array of viruses including those that cause infections in humans. Inoculation of embryonated chicken eggs is still the most convenient method for cultivating high concentrations of viruses and, thus, this technique of viral cultivation (i.e. the embryonated chicken egg technique) is still being used today for the production of vaccines and even for other research purposes even though that the cell culture technique of viral cultivation as aforementioned is replacing this method of viral cultivation in some quarters.
Embryonated chicken eggs are prepared prior to their usage for viral cultivation. Egg candling as shall be seen later is an important process of this preparation; and the major regions for the inoculation of the embryonated eggs with the viral particle or samples suspected of containing pathogenic virus(s) includes the chorioallantoic membrane, yolk sac, allantoic cavity and amniotic cavity (Figure 10). One of the major disadvantages associated with the use of embryonated eggs for viral cultivation is that the site of inoculation varies for different types of viruses. For example, herpes simplex virus is inoculated in the yolk sac while the allantoic cavity is for the inoculation of influenza virus, avian adenovirus, mumps virus and Newcastle disease virus. Poxvirus is inoculated in the chorioallantoic cavity. The advantages of using embryonated chicken eggs for viral inoculation are enormous.
- Embryonated chicken egg technique is cost effective and requires less labour to undertake.
- The embryonated eggs provide the ideal environment for the growth and replication of viruses – which only replicate in living cells.
- This technique is free from contamination especially from bacteria and other viral agents.
- It is widely used to cultivate viruses meant for the production of vaccines.
- Embryonated chicken egg technique is ideal for the isolation and cultivation of avian viruses.
- It provides a wide range of tissues and fluids foe the cultivation of viruses.
- The growth environment provided by this technique is sterile, and it does not produce immunity against the cultivated viral agent as is applicable in the use of animal models.
Figure 10: Routes of inoculation of embryonated egg with virus.
Embryonated chicken eggs are usually used for the isolation of some avian viruses. However, other viruses can also be cultured using this medium. Eggs provide a suitable means for the primary isolation and identification of viruses; for the maintenance of stock cultures; and for the production of vaccines. Depending on the virus of interest to be isolated or cultured, the specimen suspected to contain a virus is inoculated in the amniotic cavity, the allantoic cavity, and the yolk sac or on the chorioallantoic membrane. Viral growth is usually determined by haemagglutination (HA) technique or by immunoflourescence. The air sac is used for respiration and for pressure adjustments. The egg shell and shell membrane function both as a barrier and as an exchange system for gases and liquid molecules. The chorioallantoic sac and the allantoic fluid remove waste products produced by the developing embryo. The yolk sac is the source of nourishment for the developing embryo. The amnion is a thin membrane that encloses the embryo and protects it from physical damage.
EGG CANDLING TECHNIQUE
Egg candling is defined as a technique that is used to determine the condition of the air cell, yolk, and white component of an embryonated egg prior to its usage for viral culture. Candling detects bloody whites, blood spots, or meat spots, and enables observation of germ development. And it is mainly conducted in a dark area or room – so that the interior components of the egg to be candled can be properly illuminated and seen. The embryonated egg sample(s) to be candled is usually held before a light preferably one coming from a torch light in a vertical or horizontal direction as determined by the researcher (Figure 11). The light penetrates the embryonated egg and makes it possible to observe the inside of the egg. Preferably, the torch light or illuminating source is placed on the embryonated egg is held in a slanting position with the large end of the egg placed against the hole in the candler. The candler is a hollow-cylindrical tube or box that covers the lighted candle used for candling technique.
And the candler should be set on a box or table at a convenient height (about 38 to 44 inches from the floor), so that the illuminating light will not shine directly into the eyes of the researcher – thereby affecting his or her vision. The egg is grasped by the small end and, while held between the thumb and tips of the first two fingers, is turned quickly to the right or left. This moves the contents of the egg and throws the yolk nearer the shell. Because of the color of their shells, brown eggs are more difficult to candle than white eggs. Candling is generally used to dedifferentiate fresh embryonated egg samples from old egg samples; and it helps to detect such abnormalities as bloody whites, blood spots, meat spots, and cracked shells as aforementioned prior to viral cultivation using embryonated sample(s). Embryonated egg samples with bloody whites, blood or meat spots, and cracked shells should not be used for viral cultivation. Only those with plain air space and whose “white” moves freely (inclusive of the other aforementioned factors that candling helps to decipher) should be used for viral cultivation.
Figure 11: Candling of embryonated chicken eggs before inoculation with virus suspensions. CDC
PROCEDURE OF INOCULATING AN EMBRYONATED EGG WITH A VIRUS
Several routes exist for the inoculation of an embryonated chicken egg. The major routes of inoculating an embryonated egg with a virus or clinical sample suspected to contain a pathogenic virus include the chorioallantoic membrane, yolk sac, allantoic cavity and the amniotic sac (Figure 12). Viruses can be inoculated in several sites of an embryonated egg. They can be inoculated into the chorioallantoic membrane (e.g. herpes simplex virus and pox virus); allantoic cavity (e.g. influenza virus and rabies virus); amniotic sac (e.g. mump virus); and yolk sac (avian viruses). Certain areas of the embryonated chicken egg including the allantoic cavity, yolk sac, amniotic cavity and chorioallantoic membrane are chosen for the injection of viruses or viral samples required for virological investigations because viruses can only grow or replicate in certain parts of the embryonated egg. Viral replication outside these designated areas as shall be highlighted in this section is rare and counterproductive. These designated areas of viral inoculation in embryonated chicken eggs contain the necessary materials and substances that will drive viral replication; and thus they are the most preferred sites for viral inoculation when the use of the embryonated chicken egg technique for viral cultivation is anticipated.
- Chorioallantoic membrane: The chorioallantoic membrane is usually used for the cultivation of herpes simplex virus and poxvirus.
- Allantoic cavity: Allantoic cavity is used for the cultivation of avian viruses, yellow fever virus, rabies virus and influenza virus.
- Amniotic sac: Amniotic sac is used for the cultivation and isolation of mump virus as well as influenza virus.
- Yolk sac: Yolk sac is used for the cultivation and isolation of avian viruses; and this region also support the growth of a wide variety of other viruses. Some obligate intracellular parasites like Rickettsia and Chlamydia can also be propagated in this region.
The general steps followed in the inoculation of embryonated chicken eggs with a virus or virus-containing sample are highlighted in this section.
- Candle the eggs and place them gently on an egg rack making sure to place the eggs with the inoculation sites or routes facing up.
- Mark the inoculation site of the embryonated eggs on the egg rack.
- Disinfect the surface or inoculation site of the embryonated chicken eggs (of about 10-14 days old) to be inoculated with 70 % ethanol/alcohol or iodine.
Figure 12: Illustration of an embryonated egg for viral inoculation.
Figure 13: Illustration of egg shell punching device, an improvised punching tool used to create hole on an embryonated egg.
- A cotton wool socked in the disinfectant should be used to undertake this disinfection process. It is important to allow the disinfectant to dry off from the site of inoculation before proceeding with the technique.
- Pierce or make a hole in the end of the egg (i.e. at the inoculation site) using an egg punching device (Figure 13). Other tools used to punch the shell of an embryonated egg include dental drill. The egg shell punch is made from a copper wire.
- After making the drill or hole on the correct inoculation site, inoculate the right concentration of the viral sample or particle into the inoculation site using a sterile syringe and needle.
- Ensure the needle on the syringe penetrates well via the punctured site before dispensing its content especially for the inoculation of the allantoic cavity, yolk sac and the amniotic cavity which are deeper than the chorioallantoic membrane.
- After injecting the viral inoculum into the appropriate site, withdraw the needle gently from the embryonated egg.
- Seal the site of inoculation with paraffin or gelatin. A stationary tape can also be used for the sealing.
- Incubate the inoculated embryonated eggs in the incubator while ensuring that the temperature condition and humidity of the incubator are balanced for the optimal growth of the virus being cultivated.
- Inoculated embryonated eggs are usually incubated at 37oC for 2-3 days.
- After incubation, the eggs are broken and the virus is isolated from the tissues of the embryonated eggs.
- The formation of pocks or lesions on the membranes of the egg especially at the chorioallantoic membrane for some viruses indicates viral growth and replication. However, the death of the chicken embryo is also a strong indication of viral growth and multiplication.
DETECTION OF VIRAL REPLICATION IN A LIVING HOST CELL
The replication of viruses in living host cell can be detected in various ways. However, cytopathic effect, hemadsorption and interference are the most common techniques or approaches of doing this.
- CYTOPATHIC EFFECT
Cytopathic effect (CPE) is defined as those biochemical and/or morphological changes that occur in viral infected cells, and which indicate viral infectivity and replication in a particular host cell. They are degenerative changes (which could be microscopic or macroscopic in nature) that occur in host cells infected by virus; and they generally indicate viral replication. Cytopathic effect is also an early indication that a particular host cell is infected. And they may even start appearing before the infected cells start to die or fragment into different forms. CPE is more of an infectivity assay that allows virologists to decipher the death of a host cell due to viral infection and replication. Cytopathic effect assumes different forms including total cell lysis, cell fusion, cell detachment, production of inclusion bodies and cell rounding.
Though not all virus show CPE during their infectivity and replication (because they do not necessarily lead to the death of the infected host cell), CPE is an important aspect of detecting viral replication in infected host cells. For viruses that do not show CPE during their replication in host cells, other methods of detecting viral replication are used. CPE can be observed in viral infected cells visually with the naked eyes and macroscopically using light microscopy; and the production of CPE is a common assaying approach in cell/tissue culture technique of viral cultivation. Electron microscopy and other advanced viral detection techniques such as neutralization and immunoflourescence can be used to confirm the production of CPE in viral infected host cells. It is noteworthy that CPE can also be produced by other obligate intracellular parasites that are not viruses. Typical examples include bacteria in the genus Rickettsia and Chlamydia – which both exhibit some characteristics of viruses.
Hemadsorption is defined as the adsorption of red blood cells (erythrocytes) to the surface of virus-infected host cells. It is an important detection technique which is used to assay for or identify the synthesis of viral proteins in infected host cells. Hemadsorption is typically used to identify viruses whose replication in infected host cells cannot be easily detected by other means of viral deception. And this include some enveloped viruses in the family Orthomyxoviridae, Togaviridae and Paramyxoviridae – that are known to bud from the cell surface of the host cells they infect. In the technique of hemadsorption, red blood cells (RBCs) are added to virus infected cells in a cell/tissue culture medium during their incubation. The RBCs adhere or attach specifically to the virus infected cells; and this can be observed microscopically using the microscope.
The RBCs adhere to the virus infected cells because the viral replication of some virus (such as orthomyxoviruses and paramyxoviruses as aforesaid) leads to the outright destruction of the plasma or cell membrane of the infected cells in culture plates; and this allows erythrocytes to firmly attach to them in vitro, and thus allow their detection by the hemadsorption technique. Hemadsorption is an important detection technique for the recognition of virus infection in cultures especially for those viruses whose growth or replication produces little or no evidence of CPE. The cell attachment proteins (i.e. viral proteins) synthesized by viruses as aforementioned bind specifically to some host molecules such as sialic acid which are abundantly found on several host cells including the RBCs; and this confirms the basis for the hemadsorption technique used for detecting viral replication in infected host cells.
Interference is defined as the prevention of the replication of one virus by another. In interference, the attachment of a given virus to a host cell could be prevented by exposing the virus to another virus that destroys it or alter their ability to attach to their specific host cell. Interference is another viral detection technique aside hemadsorption and cytopathic effect that is used to assay viral replication in infected host cells. Typical examples of viruses that can be detected by viral interference test include rubella virus – which has the ability to prevent another virus from infecting the same cells it normally replicates in. Viral replication interference mechanisms can also be mediated by interferons and some antiviral drugs as well. Generally, in viral interference test, a given concentration or amount of a virus (usually the primary infecting or avirulent virus) is mixed or added to the culture fluid of another virus capable of exhibiting interference (e.g. rubella virus); and the culture is incubated. Rubella virus in this case inhibits the replication of the former virus, and this allows for their detection in virus infected samples.
Neutralization test is an antigen-antibody based test which is used to assay viral infectivity by determining whether a virus has been neutralized by a specific antibody. Neutralization test can be performed with any virus especially with those that do not form CPE or adhere to RBCs.
FACTORS THAT INCREASE THE INTENSITY (PATHOGENICITY OR VIRULENCE)
OF VIRAL INFECTIONS
The ability of a pathogenic virus to cause disease in a host (i.e. viral pathogenesis or virulence) is usually affected by several factors which are both of viral origin and host origin. For a given pathogenic virus to cause infection or disease in a host, the virus must invade the host cell, establish itself and initiate the processes that leads to the development of the disease it is known to cause; and the disease development is usually deciphered as mild or severe clinical symptoms in the host – depending on the virulent nature of the infecting pathogenic virus. The pathogenesis and/or virulence of a given pathogenic virus are dependent on the individual host infected, the dosage of viral administration as well as the particular viral species and strain involved in the invasion. The interactions of these factors mentioned above and as shall be discussed in this section will determine the nature of the infection or disease to be produced. They will also determine whether or not there will be clinical symptoms and signs of the disease in the affected host. Some of these important factors that generally determine or affect the degree of viral infection in a host are highlighted in this section.
- HOST IMMUNITY
Immunity is the ability of a host’s body to resist the development of a disease or infection cause by a pathogenic microorganism. It can be innate immunity (non-specific immune response) or acquired immunity (specific immune response). Humoral immunity (immune response that involves antibody production) and cellular immunity (immune response that involves the production of T cells inclusive of cytotoxic T cells and helper T cells) are the two major types of immunity in the body of a host; and these forms of protection are involved in the control and prevention of disease establishment by a given pathogenic virus. Thus, a strong and intact immune system is critical for the protection of a host against viral infection. Macrophages, phagocytes, and cytokines such as interferons (IFN), interleukins (IL) and tumor necrosis factor (TNF) are components of a host immune system which cooperatively attack or ward-off the untoward effects of invading pathogenic viruses in the body of a host. Nevertheless, some pathogenic viruses have developed innovative ways of surmounting the immune system of their host cells or organisms; and the mutation (a change in the genetic makeup of an organism) of some viral strains – which have allowed mutant forms of some pathogenic viruses have given impetus to this phenomenon.
Pathogenic viruses (such as the influenza viruses and coronaviruses that cause viral flu and severe acute respiratory syndrome, SARS respectively) undergo mutation to change their antigenic structure or proteins, and this development allows them to remain masked within their host – so that they will remain undetected by the host immune system while causing or establishing disease in the individual. In addition, some pathogenic viruses such as the human immunodeficiency virus (HIV) which causes AIDS (acquired immunodeficiency syndrome) have the ability to weaken the host immune system to an extent that it becomes less functional in attacking other pathogens that invades the host. HIV infects the cells of the immune system (particularly the T helper cells) and thus deprives the host immune system to carry out its normal function of protecting the body from the attacks of pathogens that invades it to cause disease.
- ROUTE OF VIRAL SPREAD
The route of viral spread is another factor that affects the pathogenicity of a given pathogenic virus. Pathogenic viruses that infect humans are mainly spread via blood inclusive of the serum and plasma. Semen, saliva, lymph fluids and the cerebrospinal fluids are other host’s materials through which a pathogenic virus could be transmitted from one individual to another. Because blood attains a general circulation in the body, it is often the most common means via which pathogenic viruses can be spread within a defined human host.
- INFECTIVITY OF THE VIRUS
A pathogenic virus must be highly infectious before it can establish an infection or disease in its host. Infectivity is the ability of a pathogenic virus to enter a host cell and thus initiate the process of diseases development. Pathogenic viruses with higher infectivity produces more sever clinical symptoms of the disease than viruses with lesser form of infectivity. A pathological state can only be established with a virus and its host in vivo when the invading pathogenic virus is infectious in nature.
- DOSAGE OF VIRUS IN THE HOST
The dosage of pathogenic viruses that invaded a given host is another factor that affects the development of disease as well as the pathogenicity or virulence of the invading pathogen. The higher the infectious dose of the pathogenic virus in the host organism, the more intense the production of viral progeny. New virions are critical for the maintenance or sustenance of the viral infection in the host; and when new viral progeny are not turned out via active viral replication, the viral disease process will be halted and possibly broken. The production of low viral progeny may also be associated with the low infectious dose of the invading pathogenic virus.
When the pathogenic virus that invaded a particular host cell fails to produce new virions or viral progeny within then host cell, the infection is said to be abortive. But when new viral progeny are produced after invasion within the infected host cell, the infection is said to be a productive viral infection. However, some pathogenic viruses may assume a latency phase – during which they do not produce new viral progeny after invasion of the host cell. But for such latent viral infections, the production of new virions is activated at a later stage in the host; and this is because such pathogenic viruses still posses the ability to initiate productive viral infection at a later time during the disease or infection process.
- LOCATION OF SUSCEPTIBLE CELLS FOR VIRAL INVASION
The location of susceptible host cells for viral invasion within the host is another important factor that affects the pathogenicity and/or virulence of pathogenic viruses. The pathogenicity of a given pathogenic virus may not be fully established if the target organ, tissue or cell of the virus within the host is susceptible to the invading virus. After invasion or entry, pathogenic viruses must be able to locate and reach their specific sites within the host and thus bind specifically to the receptors on the host cells. This process of invasion and attachment to receptors on the host cell is a critical step in viral replication mechanism. It is noteworthy that the closer the proximity of the invading pathogenic virus to its target site or receptor within the host, the lesser the time it will take the pathogen to reach its target organ and thus establish the disease process.
OVERVIEW OF VIRAL PATHOGENESIS AND VIRULENCE
Pathogenesis is simply defined as the mechanism of disease development in a host. The interaction of pathogenic viruses (inclusive of DNA and RNA containing viruses) with host molecules that result in the production of infection or disease is known as viral pathogenesis. Virulence is simply defined as the degree or intensity of the pathogenicity of a given pathogenic microorganism. It is the ability of a microbe (in this case, a pathogenic virus) to invade a host and produce or cause disease or infection. Virulence is a combination of the infectivity and pathogenicity of a pathogenic organism. The virulence of a pathogenic virus depends on the strain of the given virus that invades the host. Two strains of a given virus in the same viral family may vary in their virulence even though they are both pathogenic to the host. Thus, the viral strain that produces a more severe form of the disease is more virulent than the strain that produces a benign disease or infection.
A highly virulent strain of a pathogenic virus causes severe clinical symptoms of the disease than a less virulent strain of the same virus – which usually produce relatively less severe symptoms. The virulence of a virus is different from its infectivity. The infectivity of a pathogenic virus is the ability of the virus to invade a host and establish itself within the cells or tissues of the invaded host. And infectivity is a prerequisite to the development of disease (i.e. viral pathogenesis) within a given host. While some viral infections are subclinical or covert in nature, others are overt – and produces clinical signs and symptoms of the disease that they cause. Subclinical viral infections are those viral infections that produce little or no apparent clinical signs and symptoms in the affected host; and they can also be known as inapparent viral infections. Most viral infections are inapparent in nature, and thus they are self-limiting and are countered by the host immune system as aforementioned. In such scenarios, there may be infection without the appearance of clinical illness (i.e. asymptomatic infection), and the host may be exposed to the pathogenic virus without the development of infection or disease.
However, death and severe clinical symptoms may occur in certain viral infections caused by virulent strains of the virus – as is the case for some mutant forms of influenza virus and coronaviruses. Viral infections can also be chronic, acute or latent in occurrence. Chronic viral infections refer to those viral infections that last for a very long time in a host, and they can also be called persistent viral infections. In chronic or persistent viral infections, there is usually a continuous viral replication going on within the host but in a slow progressive manner; and chronic viral infections can be experienced during the latency stage of infection as aforementioned. Chronic viral infections are characterized by the prolonged survival of the invading viral pathogen within the host even long after the host immune system is actually expected to clear the virus from the body. In chronic/persistent viral infections, there is usually little or no appearance of clinical signs and symptoms of the viral disease in the affected host.
Hepatitis B virus (HBV), rubella virus, measles virus, and cytomegalovirus (CMV) are some examples of viral pathogens that establish chronic/persistent viral infections in humans. Acute viral infections are characterized by the rapid onset of disease development; and there is apparent production of clinical signs and symptoms associated with the viral disease in the affected host. It is usually the first stage of viral infections; and most acute infections are normally cleared or warded-off by the immune system of the affected host. Examples of human pathogenic viruses that show acute infection include adenoviruses, influenza viruses and respiratory synctial viruses (RSV) amongst other viral pathogens that attack the respiratory tract of humans and animals. In latent viral infections, there is usually a slow but progressive development of viral infection or disease development in the affected host.
Latent viral infections are infections in which the invading pathogenic virus assumes a covert form, and only produces clinical signs and symptoms of the disease when the viral pathogen becomes reactivated to do so – especially when the host’s immune system is compromised or suppressed. Herpes simplex viruses and varicella-zoster viruses (VZV) are typical examples of some human viruses that assume a latent stage of viral infection. In summary, it should be noted that the steps involved in viral pathogenesis is as the same steps involved in the replication of a virus within a living host as aforementioned. Prior to the development of disease within the host, the pathogenic virus must first gain entry to into the host’s body (viral entry), and thus initiate a primary replication once it has entered a suitable host. Viral entry and cell penetration is vital to the disease development of viral infections.
Pathogenic viruses exhibit cell/tissue tropism – in which a given virus shows affinity to specific host cells or tissues after entry. Tropism is defined as the movement of a pathogenic virus to specific cells or tissues of the host’s body in which they replicate. And it is an important process in viral pathogenesis that occurs immediately after viral entry. For example, Rabies virus that causes rabies in humans has tropism for the nerve cells of the body – which is why the disease (rabies) is characterized by muscular impairment and paralysis in human or animal hosts affected by the virus. After tropism, there is usually the development of clinical features of the disease caused by the invading viral pathogen. And this development is usually countered by the host’s immune system in individuals whose immune system is still intact. However, immunocompromised hosts and those whose immune system is suppressed by chemotherapy or a debilitating disease usually show severe forms of the disease.
There is usually viral shedding during the disease process; and it is at this stage (i.e. viral shedding) that new infectious virions of the invading pathogenic virus are produced. The shedding of infectious viral particles into the environment either through the blood, saliva or feaces marks the last stage of viral pathogenesis. During viral shedding, infected individuals are highly infectious and they could serve as routes via which the pathogenic virus can pass on from an infected individual to a susceptible and non-infected host. Viral shedding allows pathogenic viruses to be maintained within a given population so that the disease they cause can be perpetuated over some certain period of time until a time when sustainable containment and preventive measures are instituted to break the spread and transmission of the disease or disease agents amongst susceptible hosts.
Some pathogenic viruses such as the rabies virus that causes rabies are not actually perpetuated within the population and from an infected individual to a non-infected individual. This is because rabies virus can only infect humans from animals. In other words, animals are the main reservoirs of rabies virus, and humans become infected with the agent when they come in contact with the body fluids of infected animals including dog, fox and wolfs. Humans bitten by such animals can also transmit the virus to their victims. The saliva of such animals is a typical body fluid through which the rabies virus can be transmitted within a defined human population. Thus, there is no viral shedding for rabies infection in humans because humans are the dead end host for such viruses.
Humans infected with rabies virus that fails to recover from the disease eventually die from the infection. Pathogenic viruses affect different parts of the human body including the respiratory tract, gastrointestinal tract, the unborn foetus, the skin and the central nervous systems amongst others; and the progression of the disease varies from one pathogenic virus to another. The good news is that some viral infections are self-limiting and often subsides even without formal treatment. However, antiviral drugs and vaccines are available for the treatment and prevention of human viral infections; and vaccine and drug development is also underway for those viral infections or disease without potent antiviral drugs and vaccines.
OVERVIEW OF VIRAL HAEMORRHAGIC FEVERS
There is actually no consensus to the definition of viral haemorrhagic fevers. However, this section is aimed at letting the reader know the general concept regarding this dreaded disease of mankind. Viral haemorrhagic fevers are viral infections or diseases generally characterized by haemorrhage (bleeding) resulting from various forms of capillary damage of organs of the body. This type of viral disease is caused by a consortium of distinct viral groups generally known as viral haemorrhagic fevers of humans. And they include Ebola virus, Lassa virus, Marburg virus, Chikungunya virus and rift valley virus amongst others. It is noteworthy that the viruses responsible for causing viral haemorrhagic fevers in humans are mainly found in only four (4) viral families, they include: Flaviviridae family, Arenaviridae family, Filoviridae family and Bunyaviridae family.
Viral haemorrhagic fevers (VHFs) are also characterized by high fever aside other peculiar clinical symptoms and signs of each viral disease agent that cause VHF. Human infections with the causative agents of VHFs usually occur when humans venture into the ecological domain of the natural hosts of these viruses. Rodents, primates, chimpanzees and arthropods are the main natural hosts or reservoirs of viral haemorrhagic fever viruses of humans. VHF is a multisystem syndrome – because VHF affects several organs of the human body; and the disease leads to sporadic internal bleeding from the vascular systems and capillaries of the affected organs. Viral haemorrhagic fever viruses rarely cause mild viral infections; and most of the diseases caused by these pathogens are usually life-threatening and they also have very high mortality rate. The Ebola outbreak of 2014 in some parts of West Africa attests to the severity of infections caused by viral haemorrhagic fever viruses.
As aforementioned, viral haemorrhagic fever viruses are usually acquired when humans venture into the ecological niche of animals reservoirs of these viruses. Direct contact with an infected individual is one of the major routes of transmission of the disease within a defined human population. Inadequate infection control practices in healthcare facilities as well as contact with the body fluids of infected persons or cadavers of VHF victims are common routes of transmitting the disease between the hospital and community. The transmission of viral haemorrhagic fever in a healthcare setting is frequent amongst healthcare workers, and they also serve as medium through which the disease can spread from the hospital to the community.
The natural reservoirs or hosts of viral haemorrhagic fever viruses include bats, monkeys, chimpanzees, arthropods such as mosquitoes and primates. Viral haemorrhagic fever viruses can also spread from person to person via direct contact with body fluids of infected persons and also via contaminated hospital equipments or instruments including syringes and needles. Contaminated syringes and needles plays an important role in the transmission of viral haemorrhagic fever viruses especially in hospital settings where disease outbreak is likely to occur.
Humans are not the natural reservoirs of viral haemorrhagic fever viruses. Human infection with these pathogens only occurs when humans come into contact with infected humans hosts or any of the natural reservoirs of the disease agent. It is worthy of note that the viral haemorrhagic fever viruses that causes VHF in humans are all RNA-containing viruses (i.e. they are RNA viruses). And they are all enveloped viruses as well. Because the causative agents of VHF in humans are mainly reserved in animals, VHF can also be referred to as zoonotic viral infection or disease – since they are usually transmitted from animals to humans. The hosts of some viral haemorrhagic fever viruses such as Marburg virus and Ebola virus is still unknown even though they are believed to be transmitted to humans via primates, monkeys or chimpanzees as well as infected/contaminated bush meats. Most of the VHFs are geographically restricted to some regions of the world, and this has been used in the classification or naming of some of the viral agents that cause VHFs in humans. Some of the VHFs only occur in countries or regions of the world where the natural host of the virus seems to occur.
Most of the VHFs are named after the country, region or town where they first occurred. For example, Marburg haemorrhagic fever first occurred in Marburg and Frankfurt in Germany; and Lassa fever was named after the town in northern Nigeria where the first disease outbreak occurred in 1969. Contamination of the food of humans by the excreta or urine of rodents harbouring any of these viruses and the consumption of bush meats infected by the virus is also another common means via which human infection can occur. And for those viral haemorrhagic fevers whose natural hosts are arthropods (such as the yellow fever virus), human infections with such viruses usually occur following insect bite during blood meal. The clinical presentation of VHFs varies from one VHF to another. However, the commonest symptoms of VHF as aforementioned include bleeding and sudden onset of fever, myalgias, and prostration as well as general body weakness. VHF is a notifiable disease and must be reported to local health authorities who will take appropriate containment measures to stop the spread of the disease.
The use of PCR, antigen detection tests and virus isolation are the commonest means of diagnosing the disease, but clinical samples for VHF investigations are normally carried out in reference laboratories with suitable biosafety level 4 (BSL-4) equipments and facility for the management and processing of such highly infectious agents. Some VHF has no specific antiviral drug for treatment but Ribavirin is used for the effective treatment of Lassa fever and some Arenaviruses that cause VHF in humans. Early diagnosis and treatment is vital to avert death; and avoiding contact with the natural host or vectors in endemic countries is crucial to the prevention of VHF. Vaccines for the prevention or protection of susceptible human population against the contraction of viral haemorrhagic fever viruses does not exist except for yellow fever (caused by yellow fever virus transmitted via mosquito bite) for which a vaccine exist. However, vaccine development for some life-threatening viral haemorrhagic fevers such as Ebola is underway.
The best mode of preventing human infection with viral haemorrhagic fevers is avoiding contact with the natural hosts of the viruses that cause these deadly diseases. Safe cleanup of our environments especially by avoiding and discouraging rodents from entering our homes and defeacating on raw food materials is critical to the prevention of the spread of the disease. And proper rodent controls should also be instituted in regions where some viral haemorrhagic fevers are endemic. For viral haemorrhagic fever viruses transmitted via arthropods (e.g. yellow fever virus), the proper control of insect vectors that harbour these pathogens will help to preventing human infection with the disease agent. Proper infection control practices should be instituted in hospitals located in regions where these viral agents are endemic; and when disease outbreak due to any of these agents occur, prevention should be focused on avoiding human contacts with already infected people.
Lassa fever or lassa is an acute haemorrhagic viral disease caused by lassa virus (LASV), an Arenavirus in the viral family Arenaviridae. The natural host of LASV is the multimammate rat known as Mastomys natalensis. M. natalensis is the rodent species that naturally harbour LASV; and the rat is found throughout West Africa – thus making the disease to be endemic in the region. Lassa fever is geographical distributed in West Africa (including Nigeria, Liberia, Sierra Leone and Guinea); and the causative agent of the disease is responsible for several outbreak of Lassa fever in the region. The disease can be mild to severe in occurrence, and in some cases it can be fatal and thus cause the death of infected victims. Lassa fever is characterized by severe systemic febrile illness with changes in vascular permeability and vasoregulation; and the disease is also associated with bleeding as is obtainable with Ebola virus disease. The case fatality rate (CFR) of Lassa fever is 15-20 % but it can reach 50 % depending on the severity of the outbreak.
Lassa virus was first discovered in 1969 in a town known as Lassa in the Northern part of Nigeria. The first case of Lassa fever originated in this town, and thus the disease was named after the town. Lassa fever killed two missionary nurses stationed in the region (Lassa town) where the disease was first discovered. Lassa virus (LASV) like the Ebola virus is zoonotic in nature (i.e. it is animal-borne and can be transmitted from infected animals to humans). Lassa fever has caused quite a few epidemic outbreaks in several West African countries. According to the World Health Organization (WHO) and the Center for Disease Prevention and Control (CDC), about 100,000 to 300,000 people are supposed to be infected annually by LASV in the West African sub region. Also, over 5000 deaths due to LASV infection occur annually in West African countries.
PATHOGENESIS OF LASSA FEVER
The symptoms of Lassa fever include malaise, back pain, sore throat, diarrhea, vomiting, unexplained fever, conjunctivitis, severe prostration, swelling of the face and mucosal bleeding. Human infection with LASV occurs when human have direct contact with the body fluids of the rodent reservoir of the virus such as urine and animal droppings. The rodent rat urinates and defecates as it moves around, and its fecal droppings and urine can contaminate raw food meant for human consumption especially when the foods are left uncovered. Though close body contacts are possibly required for the case-to-case transmission of LASV amongst human population; casual body contact with lassa virus infected individuals is not a usual route of disease transmission. However, blood contacts with infected individuals can aid in the transmission of LASV. Another source of transmission of LASV is via the inhalation of contaminated air especially in rat infested homes. Nosocomial transmission of LASV amongst healthcare workers and patients is possible through the use of hospital equipments and reused invasive devices such as needles contaminated by body fluids of infected patients. The incubation period of the infection is usually 7 to 21 days. LASV has a broad tissue/cell tropism i.e. it affects several tissues/cells of the host body including the liver, kidney and the nervous system.
LABORATORY DIAGNOSIS OF LASSA FEVER
The prompt and accurate diagnosis of LASV infection is critical in administering the proper type of therapy to affected individuals since the pathogenesis of the disease is usually sporadic and unpredictable in occurrence. The laboratory diagnosis of Lassa fever is usually done using enzyme linked immunosorbent assay (ELISA). ELISA is used to detect immunoglobulins (particularly IgM and IgG antibodies) produced by the host’s immune system against the virus. It can also be used to detect lassa virus antigens. Tissue/cell culture techniques can also be employed in the laboratory diagnoses of LASV infection. And LASV can also be directly detected from clinical samples such as blood using reverse transcription-polymerase chain reaction (RT-PCR). Nevertheless, RT-PCR is mainly used in reference laboratories for research purposes aimed at studying and understanding the pathogenesis and/or pathology of LASV infection in humans.
TREATMENT OF LASSA FEVER
LASV infection is usually treated with the antiviral drug ribavirin. Ribavirin (which is chemically known as: 1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide) is a synthetic nucleoside analog of guanosine. Its mode or mechanism of action is based on the ability of the agent to interfere with the biosynthesis of guanylic acid nucleotides in the pathogenic virus; and thus interfere with the synthesis of RNA in the organism. Ribavirin is generally an inhibitor of RNA synthesis; and the drug is also used to treat other viral infections including those caused by parainfluenza viruses, Influenza viruses, Bunyaviruses, Picornaviruses and other Arenaviruses. The trade name for ribavirin in the market is virazole. Ribavirin also increases the rate of mutation in the RNA of the infecting virus; and the drug is most active when it is given at the early stage of LASV infection. No vaccine currently exists for the prevention of LASV infection in humans, but vaccine development for the effective control and prevention of the disease is still underway.
Human convalescent serum has also been found to be effective in the treatment of LASV infection in humans. As is applicable with Ebola virus infection, LASV infection in humans also require additional supportive therapy aside the administration of the antiviral drug ribavirin to the infected patient. Since LASV infected patients undergo severe gastrointestinal disorder leading to the loss of excess fluids form the body, it is important to ensure proper fluid and electrolyte replacement to avoid hypotension and shock that may cause the death of the affected individuals. Any other secondary or complicating infections should be treated alongside the Lassa fever disease; and patients infected with LASV should be placed on proper oxygenation and their blood pressure should be regularly checked as part of the supportive therapy administered.
PREVENTION AND CONTROL OF LASSA FEVER INFECTION
The people that are most at risk at getting the LASV infection are those who live in endemic regions where the disease is widespread especially in regions with high population of the natural host of the virus (i.e. M. natalensis multimammate rat). Hospital personnel’s who observed the requisite preventive measures when handling LASV infected individuals are less likely to become infected. The main preventive measure is avoiding contact with the multimammate rat that naturally harbours the virus. Barrier nursing techniques and proper isolation methods as well as using the correct personal protective equipments (including eye goggles, hand gloves, masks and gowns) both in the hospitals and in the field in the event of LASV disease outbreak is paramount to prevent outbreak or disease spread amongst healthcare workers. Food meant for human consumption should be put away or stored in rodent-proof containers so that the rat does not defecate or urinate on them.
Though the complete eradication of the multimammate rat in West Africa may not be feasible due to its high population in the region; the use of traps around the house can help to reduce their population in a given area. And homes especially in the rural areas should always be kept clean so that these rats do not find a haven to stay and transmit the LASV. In addition, the proper enlightenment and public awareness of people in LASV endemic regions in West Africa will go a long way in preventing and controlling the disease especially in the area of educating the people about sustainable measures that can be employed in keeping the natural reservoir of LASV (i.e. M. natalensis multimammate rats) at bay. Such measures aimed at reducing the population of the rats around residential areas will help to reduce the spread of the virus in LASV endemic regions.
OVERVIEW OF VACCINES
Vaccines are biological substances that contain attenuated microorganisms and/or antigens to a particular infectious disease, and which are used to protect an individual against the disease in future. They are usually suspensions of weakened, killed or fragmented microorganisms or toxins administered primarily to prevent diseases in living systems including man and animals. The medical procedure of administering these vaccines to humans or animals is generally known as vaccination. Vaccination is simply defined as the administration of vaccines to a living host. It is the most effective way to prevent killer diseases especially amongst children between the ages of 0-12 years old. The term vaccination is usually used synonymously or interchangeably with immunization. However, immunization is generally the procedure of making a person immune to a particular infectious disease. And it can be achieved through several processes which include but not limited to:
- Passive immunization – which a newborn usually acquires from the mother on birth before the baby starts developing or building its own active immunity.
- Injecting an antiserum or vaccine into the body.
- Oral administration of vaccines in tablet forms.
- Direct inoculation of a weakened or live organism into the body (this method is archaic, and it is not practiced again).
Generally, vaccines are pathogen-imposters – since they act as the carbon copy of the actual pathogen in vivo, thereby stimulating the host’s immune system to produce potent antibodies and other immune system molecules against the invading microbe in advance. Vaccines prepare and stimulate the body to fight against invading infectious disease agents in advance. According to the World Health Organization (WHO), the widespread immunity due to vaccination is largely responsible for the worldwide eradication of smallpox and the restriction of diseases such as polio, measles and tetanus from most parts of the world. Vaccination therefore has significantly contributed to the prevention and control of many human infections and diseases.
It is noteworthy that the concept of using microbes (as vaccines) especially in their attenuated/weakened forms to treat microbial diseases in humans was first established and conceptualized by Louis Pasteur. Louis Pasteur was the first to develop a potent human vaccine (rabies vaccine in particular) in 1881. Pasteur’s work showed that pathogens can be isolated, inactivated and administered in their weakened or live forms to humans to prevent them from acquiring a particular infectious disease. Though the field of vaccinology as we know it today was first introduced into clinical medicine by Edward Jenner in 1796 – when he inoculated a 13 year old boy with vaccinia virus obtained from a woman accidentally infected with cowpox. Jenner’s method of vaccination is generally known as inoculation – since it involved the direct introduction of live organisms into the body.
However, this practice has become obsolete and is no longer practiced in clinical medicine. It was later discovered by Jenner that the young lad developed immunity to smallpox infection – which caused the death of millions of people worldwide. The immunity developed in the young man against smallpox disease was due to a challenge with variola (vaccinia) virus which Edward Jenner administered into the boy. Through vaccination and/or immunization, a large population of humans can be protected from becoming infected by an infectious disease. This is because, the vaccination of a large human population against a given infection can develop herd immunity in the population – since contacts amongst people in that population will be more with protected and vaccinated individuals instead of with sick and infected persons. Herd immunity helps to reduce the spread and circulation of infectious agents within a particular human population. Most of the vaccine-preventable diseases including polio have been significantly reduced and contained in the developing parts of the world.
All the vaccines used today for the vaccination and/or immunization of humans are based o the use of killed or live forms of microorganisms or their purified subunits. Purified antigens of pathogens, toxins and their conjugated protein or polysaccharide molecules can also serve the purpose of vaccination in humans depending on the type of infectious disease they are used to prevent. For human vaccines to be available on a global scale, complex production methods, meticulous quality control measures and reliable distribution channels are needed to ensure that the products are safe, potent and effective for human use. A vaccine can confer active immunity against a specific harmful agent by stimulating the immune system to attack the agent. Once stimulated by a vaccine, the antibody producing cells known as B- lymphocytes remain sensitized and ready to respond should it ever gain enter into the body the body of the vaccinated human host. Even though no vaccine is entirely safe or completely effective, their use is strongly supported by their benefit to risk ratio. That is, the benefit of using them far outweighs any risk associated to the application in humans.
TYPES OF VACCINES
- LIVE ATTENUATED VACCINES
Live attenuated vaccines are composed of live, attenuated microorganisms that cause limited or no infection in their host upon administration. The causative agent or microorganism used for the development of live attenuated vaccine is live but it has lost its natural ability to cause the disease it is known for. Microbes used for live attenuated vaccines are sufficient enough to induce an immune response but insufficient to cause any infectious disease in the vaccinated individual. Live attenuated vaccines are made by passing the disease causing virus through a series of cell culture techniques or embryonated chicken egg method (e.g. chicken embryo) under controlled laboratory conditions that make the organism less virulent. Most of the pathogens used for the production of live attenuated vaccines are active viruses that have been cultivated under conditions that disable or inactivate their virulent properties. In some cases, closely related but less dangerous or less-pathogenic microbe can be used to produce immune response in the individual. Live attenuated vaccines are very efficacious; and they induce a protective form of immunity in the individual being vaccinated. Examples of live attenuated vaccines include: oral polio vaccine, yellow fever vaccine, and MMR vaccine (a combination of measles, mumps and rubella vaccine).
MERITS OF LIVE ATTENUATED VACCINES
- Very small doses (usually a single dose) are required to induce immunity.
- Booster doses are not needed for live attenuated vaccines.
- Antibody formation is very fast within the first seven (7) days of administration of live attenuated vaccines; and the antibodies formed can stay for a long time and can last for a lifetime. This is because, as the virus continues to replicate in its host, so will it continue to induce the immune system to produce potent antibodies in advance.
DEMERITS OF LIVE ATTENUATED VACCINES
- A major concern that must be considered in the use of live attenuated vaccine is the possibility of the virus used for the vaccine development to revert to a virulent form capable of causing disease in the vaccinated individual. Mutations can occur when the vaccine virus replicates in the host (especially in immunocompromised individuals – whose immune system have been weakened), and this may result in a more virulent form or strain of the organism that attacks the host.
- Since they are live and their activity depends on their viability, the storage conditions must be strictly adhered to (i.e. maintenance of cold chain of temperatures between 2oC- 8 oC).
- Administration of live vaccines to immunosuppressed individuals may cause serious illness or even death. And thus their usage is usually restricted in immunocompromised individuals.
- KILLED INACTIVATED VACCINES
One alternative to using live organisms in vaccine development is killing or inactivating the microbe. Killed inactivated vaccines are very efficacious like the live attenuated vaccines. The causative agent or microbe used for the development of killed inactivated vaccines is usually inactivated by physical and chemical treatments. Vaccines of this type are generally created by inactivating a pathogen, typically using heat or chemicals such as formaldehyde or formalin as aforementioned; and such chemical and physical treatment makes the organism used to be non-pathogenic. This destroys the pathogen’s ability to replicate in a human host. Examples of killed inactivated vaccines include: Inactivated Polio Vaccine (IPV), oral cholera vaccine, rabies vaccine and seasonal influenza vaccine. Toxoids vaccines are also prepared from inactivated toxic compounds of microbes. Some bacterial diseases are not caused by the bacterium itself, but by the toxins that they produce. Examples of such infections include tetanus caused by the neurotoxins produced by Clostridium tetani.
MERITS OF KILLED INACTIVATED VACCINES
- Killed inactivated vaccines are safe to use since they cannot revert to a more virulent form capable of causing disease in the individual. They can be administered to immunodeficient persons and pregnant women.
- They are cheaper than live attenuated vaccines.
DEMERITS OF KILLED INACTIVATED VACCINES
- They provide a short length of protection than the live vaccine.
- Periodic booster doses are required to maintain long term immunity.
- Inactivation, such as by formaldehyde in the case of Salk vaccine, may alter the antigenicity of the vaccine virus. And this might lead to delayed activation of the host’s immune system.
- SUBUNIT VACCINES
Subunit vaccines contain parts or fragments of the target pathogen instead of the whole organisms – as is applicable in live attenuated vaccines and killed inactivated vaccines. They are usually made by isolating a specific protein from a pathogen and presenting it as an antigen on its own. Subunit vaccines generally contain the purified portions of the microbe being vaccinated against. The antigenic properties of the pathogen of interest (usually in the form of protein molecules and carbohydrate molecules) isolated in their pure forms are used in the development of subunit vaccines; and these molecules are expected to stimulate the immune system of the host in advance. Unlike in live attenuated vaccine in which there is possibility of reactivation of the microbe into a pathogenic organism, there is no risk that subunit vaccine (usually known as toxoids) can provoke the disease in the vaccinated individual. One of the major drawbacks in the development of subunit vaccine is the difficulty in identifying potential protective or antigenic molecules out of the complex protective molecules in pathogens – that could be used as starting materials for the development of the vaccines. Examples of subunit vaccine are Diphtheria toxoid, Tetanus toxoid, Pertussis toxoid and Hepatitis B vaccine.
- CONJUGATE VACCINES
Conjugate vaccines (which can also be known as subunit-conjugated vaccines) are developed by linking or joining the polysaccharide component of the causative agent to a protein carrier molecule that augment the immunogenicity and/or antigenicity of the microbe’s polysaccharide molecule when used as a vaccine candidate. Conjugate vaccines are primarily developed against capsulated bacteria (i.e. pathogenic bacteria that forms capsules as microbial resistant forms). Certain pathogenic bacteria have polysaccharide outer coats that are poorly immunogenic. These polysaccharide outer coats are chemically linked to a carrier protein molecule to form a combination molecule that is antigenic and one that can generate immunity against a given infection in the individual being vaccinated. Examples of conjugate vaccines include Haemophilus influenzae Type B vaccine, Meningococcus A, C, Y, W135 and pneumococcus vaccine.
- RECOMBINANT VACCINES
Recombinant vaccines are genetically generated vaccines that are prepared using recombinant DNA technology or genetic engineering techniques. In the preparation of recombinant vaccines, the genes for desired antigens of a pathogenic organism are inserted into a vector. This method is complex and capital intensive. An example of a recombinant vaccine is the Hepatitis B vaccine (HBV) used against Hepatitis B virus (HBV) infection. Hepatitis B surface antigen is produced from a gene transfected into yeast cells (particularly Saccharomyces cerevisiae) and genetically purified for injection into a host.
- EDIBLE VACCINES
Edible vaccines are vaccines currently being developed and are prepared by introducing the genes responsible for the antigenic determinant of the virus into crops (especially cereals like maize and rice) that can be eaten or consumed by humans. Eating these crops is known to induce some of form of immunity in the host. In edible vaccines, the antigenic protein molecule is engineered into an edible plant; and after ingestion, the protein is uncloaked and recognized by the host’s immune system which produces antibodies in advance. Edible vaccine development is still under development, and it holds potential in revolutionizing the technology vaccination in the future.
GENERAL STEPS EMPLOYED IN THE PRODUCTION OF VACCINES
The basic steps involved in vaccine production are summarized in this section.
- Generation of the antigen: The first step in order to produce a vaccine is generating the antigen that will trigger the immune response in the host organism. For this purpose, the pathogen’s proteins or DNA molecules need to be grown and harvested in their pure forms; and some of the techniques used in cultivating these organisms are elaborated as follows:
- Viruses are grown on primary cells from chicken embryo or fertilized eggs or cell lines that reproduce repeatedly. Hepatitis A vaccine and influenza vaccine are generated this way.
- Bacteria are grown in bioreactors which are devices that use a particular growth medium that optimizes the production of antigens. Haemophilus influenza vaccines are produced this way.
- Recombinant proteins derived from the pathogen can be generated either in yeast bacteria or cell cultures.
- Isolation of the antigens: The aim of isolating the antigen from the pathogen is to release as much viral or bacterial particles as possible for the development of the vaccine. To achieve this, the antigen will be separated from the microbial cells used to generate it and isolated from the proteins and other parts of the growth medium that are still present. After this stage, the isolated molecules are purified prior to further development.
- Purification: Here, the antigens isolated are then purified in order to produce a high quality purified product. Purification can be done using different techniques for protein purification such as high performance liquid chromatography (HPLC). Recombinant proteins need many operations involving ultrafiltration and column chromatography for its purification.
- Addition of other supporting/carrier molecules: This step involves addition of other supporting components to the purified antigenic molecules. The supporting components usually added to the purified protein or microbial substances required for vaccine production include: adjuvants, stabilizers, and preservatives. These substances are added to vaccines being produced in order to boost their activity in vivo as well as preserve them prior to usage. Adjuvants enhance the recipient’s immune response to an antigen while stabilizers increase the storage life of the vaccines. Preservatives allow the use of multidose vials of a vaccine as required (i.e. it allows a particular vaccine to be used over certain period of time because it contains substances that prevent their spoilage). All the supporting components that constitute the final vaccine are combined and mixed uniformly in a single vial or syringe that can be administered to humans for the prevention of a particular infectious disease.
- Vaccine packaging: Once the vaccine is put in the recipient vessel (either a vial or a syringe), it is sealed with sterile stoppers, and thus ready for usage. The vaccine is finally labeled and distributed worldwide. Several quality control and quality assurance practices (as part of the good manufacturing practices, GMPs employed in vaccine development) are put in place in the production of vaccines since these molecules are biological substances that interact with the internal organs, cells and tissues of living organisms. The safety, efficacy and quality of every vaccine are thoroughly checked and approved by the relevant health authorities before they are released for usage by the general public or in clinical medicine.
EXCIPIENTS USED FOR VACCINE PRODUCTION
Excipients are substances added to drug or vaccine in order to make into an actual pill for administration. Besides the active vaccine itself, several excipients exist that are incorporated into the vaccine during their development. And these excipients include antibiotics, formaldehyde, aluminium gels or salts and preservatives amongst others.
- Aluminium salts or gels are added as adjuvants. Adjuvants are added to promote or enhance the immune response to the vaccine thereby allowing for a lower vaccine dosage.
- Antibiotics are also added to some vaccines to prevent the growth of bacteria during the production and storage of the vaccine.
- Formaldehyde is usually added to inactivate bacterial products for toxoid vaccines. It is also used to inactivate unwanted viruses that might contaminate the vaccine during production.
- Monosodium glutamate (MSG) and 2- phenoxy ethanol are used as stabilizers in a few vaccines to help the vaccine remain unchanged even when exposed to heat, light, acidity or humidity.
- Thiomersal is an antiseptic and an antifungal agent containing mercury that acts as a preservative in vaccines. It is usually added to the vials and/or phials of vaccine that contains more than one dose. The essence of adding these substances is for no other reason other than to prevent the contamination and growth of potentially harmful organisms in the vaccines. But due to the concerns of mercury poisoning, thiomersal is no longer used as a preservative in vaccines especially in childhood vaccines. Thiomersal is still a component of tetanus shots.
GENERAL GUIDELINES ON VACCINE USAGE
Some of the basic precautions observed in vaccine usage are highlighted in this section. To ensure safety and efficacy of the vaccines being used for vaccination, it is vital to imbibe these safety measures o avoid predicament that may arise from vaccine usage.
Do not administer vaccine if a child is sick or has fever.
- Shake vaccine vials well to obtain a uniform, homogenous suspension prior to its actual administration into the body.
- Do not use the vaccine if the suspension cannot be re suspended upon usage.
- Store vaccine vials and syringes in the refrigerator at 2oC – 8oC, and as recommended.
- Vaccines should not be frozen. And frozen vaccines should not be used for vaccination purposes but should be discarded.
- Rotate the vaccine vial or syringe in the palm to bring it to room temperature before administration.
- Spent and expired vaccine vials should be properly discarded according to the local waste management guidelines.
OVERVIEW OF ZIKA VIRUS INFECTION
Zika virus (abbreviated as: ZIKV) belongs to the viral genus Flavivirus. It is an icosahedral, enveloped, single-stranded RNA virus that causes Zika virus disease (Zika). The lipid envelope of Zika virus is covered with dense projections that consist of a membrane and envelope glycoproteins. Zika (Zika virus disease) is a disease caused by Zika virus that is spread to people primarily through the bite of an infected arthropod (mosquito). Zika disease virus bears clinical resemblances to other Flavivirus infections or diseases such as dengue fever and Chikungunya – which are also transmitted to man via the bite of infected arthropods (mosquitoes). The mosquito species that transmit the Zika virus to man is known as Aedes mosquito. The species of Aedes mosquito that transmit the Zika virus to man via mosquito bite include Aedes aegypti, Aedes africanus, Aedes luteocephalus, Aedes albopictus, Aedes vittatus, Aedes furcifer, Aedes hensilli, and Aedes apicoargenteus. It has also been reported that sexual transmission of Zika virus amongst humans is possible.
The name Zika was from Zika forest in Uganda where the disease was first discovered. Zika virus was first discovered in 1947 and is named after the Zika forest in Uganda. Specifically, Zika virus was first described in a febrile rhesus monkey in the Zika forest of Entebbe, Uganda. Though Zika virus was first discovered in Africa, the disease and/or virus have spread beyond the African continent to other parts of the world. Several outbreak of Zika virus disease has occurred around the world since the first human outbreak of the disease was reported in 1952. Tropical Africa, Southeast Asia, and the Pacific Islands are parts of the world where Zika has been previously reported at varying prevalence’s. The first reported outbreak of Zika disease virus (Zika) in South America (precisely Brazil) was recorded on May, 2015; and this viral disease has been declared by the World Health Organization (WHO) as a public health emergency of international concern – that is likely to spread international from region to region.
The areas of active transmission of Zika virus according to the Center for Disease prevention and Control (CDC) include Barbados, Bolivia, Brazil, Colombia, Puerto Rico, Costa Rica, Curacao, Dominican Republic, Ecuador, El Salvador, French Guiana, Guadeloupe, Guatemala, Guyana, Haiti, Honduras, Jamaica, Martinique, Mexico, Nicaragua, Panama, Paraguay, Saint Martin, Suriname, US Virgin Islands, Venezuela, American Samoa, Samoa, Tonga, and Cape (Figure 14.20). Fever, rash, joint pain, and conjunctivitis or red eyes are the most common symptoms of Zika virus disease; and the clinical symptoms of the disease usually last for several days to a week after an infected person has been bitten by an infected Aedes mosquito. Zika is a mild disease and it rarely leads to death. Prior infection is likely to protect previously infected individuals from futuristic infection, and the benign nature of the disease makes most infected people especially in endemic regions not to be aware that they have been infected.
Figure 14: All countries and territories with active Zika virus transmission. CDC.
Epidemiologically, the global prevalence of Zika virus infection (Zika) has not been widely reported due to the asymptomatic clinical course of the disease and the clinical resemblance of Zika to other Flavivirus infection such as dengue fever and Chikungunya infections which are also transmitted to man via the bits of infected arthropods (Aedes mosquitoes). In addition, the diagnosis of Zika virus infection is also associated with some ambiguities. The ambiguity or difficulty associated with the accurate diagnosis of Zika is because most cases of Zika virus infection are mild and self-limited; and this makes most cases of the disease to go unnoticed. The signs and symptoms of Zika virus (ZIKV) infection are nonspecific and mimic other viral infections such as dengue fever, malaria, rickettsial infection and yellow fever. Zika virus infection is currently diagnosed based on the detection and isolation of Zika virus RNA from serum using reverse-transcriptase polymerase chain reaction (RT-PCR) in serological investigations; and the highest sensitivity of PCR testing is during the initial week of illness (i.e. at the onset of the disease.
Zika virus infection is usually characterized by high viraemia (i.e. high amount of viral particles in the blood of infected individuals). Enzyme-linked immunosorbent assay (ELISA) can also be employed to serologically test for the virus-specific immunoglobulin M (IgM) and neutralizing antibodies against Zika virus infection in patients sample (e.g. blood). Zika virus infection has been reported in various hosts including humans, primates, and mosquitoes; and these reports are based on sporadic case reports, entomological surveys, and seroprevalence surveys of the disease carried out in Zika-endemic regions of the world including Africa, Oceania and Asia – where varying prevalence’s of Zika has been previously reported. Zika virus infection is now becoming a pandemic disease – since it has spread to several continents of the world (with the latest outbreak occurring in the Americas – Brazil).
One of the most debilitating effects of Zika virus infection in humans especially pregnant women (as experienced in the lasts outbreak of the disease in the Americas – Brazil) is the congenital malformations due to transplacental transmission of Zika virus from mother to child; and other serious clinical episodes such as microcephaly, Guillain-Barré syndrome and various ophthalmologic abnormalities have been linked to Zika virus infection – which is why the disease is now a notifiable disease and a pandemic. Guillain-Barré syndrome (GBS) can be described as a collection of clinical syndromes that manifests as an acute inflammatory polyradiculoneuropathy with resultant weakness and diminished reflexes. The classic description of GBS is that of a demyelinating neuropathy with ascending weakness.
Currently, no prophylactic treatment, drug or vaccine is available for the treatment and prevention/control of Zika virus infection; and there is currently no commercially available test for Zika virus infection. However, research is ongoing to develop and make available test kits, drugs and vaccines for the accurate diagnosis, treatment and prevention of Zika virus infection in human population. Public awareness for vector control and eradication of mosquito breeding grounds in Zika-endemic regions is critical to the prevention and control of the Zika virus infection. Tourists and travelers visiting Zika-endemic countries should avoid mosquito bites as much as possible and where protective clothing’s when outdoors. Nevertheless, the best method for preventing Zika virus infection is to avoid travel to areas with active Zika virus transmission.
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