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Virology
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Gamma phage, an example of virus particles (visualised by electron microscopy)

Virology is the scientific study of biological viruses. It is a subfield of microbiology that focuses on their detection, structure, classification and evolution, their methods of infection and exploitation of host cells for reproduction, their interaction with host organism physiology and immunity, the diseases they cause, the techniques to isolate and culture them, and their use in research and therapy.

The identification of the causative agent of tobacco mosaic disease (TMV) as a novel pathogen by Martinus Beijerinck (1898) is now acknowledged as being the official beginning of the field of virology as a discipline distinct from bacteriology. He realized the source was neither a bacterial nor a fungal infection, but something completely different. Beijerinck used the word "virus" to describe the mysterious agent in his 'contagium vivum fluidum' ('contagious living fluid'). Rosalind Franklin proposed the full structure of the tobacco mosaic virus in 1955.

One main motivation for the study of viruses is because they cause many infectious diseases of plants and animals.[1] The study of the manner in which viruses cause disease is viral pathogenesis. The degree to which a virus causes disease is its virulence.[2] These fields of study are called plant virology, animal virology and human or medical virology.[3]

Virology began when there were no methods for propagating or visualizing viruses or specific laboratory tests for viral infections. The methods for separating viral nucleic acids (RNA and DNA) and proteins, which are now the mainstay of virology, did not exist. Now there are many methods for observing the structure and functions of viruses and their component parts. Thousands of different viruses are now known about and virologists often specialize in either the viruses that infect plants, or bacteria and other microorganisms, or animals. Viruses that infect humans are now studied by medical virologists. Virology is a broad subject covering biology, health, animal welfare, agriculture and ecology.

History

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Main articles: History of virology and Social history of viruses
An old, bespectacled man wearing a suit and sitting at a bench by a large window. The bench is covered with small bottles and test tubes. On the wall behind him is a large old-fashioned clock below which are four small enclosed shelves on which sit many neatly labelled bottles.
Martinus Beijerinck in his laboratory in 1921

Louis Pasteur was unable to find a causative agent for rabies and speculated about a pathogen too small to be detected by microscopes.[4] In 1884, the French microbiologist Charles Chamberland invented the Chamberland filter (or Pasteur-Chamberland filter) with pores small enough to remove all bacteria from a solution passed through it.[5] In 1892, the Russian biologist Dmitri Ivanovsky used this filter to study what is now known as the tobacco mosaic virus: crushed leaf extracts from infected tobacco plants remained infectious even after filtration to remove bacteria. Ivanovsky suggested the infection might be caused by a toxin produced by bacteria, but he did not pursue the idea.[6] At the time it was thought that all infectious agents could be retained by filters and grown on a nutrient medium—this was part of the germ theory of disease.[7]

In 1898, the Dutch microbiologist Martinus Beijerinck repeated the experiments and became convinced that the filtered solution contained a new form of infectious agent.[8] He observed that the agent multiplied only in cells that were dividing, but as his experiments did not show that it was made of particles, he called it a contagium vivum fluidum (soluble living germ) and reintroduced the word virus. Beijerinck maintained that viruses were liquid in nature, a theory later discredited by Wendell Stanley, who proved they were particulate.[6] In the same year, Friedrich Loeffler and Paul Frosch passed the first animal virus, aphthovirus (the agent of foot-and-mouth disease), through a similar filter.[9]

In the early 20th century, the English bacteriologist Frederick Twort discovered a group of viruses that infect bacteria, now called bacteriophages (or commonly 'phages'), in 1915.[10][11] The French-Canadian microbiologist Félix d'Herelle announced his independent discovery of bacteriophages in 1917. D'Herelle described viruses that, when added to bacteria on an agar plate, would produce areas of dead bacteria. He accurately diluted a suspension of these viruses and discovered that the highest dilutions (lowest virus concentrations), rather than killing all the bacteria, formed discrete areas of dead organisms. Counting these areas and multiplying by the dilution factor allowed him to calculate the number of viruses in the original suspension.[12] Phages were heralded as a potential treatment for diseases such as typhoid and cholera, but their promise was forgotten with the development of penicillin. The development of bacterial resistance to antibiotics has renewed interest in the therapeutic use of bacteriophages.[13]

By the end of the 19th century, viruses were defined in terms of their infectivity, their ability to pass filters, and their requirement for living hosts. Viruses had been grown only in plants and animals. In 1906 Ross Granville Harrison invented a method for growing tissue in lymph, and in 1913 E. Steinhardt, C. Israeli, and R.A. Lambert used this method to grow vaccinia virus in fragments of guinea pig corneal tissue.[14] In 1928, H. B. Maitland and M. C. Maitland grew vaccinia virus in suspensions of minced hens' kidneys. Their method was not widely adopted until the 1950s when poliovirus was grown on a large scale for vaccine production.[15]

Another breakthrough came in 1931 when the American pathologist Ernest William Goodpasture and Alice Miles Woodruff grew influenza and several other viruses in fertilised chicken eggs.[16] In 1949, John Franklin Enders, Thomas Weller, and Frederick Robbins grew poliovirus in cultured cells from aborted human embryonic tissue,[17] the first virus to be grown without using solid animal tissue or eggs. This work enabled Hilary Koprowski, and then Jonas Salk, to make an effective polio vaccine.[18]

The first images of viruses were obtained upon the invention of electron microscopy in 1931 by the German engineers Ernst Ruska and Max Knoll.[19] In 1935, American biochemist and virologist Wendell Meredith Stanley examined the tobacco mosaic virus and found it was mostly made of protein.[20] A short time later, this virus was separated into protein and RNA parts.[21] The tobacco mosaic virus was the first to be crystallised and its structure could, therefore, be elucidated in detail. The first X-ray diffraction pictures of the crystallised virus were obtained by Bernal and Fankuchen in 1941. Based on her X-ray crystallographic pictures, Rosalind Franklin discovered the full structure of the virus in 1955.[22] In the same year, Heinz Fraenkel-Conrat and Robley Williams showed that purified tobacco mosaic virus RNA and its protein coat can assemble by themselves to form functional viruses, suggesting that this simple mechanism was probably the means through which viruses were created within their host cells.[23]

The second half of the 20th century was the golden age of virus discovery, and most of the documented species of animal, plant, and bacterial viruses were discovered during these years.[24] In 1946, bovine viral diarrhoea (a pestivirus) was first described. In 1957 equine arterivirus was discovered. In 1963 the hepatitis B virus was discovered by Baruch Blumberg.[25] In 1965 Howard Temin described the first retrovirus. Reverse transcriptase, the enzyme that retroviruses use to make DNA copies of their RNA, was first described in 1970 by Temin and David Baltimore independently.[26] In 1983 Luc Montagnier's team at the Pasteur Institute in France, first isolated the retrovirus now called HIV.[27] In 1989 Michael Houghton's team at Chiron Corporation discovered hepatitis C.[28][29]

Detecting viruses

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An electron microscope

There are several approaches to detecting viruses and these include the detection of virus particles (virions) or their antigens or nucleic acids and infectivity assays.

Electron microscopy

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Electron micrographs of viruses. A, rotavirus; B, adenovirus; C, norovirus; and D, astrovirus.

Viruses were seen for the first time in the 1930s when electron microscopes were invented. These microscopes use beams of electrons instead of light, which have a much shorter wavelength and can detect objects that cannot be seen using light microscopes. The highest magnification obtainable by electron microscopes is up to 10,000,000 times[30] whereas for light microscopes it is around 1,500 times.[31]

Virologists often use negative staining to help visualise viruses. In this procedure, the viruses are suspended in a solution of metal salts such as uranium acetate. The atoms of metal are opaque to electrons and the viruses are seen as suspended in a dark background of metal atoms.[30] This technique has been in use since the 1950s.[32] Many viruses were discovered using this technique and negative staining electron microscopy is still a valuable weapon in a virologist's arsenal.[33]

Traditional electron microscopy has disadvantages in that viruses are damaged by drying in the high vacuum inside the electron microscope and the electron beam itself is destructive.[30] In cryogenic electron microscopy the structure of viruses is preserved by embedding them in an environment of vitreous water.[34] This allows the determination of biomolecular structures at near-atomic resolution,[35] and has attracted wide attention to the approach as an alternative to X-ray crystallography or NMR spectroscopy for the determination of the structure of viruses.[36]

Cryoelectron micrograph of a rotavirus

Growth in cultures

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Viruses are obligate intracellular parasites and because they only reproduce inside the living cells of a host these cells are needed to grow them in the laboratory. For viruses that infect animals (usually called "animal viruses") cells grown in laboratory cell cultures are used. In the past, fertile hens' eggs were used and the viruses were grown on the membranes surrounding the embryo. This method is still used in the manufacture of some vaccines. For the viruses that infect bacteria, the bacteriophages, the bacteria growing in test tubes can be used directly. For plant viruses, the natural host plants can be used or, particularly when the infection is not obvious, so-called indicator plants, which show signs of infection more clearly.[37][38]

Cytopathic effect of herpes simplex virus. The infected cells have become round and balloon-like.

Viruses that have grown in cell cultures can be indirectly detected by the detrimental effect they have on the host cell. These cytopathic effects are often characteristic of the type of virus. For instance, herpes simplex viruses produce a characteristic "ballooning" of the cells, typically human fibroblasts. Some viruses, such as mumps virus cause red blood cells from chickens to firmly attach to the infected cells. This is called "haemadsorption" or "hemadsorption". Some viruses produce localised "lesions" in cell layers called plaques, which are useful in quantitation assays and in identifying the species of virus by plaque reduction assays.[39][40]

Viruses growing in cell cultures are used to measure their susceptibility to validated and novel antiviral drugs.[41]

Serology

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Viruses are antigens that induce the production of antibodies and these antibodies can be used in laboratories to study viruses. Related viruses often react with each other's antibodies and some viruses can be named based on the antibodies they react with. The use of the antibodies which were once exclusively derived from the serum (blood fluid) of animals is called serology.[42] Once an antibody–reaction has taken place in a test, other methods are needed to confirm this. Older methods included complement fixation tests,[43] hemagglutination inhibition and virus neutralisation.[44] Newer methods use enzyme immunoassays (EIA).[45]

In the years before PCR was invented immunofluorescence was used to quickly confirm viral infections. It is an infectivity assay that is virus species specific because antibodies are used. The antibodies are tagged with a dye that is luminescencent and when using an optical microscope with a modified light source, infected cells glow in the dark.[46]

Polymerase chain reaction (PCR) and other nucleic acid detection methods

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PCR is a mainstay method for detecting viruses in all species including plants and animals. It works by detecting traces of virus specific RNA or DNA. It is very sensitive and specific, but can be easily compromised by contamination. Most of the tests used in veterinary virology and medical virology are based on PCR or similar methods such as transcription mediated amplification. When a novel virus emerges, such as the covid coronavirus, a specific test can be devised quickly so long as the viral genome has been sequenced and unique regions of the viral DNA or RNA identified.[47] The invention of microfluidic tests as allowed for most of these tests to be automated,[48] Despite its specificity and sensitivity, PCR has a disadvantage in that it does not differentiate infectious and non-infectious viruses and "tests of cure" have to be delayed for up to 21 days to allow for residual viral nucleic acid to clear from the site of the infection.[49]

Diagnostic tests

[edit]

In laboratories many of the diagnostic test for detecting viruses are nucleic acid amplification methods such as PCR. Some tests detect the viruses or their components as these include electron microscopy and enzyme-immunoassays. The so-called "home" or "self"-testing gadgets are usually lateral flow tests, which detect the virus using a tagged monoclonal antibody.[50] These are also used in agriculture, food and environmental sciences.[51]

Quantitation and viral loads

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Main article: Virus quantification

Counting viruses (quantitation) has always had an important role in virology and has become central to the control of some infections of humans where the viral load is measured.[52] There are two basic methods: those that count the fully infective virus particles, which are called infectivity assays, and those that count all the particles including the defective ones.[30]

Infectivity assays

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Plaques in cells caused herpes simplex virus. The cells have been fixed and stained blue.

Infectivity assays measure the amount (concentration) of infective viruses in a sample of known volume.[53] For host cells, plants or cultures of bacterial or animal cells are used. Laboratory animals such as mice have also been used particularly in veterinary virology.[54] These are assays are either quantitative where the results are on a continuous scale or quantal, where an event either occurs or it does not. Quantitative assays give absolute values and quantal assays give a statistical probability such as the volume of the test sample needed to ensure 50% of the hosts cells, plants or animals are infected. This is called the median infectious dose or ID 50.[55] Infective bacteriophages can be counted by seeding them onto "lawns" of bacteria in culture dishes. When at low concentrations, the viruses form holes in the lawn that can be counted. The number of viruses is then expressed as plaque forming units. For the bacteriophages that reproduce in bacteria that cannot be grown in cultures, viral load assays are used.[56]

Immunoflourescence: Cells infected by rotavirus (top) and uninfected cells (bottom)

The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using fluorescently labeled antibodies specific for a viral antigen to detect infected host cells and infectious virus particles before an actual plaque is formed. The FFA is particularly useful for quantifying classes of viruses that do not lyse the cell membranes, as these viruses would not be amenable to the plaque assay. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours) under a semisolid overlay medium that restricts the spread of infectious virus, creating localized clusters (foci) of infected cells. Plates are subsequently probed with fluorescently labeled antibodies against a viral antigen, and fluorescence microscopy is used to count and quantify the number of foci. The FFA method typically yields results in less time than plaque or fifty-percent-tissue-culture-infective-dose (TCID50) assays, but it can be more expensive in terms of required reagents and equipment. Assay completion time is also dependent on the size of area that the user is counting. A larger area will require more time but can provide a more accurate representation of the sample. Results of the FFA are expressed as focus forming units per milliliter, or FFU/mL.[57]

Viral load assays

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Main article: Viral load

When an assay for measuring the infective virus particle is done (Plaque assay, Focus assay), viral titre often refers to the concentration of infectious viral particles, which is different from the total viral particles. Viral load assays usually count the number of viral genomes present rather than the number of particles and use methods similar to PCR.[58] Viral load tests are an important in the control of infections by HIV.[59] This versatile method can be used for plant viruses.[60][61]

Molecular biology

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Molecular virology is the study of viruses at the level of nucleic acids and proteins. The methods invented by molecular biologists have all proven useful in virology. Their small sizes and relatively simple structures make viruses an ideal candidate for study by these techniques.

Purifying viruses and their components

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Caesium chloride (CsCl) solution and two morphological types of rotavirus. Following centrifugation at 100,000 g a density gradient forms in the CsCl solution and the virus particles separate according to their densities. The tube is 10 cm tall. The viruses are the two "milky" zones close together.[62]

For further study, viruses grown in the laboratory need purifying to remove contaminants from the host cells. The methods used often have the advantage of concentrating the viruses, which makes it easier to investigate them.

Centrifugation

[edit]

Centrifuges are often used to purify viruses. Low speed centrifuges, i.e. those with a top speed of 10,000 revolutions per minute (rpm) are not powerful enough to concentrate viruses, but ultracentrifuges with a top speed of around 100,000 rpm, are and this difference is used in a method called differential centrifugation. In this method the larger and heavier contaminants are removed from a virus mixture by low speed centrifugation. The viruses, which are small and light and are left in suspension, are then concentrated by high speed centrifugation.[63]

Following differential centrifugation, virus suspensions often remain contaminated with debris that has the same sedimentation coefficient and are not removed by the procedure. In these cases a modification of centrifugation, called buoyant density centrifugation, is used. In this method the viruses recovered from differential centrifugation are centrifuged again at very high speed for several hours in dense solutions of sugars or salts that form a density gradient, from low to high, in the tube during the centrifugation. In some cases, preformed gradients are used where solutions of steadily decreasing density are carefully overlaid on each other. Like an object in the Dead Sea, despite the centrifugal force the virus particles cannot sink into solutions that are more dense than they are and they form discrete layers of, often visible, concentrated viruses in the tube. Caesium chloride is often used for these solutions as it is relatively inert but easily self-forms a gradient when centrifuged at high speed in an ultracentrifuge.[62] Buoyant density centrifugation can also be used to purify the components of viruses such as their nucleic acids or proteins.[64]

Electrophoresis

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Polyacrylamide gel electrophoresis of rotavirus proteins stained with Coomassie blue

The separation of molecules based on their electric charge is called electrophoresis. Viruses and all their components can be separated and purified using this method. This is usually done in a supporting medium such as agarose and polyacrylamide gels. The separated molecules are revealed using stains such as coomasie blue, for proteins, or ethidium bromide for nucleic acids. In some instances the viral components are rendered radioactive before electrophoresis and are revealed using photographic film in a process known as autoradiography.[65]

Sequencing of viral genomes

[edit]
Main article: DNA sequencing

As most viruses are too small to be seen by a light microscope, sequencing is one of the main tools in virology to identify and study the virus. Traditional Sanger sequencing and next-generation sequencing (NGS) are used to sequence viruses in basic and clinical research, as well as for the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, and drug-resistance testing. There are more than 2.3 million unique viral sequences in GenBank.[66] NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes.[66] Viral genome sequencing as become a central method in viral epidemiology and viral classification.

Phylogenetic analysis

[edit]

Data from the sequencing of viral genomes can be used to determine evolutionary relationships and this is called phylogenetic analysis.[67] Software, such as PHYLIP, is used to draw phylogenetic trees. This analysis is also used in studying the spread of viral infections in communities (epidemiology).[68]

Cloning

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Main article: Cloning vector

When purified viruses or viral components are needed for diagnostic tests or vaccines, cloning can be used instead of growing the viruses.[69] At the start of the COVID-19 pandemic the availability of the severe acute respiratory syndrome coronavirus 2 RNA sequence enabled tests to be manufactured quickly.[70] There are several proven methods for cloning viruses and their components. Small pieces of DNA called cloning vectors are often used and the most common ones are laboratory modified plasmids (small circular molecules of DNA produced by bacteria). The viral nucleic acid, or a part of it, is inserted in the plasmid, which is the copied many times over by bacteria. This recombinant DNA can then be used to produce viral components without the need for native viruses.[71]

Phage virology

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The viruses that reproduce in bacteria, archaea and fungi are informally called "phages",[72] and the ones that infect bacteria – bacteriophages – in particular are useful in virology and biology in general.[73] Bacteriophages were some of the first viruses to be discovered, early in the twentieth century,[74] and because they are relatively easy to grow quickly in laboratories, much of our understanding of viruses originated by studying them.[74] Bacteriophages, long known for their positive effects in the environment, are used in phage display techniques for screening proteins DNA sequences. They are a powerful tool in molecular biology.[75]

Genetics

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All viruses have genes which are studied using genetics.[76] All the techniques used in molecular biology, such as cloning, creating mutations RNA silencing are used in viral genetics.[77]

Reassortment

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Reassortment is the switching of genes from different parents and it is particularly useful when studying the genetics of viruses that have segmented genomes (fragmented into two or more nucleic acid molecules) such as influenza viruses and rotaviruses. The genes that encode properties such as serotype can be identified in this way.[78]

Recombination

[edit]

Often confused with reassortment, recombination is also the mixing of genes but the mechanism differs in that stretches of DNA or RNA molecules, as opposed to the full molecules, are joined during the RNA or DNA replication cycle. Recombination is not as common as reassortment in nature but it is a powerful tool in laboratories for studying the structure and functions of viral genes.[79]

Reverse genetics

[edit]

Reverse genetics is a powerful research method in virology.[80] In this procedure complementary DNA (cDNA) copies of virus genomes called "infectious clones" are used to produce genetically modified viruses that can be then tested for changes in say, virulence or transmissibility.[81]

Virus classification

[edit]
For how viruses are classified with relation to other living things, see Tree of life (biology).
Main article: Virus classification

A major branch of virology is virus classification. It is artificial in that it is not based on evolutionary phylogenetics but it is based shared or distinguishing properties of viruses.[82][83] It seeks to describe the diversity of viruses by naming and grouping them on the basis of similarities.[84] In 1962, André Lwoff, Robert Horne, and Paul Tournier were the first to develop a means of virus classification, based on the Linnaean hierarchical system.[85] This system based classification on phylum, class, order, family, genus, and species. Viruses were grouped according to their shared properties (not those of their hosts) and the type of nucleic acid forming their genomes.[86] In 1966, the International Committee on Taxonomy of Viruses (ICTV) was formed. The system proposed by Lwoff, Horne and Tournier was initially not accepted by the ICTV because the small genome size of viruses and their high rate of mutation made it difficult to determine their ancestry beyond order. As such, the Baltimore classification system has come to be used to supplement the more traditional hierarchy.[87] Starting in 2018, the ICTV began to acknowledge deeper evolutionary relationships between viruses that have been discovered over time and adopted a 15-rank classification system ranging from realm to species.[88] Additionally, some species within the same genus are grouped into a genogroup.[89][90]

ICTV classification

[edit]

The ICTV developed the current classification system and wrote guidelines that put a greater weight on certain virus properties to maintain family uniformity. A unified taxonomy (a universal system for classifying viruses) has been established. Only a small part of the total diversity of viruses has been studied.[91] As of 2021, 6 realms, 10 kingdoms, 17 phyla, 2 subphyla, 39 classes, 65 orders, 8 suborders, 233 families, 168 subfamilies, 2,606 genera, 84 subgenera, and 10,434 species of viruses have been defined by the ICTV.[92]

The general taxonomic structure of taxon ranges and the suffixes used in taxonomic names are shown hereafter. As of 2021, the ranks of subrealm, subkingdom, and subclass are unused, whereas all other ranks are in use.[92]

Realm (-viria)
Subrealm (-vira)
Kingdom (-virae)
Subkingdom (-virites)
Phylum (-viricota)
Subphylum (-viricotina)
Class (-viricetes)
Subclass (-viricetidae)
Order (-virales)
Suborder (-virineae)
Family (-viridae)
Subfamily (-virinae)
Genus (-virus)
Subgenus (-virus)
Species

Baltimore classification

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Main article: Baltimore classification
A diagram showing how the Baltimore Classification is based on a virus's DNA or RNA and method of mRNA synthesis
The Baltimore Classification of viruses is based on the method of viral mRNA synthesis.

The Nobel Prize-winning biologist David Baltimore devised the Baltimore classification system.[93]

The Baltimore classification of viruses is based on the mechanism of mRNA production. Viruses must generate mRNAs from their genomes to produce proteins and replicate themselves, but different mechanisms are used to achieve this in each virus family. Viral genomes may be single-stranded (ss) or double-stranded (ds), RNA or DNA, and may or may not use reverse transcriptase (RT). In addition, ssRNA viruses may be either sense (+) or antisense (−). This classification places viruses into seven groups:

References

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[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Virology

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from Grokipedia
Virology is the scientific discipline dedicated to the study of viruses and the diseases they cause, encompassing their structure, replication, pathogenesis, and interactions with host organisms. Viruses are acellular, obligate intracellular parasites that consist of a nucleic acid genome—either DNA or RNA—enclosed within a protective protein capsid, and in some cases, an outer lipid envelope derived from the host cell membrane. These infectious agents are incapable of independent replication and must hijack the cellular machinery of living host cells, such as those in bacteria, plants, animals, or humans, to produce progeny virions. Ranging in size from 20 to 300 nanometers, viruses represent the smallest known pathogens and can infect every type of organism on Earth. The structure of viruses is remarkably diverse yet follows fundamental principles. A complete virus particle, known as a virion, serves primarily to deliver its genome into a susceptible host cell. Genomes vary as single- or double-stranded DNA or RNA, linear or circular, and may be monopartite (one segment) or multipartite (multiple segments). Capsids exhibit symmetrical arrangements, such as helical (rod-like filaments, as in tobacco mosaic virus) or icosahedral (20-faced polyhedrons, as in adenoviruses with 252 capsomeres). Enveloped viruses, like influenza or HIV, acquire a lipid bilayer during assembly, studded with viral glycoproteins that facilitate attachment to specific host cell receptors. Classification systems, such as the Baltimore classification, organize viruses into seven groups based on genome type and replication strategy, while the International Committee on Taxonomy of Viruses (ICTV) uses morphological, genetic, and biological criteria to define families like Picornaviridae (non-enveloped RNA viruses) and Herpesviridae (enveloped DNA viruses). Virus replication is a precisely orchestrated that exploits host resources, distinguishing viruses from other microbes. Upon attachment via receptor binding, the virion enters the cell through or fusion, releasing its . The viral then directs the synthesis of viral proteins and replication of nucleic acids using host ribosomes, polymerases, and energy sources, often leading to the assembly of hundreds of new virions within hours. viruses, comprising about 70% of known viruses, exhibit high rates (up to 10⁻⁴ per ) due to error-prone polymerases, driving rapid evolution and antigenic drift. Infections can be lytic (causing host cell and release of virions), latent (dormant integration into the host , as in herpesviruses), or persistent (chronic low-level replication), with outcomes ranging from to severe disease. Virology's historical development traces back to ancient observations of unexplained plagues, evolving through the germ theory era with the discovery of filterable agents in the late and milestones like the first virus crystallization and development in the . Today, virology addresses critical global challenges, including emerging pathogens like and , through advancements in , antiviral therapies targeting replication steps, and that have eradicated diseases such as . Key research areas include host-virus interactions, immune evasion mechanisms, and viral oncogenesis, underscoring virology's role in , , and .

Fundamentals

Definition and Characteristics

Viruses are defined as intracellular parasites that depend entirely on the cellular machinery of host organisms for their replication and propagation. Unlike or other microbes, viruses cannot reproduce independently and must infect a living host cell to hijack its metabolic processes and biosynthetic pathways. This parasitic lifestyle distinguishes viruses as non-autonomous entities that exist extracellularly as inert particles until they encounter a suitable host. Viruses are acellular, lacking the organelles, cytoplasm, and metabolic capabilities found in cellular life forms, including ribosomes for protein synthesis and independent energy production. They typically range in size from 20 to 300 nanometers, rendering them ultramicroscopic and invisible under standard light microscopy, which requires electron microscopy for visualization. This acellular nature and small scale underscore their inability to grow or carry out metabolic functions outside a host, positioning them as fundamentally distinct from prokaryotes and eukaryotes. The classification of viruses as living or non-living organisms remains a subject of debate among biologists, centered on established criteria for such as cellular organization, , , growth, response to stimuli, , and . Viruses possess genetic material capable of , through , and transmission of heritable information, fulfilling some criteria. However, their lack of autonomous —relying instead on host cells—and absence of independent lead most scientists to classify them as non-living, though they blur the boundary between biotic and abiotic entities. At a high level, the viral life cycle consists of , where the virus attaches to and enters a host cell; replication of the viral genome using host resources; assembly of new viral particles; and release, often through cell or , to disseminate to other cells. This cycle enables viruses to propagate efficiently while exploiting host biology without contributing to cellular .

Viral Components

Viruses are acellular entities composed of a limited set of molecular building blocks, distinguishing them from cellular by their minimalistic design focused on efficient propagation. The core components include a genome encased in a protective protein , with some viruses featuring an outer lipid envelope and specialized appendages. These elements collectively enable genome protection, host recognition, and delivery, while the overall chemical makeup emphasizes proteins as the dominant structural material. The forms a robust protein shell that encases and safeguards the viral genome from , such as nucleases and physical stress. Composed of multiple copies of one or a few virus-encoded proteins arranged with either icosahedral or helical , the also facilitates initial attachment to host cells via surface-exposed regions. For instance, icosahedral capsids, seen in adenoviruses, consist of 252 capsomeres forming a near-spherical structure approximately 70-90 nm in diameter, while helical capsids, as in , wind around the genome in a rod-like configuration. The viral genome, serving as the hereditary material, consists of either DNA or RNA, which can be single-stranded or double-stranded, linear or circular, and monopartite or segmented. Genome sizes vary widely, from approximately 7.5 kb in single-stranded RNA picornaviruses like poliovirus to over 1.2 Mb in double-stranded DNA mimiviruses, reflecting diverse coding capacities from a handful to over 900 proteins. These genome types underpin the Baltimore classification system, grouping viruses by replication strategies. Many viruses acquire an , a derived from modified host cell membranes that surrounds the , providing additional stability and aiding in immune evasion. Embedded in this envelope are virus-encoded glycoproteins that project as , crucial for specific recognition and binding to host receptors; examples include the and neuraminidase spikes in viruses or the envelope glycoproteins gp120 and in . Non-enveloped viruses, such as , lack this layer and rely solely on the for protection. In enveloped viruses, matrix proteins lie beneath the envelope, bridging it to the and coordinating assembly by linking glycoproteins to the nucleocapsid core. Certain viruses possess additional specialized structures beyond the basic nucleocapsid or enveloped form. Bacteriophages often feature tails and : a tubular tail for injection into bacterial hosts, as in T4 phage with its contractile tail, and tail or fibers for receptor binding to initiate . These appendages interrupt capsid symmetry and are absent in most animal viruses but exemplify structural diversity in prokaryotic pathogens. Chemically, viral particles are predominantly proteinaceous, with proteins comprising 70-90% of the dry weight in non-enveloped viruses to form the and any internal scaffolds. Nucleic acids account for 5-30% of the mass, varying inversely with ; for example, RNA constitutes about 10% in picornaviruses. Enveloped viruses incorporate , typically 30-35% of dry weight, primarily phospholipids and derived from the host, alongside minor carbohydrates in glycoproteins. This composition underscores the parasitic nature of viruses, hijacking host resources for structural elements while minimizing their own synthetic burden.

History

Early Observations

The concept of infectious agents smaller than emerged from studies on diseases like and during the 18th and 19th centuries, which laid groundwork for understanding viral transmission through observational experiments on contagion and . Edward Jenner's 1796 demonstration that exposure to material could prevent infection highlighted the role of transmissible agents in disease spread, influencing later ideas about invisible pathogens. Similarly, 19th-century investigations into outbreaks in the Americas and Europe revealed patterns of human-to-human transmission via close contact or fomites, though the exact mechanisms remained elusive without knowledge of filterable agents. These efforts shifted medical thinking from toward specific contagious principles, setting the stage for virology. A pivotal advance came in 1892 when Russian scientist Dmitri Ivanovsky conducted filtration experiments on mosaic disease, a condition affecting plants that caused mottled leaves and stunted growth. He passed sap from infected plants through a fine porcelain designed to retain bacteria, yet the filtrate remained infectious when applied to healthy plants, indicating the presence of an ultrafilterable agent smaller than known microbes. This observation challenged the prevailing view that all infectious diseases were caused by visible bacteria, as identified by and . Building on Ivanovsky's findings, Dutch replicated and extended the experiments in 1898, confirming the filterable nature of the tobacco mosaic agent while demonstrating its ability to multiply in host tissues without forming bacterial colonies. Beijerinck proposed the term contagium vivum um—a "living infectious "—to describe this self-propagating, non-cellular entity that reproduced only within living cells, distinguishing it from inert chemicals or bacterial products. His work established viruses as contagious agents capable of indefinite reproduction in susceptible hosts, marking a conceptual shift toward recognizing them as distinct . In the same year, German scientists Friedrich Loeffler and Paul Frosch showed that in cattle was transmitted by a filterable agent, providing the first evidence of a viral in animals and extending the concept beyond plants. Early interpretations often misconstrued these filterable agents as bacterial s, enzymes, or fragments of disintegrated , reflecting the era's limited tools for detection and the assumption that all infections stemmed from microbial cells. For instance, some researchers viewed the tobacco mosaic agent as a soluble produced by unseen , while others speculated it consisted of bacterial too small to filter out. These misconceptions persisted until experimental accumulated, highlighting the need for new paradigms in infectious disease research. The first direct visualization of a virus occurred through electron microscopy, enabling observation of these submicroscopic particles. In 1931, and Max Knoll developed the first transmission electron microscope, achieving resolutions far beyond light microscopy and opening the door to imaging nanoscale structures. By 1939, Helmut Ruska (Ernst's brother) and colleagues captured the first electron micrographs of , revealing its rod-shaped particles approximately 300 nm long and 18 nm in diameter, confirming its particulate nature and solidifying viruses as discrete entities rather than mere fluids or toxins.

Key Milestones in Virology

In 1935, American biochemist Wendell M. Stanley achieved a groundbreaking isolation and crystallization of the (TMV), demonstrating that viruses could be purified as crystalline nucleoproteins, which blurred the distinction between living organisms and chemical entities. This work, conducted at the Rockefeller Institute, involved precipitating TMV from infected plant sap using and confirming its infectivity after recrystallization, earning Stanley the 1946 for advancing the understanding of viral structure. Between 1915 and 1917, British bacteriologist Frederick Twort and Canadian-French microbiologist Felix d'Hérelle independently discovered , viruses that infect and lyse bacteria. Twort observed a filterable agent causing bacterial colonies to dissolve, while d'Hérelle isolated similar agents from patients and coined the term "bacteriophage" (bacteria-eater), proposing their potential as antibacterial agents. These findings established phages as model organisms for studying and , influencing later virological research. A pivotal confirmation of DNA as the genetic material came in 1952 through the Hershey-Chase experiment, conducted by Alfred Hershey and Martha Chase using the T2 bacteriophage infecting Escherichia coli. By radioactively labeling phage DNA with phosphorus-32 and protein coats with sulfur-35, they showed that only the DNA entered bacterial cells to direct viral reproduction, while the protein remained outside, thus resolving debates favoring proteins as hereditary agents. This experiment, performed at Cold Spring Harbor Laboratory, provided conclusive evidence supporting DNA's role in heredity and influenced subsequent molecular biology research. The discovery of reverse transcriptase in the early 1970s revolutionized understanding of retroviruses, with Howard Temin and Satoshi Mizutani identifying the enzyme in Rous sarcoma virus virions, enabling RNA-templated DNA synthesis contrary to the central dogma. Independently, David Baltimore detected the same RNA-dependent DNA polymerase in avian myeloblastosis virus, confirming its presence across retroviral families. This 1970 breakthrough, awarded the 1975 Nobel Prize in Physiology or Medicine to Temin and Baltimore, laid the foundation for studying RNA tumor viruses and later identifying human immunodeficiency virus (HIV) in 1983 by teams led by Françoise Barré-Sinoussi, Luc Montagnier, and Robert Gallo, who isolated the retrovirus from AIDS patients. The 2008 Nobel Prize in Physiology or Medicine recognized Barré-Sinoussi and Montagnier's HIV discovery for its impact on combating the global AIDS epidemic. The invention of the (PCR) in 1983 by at transformed viral detection and molecular virology by enabling exponential amplification of specific DNA sequences from minute samples. First detailed in a 1985 paper by Randall Saiki and colleagues, PCR utilized thermostable to cycle through denaturation, annealing, and extension, revolutionizing diagnostics for viruses like and . received the 1993 for this technique, which became indispensable for viral genome sequencing and epidemiological tracking. In the , unveiled the vast viral diversity on , with estimates from global sampling suggesting approximately 10^{31} virus particles, predominantly bacteriophages in oceans and soils, far exceeding other biological entities. This 2007 quantification by Curtis Suttle, built upon by 2011 metagenomic surveys, highlighted viruses' role in dynamics and spurred discoveries of novel viral families through unbiased sequencing. Concurrently, the 2012 development of -Cas9 by , , and colleagues repurposed bacterial adaptive immunity into a programmable tool for precise , with applications in virology including targeted disruption of viral genomes in host cells and engineering antiviral therapies. Since then, has facilitated studies of viral replication cycles and vaccine development, earning Doudna and Charpentier the 2020 .

Classification

ICTV System

The International Committee on Taxonomy of Viruses (ICTV), established in 1966 as the International Committee on of Viruses (ICNV) under the Virology Division of the International Union of Microbiological Societies and renamed in 1975, maintains a universal system for classifying viruses based on their evolutionary relationships. This framework organizes viruses into a hierarchical that is periodically updated through proposals reviewed by study groups and ratified by the ICTV Executive Committee, ensuring a standardized and that reflects advances in virological research. The system emphasizes phylogenetic coherence, grouping viruses that share common ancestry while accommodating the diversity of viral forms. The ICTV taxonomy employs a Linnaean-inspired with ranks including (the highest), kingdom, , class, order, , , , , and . In 2018, the rank was formally introduced to capture deep evolutionary divergences, with established as a encompassing RNA viruses that utilize for replication, unifying diverse groups like coronaviruses and flaviviruses under a monophyletic . As of the 2025 taxonomy release (MSL #40 v2), the ICTV recognizes 7 realms, accommodating diverse viral lineages. This addition expanded the to better align with genomic evidence of ancient viral lineages, allowing for a more comprehensive partitioning of the virosphere. Classification within the ICTV system relies on multiple criteria, including similarity, virion morphology, replication strategies, and host range, with an increasing emphasis on phylogenomic analyses to delineate taxa. For instance, -based metrics such as protein-coding conservation and pairwise genetic distances are used to propose new or higher ranks, supplemented by phenotypic where available. These criteria ensure that taxa reflect shared evolutionary history rather than superficial traits, though metagenomic from uncultured viruses often requires integrative approaches to establish . Representative examples illustrate the system's application: the family Herpesviridae, comprising double-stranded DNA viruses that establish latency in mammalian and avian hosts, falls within the order and is characterized by enveloped icosahedral virions and serial propagation in . Similarly, the order Mononegavirales includes non-segmented negative-sense RNA viruses, such as the genus (with species like ), which features filamentous virions and a broad host range spanning mammals and bats. Despite its robustness, the ICTV system faces challenges from viruses' rapid evolutionary rates, which can generate significant and necessitate frequent taxonomic revisions, and from the prevalence of unculturable viruses discovered via , which lack traditional phenotypic data for robust placement. As of the 2025 taxonomy release, the ICTV recognizes over 16,000 across 3,768 genera and 368 families, reflecting ongoing efforts to incorporate vast genomic datasets while addressing these complexities.

Baltimore Classification

The Baltimore classification system, proposed by in 1971, categorizes viruses into seven groups based on the nature of their and the mechanism by which they synthesize (mRNA) during replication. This scheme adapts the —DNA to to protein—to viral life cycles, emphasizing how viruses exploit host machinery to produce mRNA for protein synthesis. Unlike taxonomic systems that prioritize evolutionary relationships, the Baltimore classification focuses on molecular replication strategies, providing a framework to predict the enzymatic requirements and potential therapeutic targets for each viral group. The seven classes are defined as follows:
ClassGenome TypemRNA Synthesis MechanismRepresentative Examples
IDouble-stranded DNA (dsDNA)Host RNA polymerase transcribes dsDNA directly into mRNAAdenoviruses, herpesviruses, poxviruses
IISingle-stranded DNA (ssDNA)Host converts ssDNA to dsDNA intermediate, then transcribes mRNAParvoviruses
IIIDouble-stranded RNA (dsRNA)Viral transcribes one strand into mRNAReoviruses
IVPositive-sense single-stranded (+ssRNA)The +ssRNA genome serves directly as mRNAPicornaviruses (e.g., ), coronaviruses
VNegative-sense single-stranded (-ssRNA)Viral transcribes -ssRNA into +ssRNA mRNAOrthomyxoviruses (e.g., ), rhabdoviruses (e.g., )
VISingle-stranded with (ssRNA-RT) converts +ssRNA to DNA, which integrates into host ; host machinery transcribes mRNA from integrated DNARetroviruses (e.g., )
VIIDouble-stranded DNA with (dsDNA-RT) partially transcribes dsDNA to RNA intermediate, then back to DNA; host transcribes mRNA from final DNAHepadnaviruses (e.g., )
In Class I viruses, the dsDNA genome resembles cellular DNA, allowing direct transcription by host RNA polymerase II into mRNA, similar to eukaryotic . Class II viruses require a host DNA polymerase to generate a dsDNA replicative form from the ssDNA genome before mRNA transcription. For RNA viruses in Classes III, IV, and V, viral polymerases are essential since host cells lack RNA-dependent RNA polymerases; Class IV uses its genome directly as mRNA, while Classes III and V involve transcription from dsRNA or -ssRNA templates, respectively. Classes VI and VII, involving reverse transcription, highlight unique adaptations where RNA serves as a template for , enabling integration into the host genome for persistent . This classification offers key advantages by linking genome type to replication needs, facilitating the design of antiviral drugs that target specific viral enzymes, such as inhibitors for Class VI viruses. For instance, understanding that (Class VI) requires reverse transcription has led to therapies like , which inhibit this step. However, a major limitation is its lack of phylogenetic insight, as viruses in the same class may not share a common ancestor, unlike the International Committee on Taxonomy of Viruses (ICTV) system, which emphasizes evolutionary relationships. Since its inception, the has undergone minor refinements, such as the addition of Class VII in the 1980s to accommodate hepadnaviruses, but the core framework remains unchanged, serving as a foundational tool in virology and .

Replication Cycle

Attachment and Entry

The attachment and entry phase represents the critical initial step in the cycle, where viruses must recognize and invade host cells to deliver their genetic material intracellularly. This process begins with the specific binding of viral surface proteins to host cell receptors, enabling the virus to adhere to the target cell surface. Successful attachment is followed by entry, during which the or merges with or penetrates the host membrane, often triggered by conformational changes in viral proteins. These mechanisms vary widely among viruses, influenced by their structural features and the host cell type, and are essential for determining viral tropism and pathogenicity. Viral attachment is mediated by interactions between viral glycoproteins or capsid proteins and specific receptors on the host cell surface. For instance, the severe acute respiratory syndrome coronavirus 2 () utilizes its spike protein to bind the (ACE2) receptor on respiratory epithelial cells, facilitating initial adhesion. Similarly, human immunodeficiency virus type 1 (HIV-1) employs its envelope glycoprotein gp120 to interact with the receptor on T lymphocytes and macrophages, a key determinant of its cellular . These receptor-ligand interactions are highly specific, often mimicking host signaling pathways to promote viral docking. Co-receptors and additional host factors further refine viral tropism by modulating attachment efficiency and specificity. In HIV-1 infection, after CD4 binding, gp120 engages chemokine co-receptors such as CCR5 or CXCR4, which are required for subsequent membrane fusion and dictate the virus's preference for different immune cell subsets. For influenza A viruses, attachment involves the hemagglutinin protein recognizing sialic acid residues on host glycans, with the linkage type (α2,3 or α2,6) influencing tissue tropism—α2,6-linked sialic acids predominate in the human upper respiratory tract, promoting human adaptation. These co-factors and glycan variations create barriers to cross-species transmission and shape epidemic potential. Once attached, viruses employ diverse entry mechanisms to breach the host membrane. Many enveloped viruses, including those using clathrin-mediated , are internalized into endocytic vesicles where low triggers conformational changes leading to fusion; for example, undergoes hemagglutinin-mediated fusion in early endosomes. In contrast, paramyxoviruses like initiate fusion directly at the plasma membrane via their fusion (F) protein, bypassing and enabling rapid entry at neutral . Non-enveloped viruses, such as adenoviruses, often penetrate the endosomal membrane directly through pore formation by penton base proteins, releasing the into the . These pathways allow viruses to exploit host trafficking machinery while avoiding immune detection. Following entry, uncoating releases the viral from its protective or , a process tightly regulated to ensure timely delivery to replication sites. For many viruses internalized via , such as and adenoviruses, uncoating is pH-dependent, occurring in acidic endosomes where induces disassembly and genome ejection. This step is crucial for transitioning from extracellular virions to intracellular components, with disruptions often leading to abortive infections. Genome types, as classified by the system, can influence uncoating requirements, such as the need for reverse transcription in retroviruses prior to nuclear import. Host cells impose physical and chemical barriers to impede viral attachment and entry, with viruses evolving countermeasures to overcome them. layers in respiratory and gastrointestinal tracts trap virions through glycan interactions, while propels them away from entry sites. A viruses counter this via neuraminidase, which cleaves linkages in mucins, enabling virion diffusion and access to underlying epithelial cells. These innate defenses, combined with viral adaptations, underscore the at the host-virus interface.

Genome Replication and Gene Expression

Viral replication and occur intracellularly following entry and uncoating, with strategies adapted to the type of and the host cell environment. These processes enable viruses to produce progeny genomes and viral proteins essential for assembly, while exploiting or modifying host machinery. The mechanisms vary significantly across virus families, reflecting the , and are tightly regulated to ensure efficient propagation. For DNA viruses, replication typically takes place in the host cell nucleus using the host's DNA-dependent DNA polymerase, though some encode their own polymerase for cytoplasmic replication. Adenoviruses, for instance, replicate their linear double-stranded DNA genomes in nuclear replication compartments, initiating at specific origins with the aid of viral proteins and host factors like Oct-1 and NFI to produce approximately 1 million genome copies within 40 hours. In contrast, poxviruses replicate their large linear double-stranded DNA genomes entirely in the cytoplasm using a virus-encoded DNA polymerase and associated factors, forming viral factories that sequester host ribosomes and enzymes. Gene expression in these viruses follows a temporal cascade: early genes, transcribed by host or viral RNA polymerase before replication, encode regulatory and replication proteins, while late genes, expressed post-replication, produce structural components. RNA viruses rely on virus-encoded RNA-dependent RNA polymerases (RdRps) for both replication and mRNA synthesis, as host cells lack enzymes for RNA-templated RNA synthesis. Positive-sense single-stranded RNA (+ssRNA) viruses, such as , use their directly as mRNA upon entry; translation of a polyprotein yields the RdRp (e.g., 3Dpol in picornaviruses), which then forms replication complexes on cytoplasmic membranes to synthesize negative-sense intermediates and new +ssRNA s. Negative-sense single-stranded RNA (-ssRNA) viruses, like , package RdRp in the virion; upon entry, it transcribes the genome into +ssRNA mRNAs for initial protein synthesis, including more RdRp, before replicating full-length antigenomes and progeny genomes in nucleocapsid-associated complexes. Some RNA viruses, including coronaviruses, generate subgenomic RNAs via discontinuous transcription to express downstream genes, allowing coordinated production of non-structural and structural proteins. Retroviruses, such as HIV, employ a unique RNA-to-DNA conversion via reverse transcriptase (RT), an error-prone enzyme that synthesizes double-stranded DNA from the +ssRNA genome using a host tRNA primer at the primer-binding site. This process involves minus-strand strong-stop DNA synthesis, RNase H-mediated RNA degradation, strand transfers via long terminal repeats (LTRs), and plus-strand synthesis primed at polypurine tracts, culminating in a linear dsDNA provirus that integrates into the host genome by viral integrase. Once integrated, the provirus is transcribed by host RNA polymerase II into full-length RNAs serving as mRNAs for Gag-Pol-Env polyproteins and genomic RNAs for packaging, with temporal regulation achieved through alternative splicing and Rev-mediated nuclear export of unspliced RNAs. Across virus types, is temporally regulated to optimize replication: early phases prioritize non-structural proteins for amplification, while late phases focus on structural proteins for virion assembly. This cascade, observed in adenoviruses and herpesviruses, relies on promoter sequences, viral transactivators, and replication-linked to switch from early to late transcription. In viruses like coronaviruses, subgenomic RNAs ensure nested expression of structural genes late in infection. RNA viruses exhibit notably high mutation rates, typically ranging from 10^{-4} to 10^{-5} substitutions per per replication cycle, due to the lack of in RdRp (except in some nidoviruses with activity), which generates essential for rapid and . DNA viruses and retroviruses have lower rates, around 10^{-6} to 10^{-8} per site, benefiting from host mechanisms. These error rates underscore the quasispecies nature of RNA virus populations, driving antigenic variation and immune evasion.

Genetics and Evolution

Genetic Variation Mechanisms

Viruses exhibit remarkable , which enables rapid to host immune responses, antiviral therapies, and environmental pressures. This diversity arises primarily through inherent molecular processes during replication and genetic exchange, distinguishing from that of cellular organisms. Key mechanisms include , recombination, and reassortment, each contributing to the generation of viral genotypes that can confer selective advantages. Mutation is a fundamental source of viral genetic variation, encompassing point mutations (substitutions of single ), insertions, and deletions that alter the sequence. In RNA viruses, mutation rates are exceptionally high, typically ranging from 10^{-3} to 10^{-5} errors per per replication cycle, due to the error-prone nature of RNA-dependent RNA polymerases lacking activity. DNA viruses generally mutate at lower rates, around 10^{-6} to 10^{-8}, as their polymerases often incorporate host mechanisms, though some, like herpesviruses, can still generate significant diversity through polymerase infidelity. These mutations can lead to synonymous changes that preserve sequences or nonsynonymous ones that modify protein function, influencing viral fitness, antigenicity, and pathogenicity; for instance, point mutations in the hemagglutinin gene of drive antigenic drift. Insertions and deletions may disrupt or create new open reading frames, as observed in coronaviruses where such events expand the and introduce accessory genes. Recombination involves the exchange of genetic material between two viral genomes, producing chimeric progeny that combine segments from parental strains. occurs between similar sequences at aligned sites, facilitating precise swaps that maintain genome integrity, and is well-documented in positive-sense RNA viruses like , where it contributes to the emergence of new variants such as severe acute respiratory syndrome 2 () recombinants. In contrast, non-homologous recombination joins dissimilar sequences at non-aligned positions, often resulting in deletions or insertions, and is prevalent in retroviruses like , where it generates drug-resistant strains during reverse transcription. , mediated by viral or host enzymes, targets particular motifs; for example, in bacteriophage lambda, integrase catalyzes recombination at attachment sites, though analogous processes in animal viruses are rarer. In viruses, intra-segmental has been detected but appears infrequent compared to other mechanisms. Reassortment, unique to viruses with segmented genomes such as influenza A, involves the random packaging of genome segments from co-infecting parental viruses into new virions, rapidly generating diverse progeny. This process played a pivotal role in historical pandemics; the 1918 H1N1 "" likely arose from reassortment between and strains, combining avian-like genes with human-adapted surface proteins. Similarly, the 2009 H1N1 pandemic virus emerged via triple reassortment in , incorporating segments from North American avian, H3N2, and classical lineages, which enhanced transmissibility and led to global spread. Reassortment's efficiency stems from the multipartite nature of segmented genomes—eight segments in influenza A—allowing up to 2^8 possible combinations per co-infection, though not all are viable. This mechanism underscores the zoonotic potential of segmented viruses, as interspecies transmission facilitates segment mixing. Reverse genetics systems enable laboratory manipulation of viral genomes to dissect the functional consequences of , allowing the synthesis of infectious viruses from cDNA plasmids. These approaches have been instrumental in creating chimeric viruses that incorporate segments from different strains, such as A/B reassortants to study host range determinants. For segmented viruses, plasmid-based facilitates targeted segment swaps, replicating natural reassortment to test potential, as demonstrated in reconstructions of the 1918 virus. In non-segmented viruses like coronaviruses, full-length cDNA clones permit precise insertions or mutations, revealing how specific changes enhance replication or immune evasion. Such engineered chimeras have advanced development and antiviral screening, confirming the adaptive roles of natural variation mechanisms. The quasispecies concept describes viral populations as dynamic swarms of closely related rather than uniform clones, arising from high mutation rates during replication. Introduced by in 1971, it posits that error-prone replication generates a centered around a master sequence, with collective fitness determined by cooperative interactions among variants. In viruses like or , quasispecies diversity enables rapid adaptation to immune pressures, as subpopulations with advantageous mutations expand under selection. This framework explains phenomena such as treatment escape, where the mutant cloud harbors pre-existing resistant variants, and highlights the limitations of targeting single genotypes in antiviral strategies.

Phylogenetic Methods

Phylogenetic methods in virology utilize genetic to reconstruct evolutionary histories of viruses, enabling inferences about , transmission, and adaptation. These approaches begin with , where homologous regions of viral genomes are aligned to identify similarities and differences, often using algorithms like ClustalW or MUSCLE integrated into software suites. From aligned sequences, phylogenetic trees are constructed using statistical models that account for evolutionary processes such as substitution rates and branch lengths. Maximum likelihood (ML) methods, implemented in tools like MEGA, estimate tree topologies by maximizing the probability of observing the under a given evolutionary model, providing robust assessments of support via bootstrap resampling. , as in BEAST software, incorporates prior probabilities and uses (MCMC) sampling to generate posterior distributions of trees, allowing integration of temporal for more nuanced evolutionary reconstructions. The hypothesis underpins time-calibrated phylogenies, assuming a relatively constant rate of to estimate divergence times from sequence differences. In virology, relaxed clock models in BEAST accommodate rate variations across lineages, crucial for rapidly evolving viruses. For instance, analysis of HIV-1 env sequences has dated the origin of the global to around the 1920s in , aligning with historical zoonotic spillover events from simian immunodeficiency viruses. Such estimates rely on calibrating clocks with known outbreak dates or records, though viral clocks often exhibit rate heterogeneity due to selection pressures. Phylogeography extends by incorporating spatiotemporal data, mapping viral spread across geographic regions to trace migration patterns and introduction events. Platforms like Nextstrain employ real-time Bayesian phylodynamics to visualize evolution, revealing multiple introductions into and subsequent global dissemination from lineages like B.1.1.7. These analyses use discrete trait models to infer location shifts along tree branches, aiding in identifying superspreader events and informing responses. Despite their power, viral phylogenetics faces challenges from recombination, which shuffles genetic material between strains and creates mosaic genomes that confound tree-like evolutionary assumptions, requiring detection tools like RDP4 to identify breakpoints. Multiple saturation, where sites accumulate so many substitutions that ancestral signals are lost, further complicates deep-time inferences, particularly in high-mutation-rate viruses like , necessitating site-specific rate models to filter . In applications, phylogenetic methods have been pivotal for outbreak investigations, such as the 2014-2016 West African , where whole-genome sequencing and ML phylogenies traced the outbreak to a single introduction from around 2004, with subsequent diversification in . This analysis, combining 99 genomes, highlighted multiple independent transmissions and informed efforts. Recent advances as of 2025 have integrated structural data into , enabling the analysis of protein evolution alongside genomic sequences. Structural phylogenetics uses tools like to predict and compare viral protein structures across species, unraveling diversification patterns in protein families with ancient origins. Additionally, databases such as Viro3D provide comprehensive resources for protein structures, facilitating studies of evolutionary relationships and accelerating molecular virology .

Structure Determination

Purification Techniques

Purification techniques in virology are essential for isolating intact viral particles or their components from complex biological samples, enabling downstream analyses such as structural studies and assays. These methods rely on physical and chemical properties like size, density, and surface charge to separate viruses from host cell debris, proteins, and nucleic acids. Common approaches include , , , and , often used in combination to achieve high purity and yield. Centrifugation is a foundational technique for purification, exploiting differences in rates under . at low speeds (e.g., 1,000–10,000 × g) initially removes large cellular and aggregates, while higher-speed pelleting (up to 100,000 × g) concentrates viral particles. gradient centrifugation, using media like or cesium chloride (CsCl), further refines separation by isopycnic banding, where viruses equilibrate at their buoyant , typically 1.1–1.5 g/cm³, with enveloped viruses lower (1.1–1.2 g/cm³) due to content and non-enveloped higher (1.3–1.4 g/cm³). gradients (10–60% w/v) are gentle and widely used for fragile viruses, while CsCl gradients provide sharper resolution but may disrupt some enveloped particles due to their hyperosmotic nature. Filtration methods, particularly , concentrate viruses from large fluid volumes by retaining viruses while allowing water and small solutes to pass through membranes with appropriate (MWCO, typically 10–100 kDa) or pore sizes (0.001–0.02 µm). Larger pore filters (0.2–0.45 µm) are used for initial clarification to remove cellular and aggregates, permitting viruses to pass while retaining larger contaminants. Tangential flow filtration variants enhance efficiency by reducing , making it suitable for processing supernatants or environmental samples. This technique is particularly valuable for initial enrichment before more selective methods. Precipitation using (PEG) is a scalable, cost-effective approach for large-scale isolation, especially bacteriophages. PEG (typically 6–10% w/v) with salts like NaCl reduces by dehydrating the particles, causing selective at 4°C overnight, followed by low-speed to pellet the viruses. This method yields high recoveries (up to 90%) for phages and some animal viruses, though it may co-precipitate impurities requiring subsequent polishing steps. Chromatographic techniques provide high-resolution purification based on molecular interactions. Size-exclusion chromatography separates viruses by hydrodynamic volume, eluting larger particles first through porous matrices like , often used post- to remove aggregates. Affinity chromatography employs specific ligands, such as antibodies or for enveloped viruses, to bind and elute target particles under controlled conditions, achieving purities exceeding 95% for recombinant viruses like A. These methods are orthogonal to centrifugation and essential for therapeutic-grade preparations. Purity and yield of virus preparations are assessed spectrophotometrically using the at 260 nm (nucleic acids) to 280 nm (proteins). For purified viral nucleic acids, near 1.8 indicate high purity, while for intact virions like adenoviruses, of 1.2–1.4 are typical, reflecting the nucleic acid-to-protein . Lower suggest protein impurities, while higher values may indicate free nucleic acids; yields are quantified by total protein or particle counts relative to input. These techniques prepare samples for applications like electron visualization.

Sequencing and Analysis

Sequencing and analysis of viral s and structural proteins typically follow purification techniques, which provide the high-quality nucleic acids and protein samples necessary for accurate data generation. The foundational method for viral genome sequencing was chain-termination sequencing, developed by , which enabled the determination of the first complete viral DNA genome: that of φX174, a 5,375-nucleotide single-stranded , in 1977. This approach relied on extension with dideoxynucleotides to generate fragments of varying lengths, separated by to read the sequence. Sanger sequencing became widely adopted for small viral genomes due to its accuracy and simplicity, though it was labor-intensive for larger ones. Next-generation sequencing (NGS) technologies, such as Illumina platforms, revolutionized viral sequencing by enabling high-throughput analysis, particularly for metagenomics where diverse viral populations are present in environmental or clinical samples. Illumina sequencing generates millions of short reads (typically 10^6 or more paired-end reads of 150 bp) from amplified DNA libraries, allowing simultaneous sequencing of multiple samples and detection of low-abundance viruses. For example, shotgun metagenomic NGS on Illumina has identified novel RNA and DNA viruses in human microbiomes by sequencing unbiased nucleic acid extracts. Following sequencing, viral genome assembly reconstructs the full from short reads using computational algorithms. De novo assembly, suitable for novel viruses without a reference, employs overlap-layout-consensus methods like SPAdes or metaSPAdes to generate contigs from raw reads, often challenged by viral genome heterogeneity and host . In contrast, reference-based assembly maps reads to a known viral genome using tools like BWA or Bowtie, facilitating variant detection in well-characterized viruses such as or . Hybrid approaches combining both methods improve completeness for complex . Analysis of structural proteins, such as components, often begins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis () to separate and visualize proteins by molecular weight after purification. reveals the purity and of viral proteins, like the VP1, VP2, and VP3 proteins in adeno-associated viruses (AAVs), where VP3 typically predominates. For precise identification, (MS) techniques, including liquid chromatography-tandem MS (LC-MS/MS), digest proteins into peptides and match them against databases, confirming post-translational modifications and sequence variants in viral envelopes or s. Functional annotation of assembled viral genomes identifies open reading frames (ORFs) using tools like Prokka or GeneMark, predicting protein-coding regions based on start/stop codons and ribosomal binding sites. Conserved motifs are then annotated via homology searches against databases like or ; for instance, (RdRp) domains in positive-sense RNA viruses feature seven catalytic motifs (A–G) in the palm and fingers subdomains, essential for nucleotide polymerization and conserved across families like Picornaviridae. These annotations provide insights into replication machinery without evolutionary inference. Recent advances in long-read sequencing, such as (PacBio) single-molecule real-time (SMRT) technology, address limitations of short-read methods by producing continuous reads up to 20 kb, enabling complete assembly of viral genomes in one contig. For , PacBio HiFi sequencing has generated highly accurate full-length genomes (∼30 kb) with >99.9% consensus accuracy, resolving structural variants and insertions missed by short reads during the 2020–2023 pandemic surveillance. This approach has become standard for complex polyploid viruses and metagenomic discovery.

Structural Imaging and Modeling

Following purification and molecular analysis, structural determination of viruses often employs imaging and computational techniques to resolve three-dimensional architectures at near-atomic resolution. Cryo-electron microscopy (cryo-EM) is a cornerstone method, flash-freezing virion samples in vitreous ice to preserve native states, then imaging with electron beams to reconstruct 3D models from thousands of 2D projections. As of 2025, cryo-EM has elucidated structures of diverse viruses, including enveloped coronaviruses and non-enveloped picornaviruses, revealing receptor-binding sites and assembly pathways. X-ray crystallography complements cryo-EM by diffracting X-rays off crystallized viral components, such as proteins or glycoproteins, to determine atomic coordinates. This technique has been pivotal for small viruses like but requires high-quality crystals, limiting its use for flexible enveloped structures. Recent computational advances, including AI-driven tools like , predict viral protein folds from sequences, accelerating structure determination without experimental crystallization. Databases such as Viro3D (launched in 2025) compile over 85,000 modeled structures from 4,400 viruses, aiding and evolutionary studies. These methods integrate with purification and sequencing to provide comprehensive insights into viral architecture.

Detection and Diagnosis

Microscopy and Culture Methods

(TEM) remains a cornerstone for direct visualization of viral particles, offering nanometer-scale resolution that enables detailed morphological analysis of viruses too small for light . The first electron micrographs of a virus were captured in 1939 by Helmut Ruska and colleagues, who imaged the using an early commercial TEM, marking a pivotal advancement in virology by confirming the submicroscopic nature of viruses. In TEM, viral samples are typically examined at magnifications up to 100,000× or higher to reveal symmetry, envelope structures, and overall particle dimensions, providing essential diagnostic and structural insights. Negative staining enhances contrast in TEM by surrounding unstained viral particles with electron-dense heavy metal salts, such as phosphotungstate, which outline the virion's shape without penetrating it. This technique is particularly useful for rapid identification of virus families based on morphology, as seen in clinical samples where it allows undirected scanning for diverse pathogens. For higher-resolution three-dimensional structural determination, cryo-electron microscopy (cryo-EM) vitrifies samples in amorphous ice to preserve native states, enabling atomic-level imaging of viral complexes; this method's development earned Jacques Dubochet, , and Richard Henderson the 2017 . Virus culture methods propagate infectious particles in host systems to study replication and pathogenicity. Cell lines, such as Vero cells derived from African green monkey kidney, support growth of a broad range of viruses including herpesviruses, paramyxoviruses, and flaviviruses due to their permissiveness and ease of maintenance. For influenza viruses, embryonated chicken eggs provide a traditional in vivo-like environment, with the chorioallantoic membrane serving as a site for viral propagation since its establishment in the 1930s. In the 2020s, cultures—three-dimensional, stem cell-derived models mimicking organ architecture—have emerged for more physiologically relevant virus studies, such as infection in intestinal or brain organoids. Plaque assays quantify infectious by exploiting cytopathic effects (CPE), where viruses lyse host cells in a , forming visible clear zones or plaques under an overlay. Each plaque typically arises from a single infectious particle, allowing calculation of plaque-forming units (PFU) per milliliter to measure , a standard for viruses like coronaviruses that induce distinct CPE. Despite their utility, and methods face significant limitations. The vast majority—estimated at over 99%—of viruses in environmental viromes remain non-culturable in standard systems, hindering comprehensive study of microbial diversity. High-risk pathogens like virus require 4 (BSL-4) containment for due to transmission risks and lack of or treatments, restricting access to specialized facilities. Purification techniques, such as ultracentrifugation, often precede these methods to isolate viruses from complex samples for clearer imaging.

Molecular and Serological Assays

Molecular assays in virology enable the direct detection of viral nucleic acids, offering high specificity and sensitivity for identifying pathogens in clinical and research samples without requiring virus cultivation. These methods, particularly (PCR) variants, have become standard for rapid diagnosis, as they amplify target genetic material to detectable levels. For viruses, reverse transcription PCR (RT-PCR) first converts to using , followed by PCR amplification; this approach was pivotal in diagnostics, where RT-PCR targeted genes like the N and E regions, achieving detection limits as low as 10 copies per microliter in nasopharyngeal swabs. Quantitative PCR (qPCR), often combined with reverse transcription as RT-qPCR, incorporates fluorescent probes or dyes to monitor amplification in real-time, allowing not only detection but also relative quantification of through cycle threshold (Ct) values, with typical sensitivities exceeding 95% for viruses like and coronaviruses when Ct values below 33 indicate active . These assays minimize false negatives by using multiple primer sets, though contamination can lead to false positives, necessitating strict laboratory controls. Alternative nucleic acid-based methods address limitations of thermal cycling in PCR, such as the need for specialized equipment. (LAMP), developed in 2000, uses a set of four to six primers and a DNA polymerase with strand displacement activity to amplify DNA isothermally at 60-65°C, completing in 30-60 minutes and detecting as few as 100 copies of viral after reverse transcription (RT-LAMP). In virology, RT-LAMP has been applied to detect RNA viruses like Zika and with sensitivities of 95-99% and specificities near 100%, offering point-of-care potential due to its simplicity and low cost, though it risks non-specific amplification leading to false positives if primers cross-react with host sequences. Next-generation sequencing (NGS), while more resource-intensive, facilitates variant detection during outbreaks by sequencing amplicons or whole viral genomes, identifying mutations in viruses like with high resolution and minimal cross-reactivity when bioinformatics filters are applied. Overall, molecular assays like PCR and LAMP provide detection thresholds of 10-100 copies per milliliter, surpassing traditional methods in speed and precision for early diagnosis. CRISPR-Cas-based diagnostics, such as SHERLOCK (using Cas13) and DETECTR (using Cas12), represent a major advancement as of 2025 for rapid, isothermal detection of viral nucleic acids at point-of-care settings. These methods leverage CRISPR-associated enzymes to cleave reporter molecules upon target recognition, producing detectable signals (e.g., or lateral flow readout) without thermal cycling. They achieve sensitivities comparable to PCR (down to 10-100 copies per reaction) and specificities >95-99%, with applications in detecting , Zika, , and other viruses in resource-limited environments; for instance, SHERLOCK detected variants with 95% sensitivity in clinical samples during outbreaks. While promising for field use, challenges include potential off-target effects and the need for optimized guide RNAs. Serological assays complement molecular methods by detecting host immune responses, such as antibodies, which indicate past or ongoing after viral clearance. Enzyme-linked immunosorbent assay () captures virus-specific immunoglobulins like IgM (early response) and IgG (long-term immunity) using immobilized viral antigens, with sensitivities up to 88% for IgG detection 21-27 days post- and specificities exceeding 99% when targeting proteins like the spike. These assays are widely used in virology for seroprevalence studies, such as tracking or dengue exposure, but false positives can arise from with related viruses, like seasonal coronaviruses. Neutralization assays evaluate functional immunity by measuring antibodies that inhibit viral entry into cells, often using pseudoviruses or live virus plaque reduction; they correlate strongly with results (sensitivities 95%, specificities 100%) and are essential for assessing efficacy against viruses like , though they require biosafety level 3 facilities. Antigen detection tests provide rapid, point-of-care alternatives by identifying viral proteins directly. Lateral flow assays, akin to tests, employ antibody-coated strips to capture in samples like , yielding results in 15-30 minutes; for , these detect nucleocapsid protein with sensitivities of 78-90% in high-viral-load samples (Ct <25) and specificities of 92-100%, though performance drops in low-prevalence settings due to higher false negative rates from lower analytical sensitivity compared to PCR. Examples include influenza and mpox antigen strips, which prioritize speed over quantification, with cross-reactivity minimized by monoclonal antibodies but still possible with closely related strains. These tests are particularly valuable in resource-limited settings, confirming presence via simple visualization, and can be verified by culture if needed.

Quantification

Infectivity Assays

Infectivity assays measure the number of functional, infectious virus particles capable of initiating replication in host cells or organisms, providing essential data for evaluating viral stocks, vaccine efficacy, and antiviral treatments. These methods distinguish viable virions from non-infectious particles or genomic material, focusing on biological activity rather than mere presence. Common assays rely on cell culture or animal systems to quantify infectivity endpoints, such as the dose required to infect 50% of test subjects. Plaque assays, first developed by Renato Dulbecco in 1952 for animal viruses, quantify infectious particles by counting visible plaques—clear zones of cell lysis—formed when a single virion infects and spreads in a monolayer of susceptible cells overlaid with a semi-solid medium like . The virus titer is expressed as plaque-forming units (PFU) per milliliter, calculated by dividing the number of plaques by the dilution factor and inoculum volume; for cytopathic viruses like or vesicular stomatitis virus, this yields direct counts of infectious units. For viruses that do not cause overt cytopathic effects, the tissue culture infectious dose 50% (TCID50) assay uses serial dilutions of virus inoculated into multi-well cell cultures, with infectivity scored by microscopic observation of cytopathic effects or other indicators after incubation. The TCID50 value, representing the dilution at which 50% of wells show infection, is determined via statistical methods like Reed-Muench interpolation, which pools data across dilutions to estimate the endpoint without assuming a normal distribution. This method, originally described in 1938, is widely used for titering viruses such as influenza or coronaviruses and can be adapted to 96-well formats for higher throughput. Focus-forming unit (FFU) assays extend plaque-like quantification to non-cytopathic viruses by infected cell foci—clusters of antigen-expressing cells—after fixation, allowing enumeration under a without relying on . Developed as a variant for viruses like or dengue, FFU titers are reported similarly to PFU and offer higher sensitivity for low-titer samples by using specific antibodies to visualize infection foci as early as 2-3 days post-inoculation. Animal models assess infectivity through the median lethal dose (LD50), the amount of virus required to kill 50% of a test population, providing insights into pathogenesis and virulence in vivo. For influenza A viruses, intranasal challenge of mice with serial dilutions determines LD50 by monitoring mortality over 14 days, often yielding values around 102-104 PFU for highly pathogenic strains like H5N1, which informs vaccine dosing and antiviral testing. Reporter viruses incorporate genes encoding fluorescent proteins, such as (GFP), to enable rapid, non-destructive quantification of infectivity via or , where each infectious particle produces a detectable signal in transduced cells. This approach, pioneered for HIV-1 in 2001, allows real-time tracking of replication-competent virions in high-throughput formats, with titers expressed as infectious units per milliliter based on the proportion of fluorescent cells. Standardization of infectivity assays follows (WHO) guidelines to ensure vaccine potency, requiring minimum titers at release and end-of-shelf-life. For live attenuated , each dose must contain at least 106.5 fluorescent focus units (FFU) of each as of 2025, verified in eggs or cell cultures, while require not less than 1000 mouse LD50 (equivalent to ~3-4 log10 PFU) per 0.5 mL dose to guarantee and safety.

Viral Load Measurements

Viral load measurements quantify the amount of viral genetic material in a patient's sample, typically using nucleic acid amplification techniques to monitor infection progression, treatment efficacy, and disease management in virology. These assays detect and enumerate viral RNA or DNA copies, providing critical data for clinical decision-making across various viral infections, such as HIV and hepatitis C virus (HCV). Unlike infectivity assays, viral load tests measure total genetic material without assessing particle viability. Quantitative (qPCR), also known as real-time PCR, is the most widely used method for quantification. In qPCR, the cycle threshold (Ct) value represents the number of amplification cycles required for the fluorescent signal to exceed background levels, serving as a semi-quantitative proxy for ; lower Ct values indicate higher initial viral concentrations, as each cycle roughly doubles the target sequence. For instance, Ct values below 25 are often associated with high s in infections like , while values above 30 suggest low loads. qPCR relies on standard curves generated from known viral copy concentrations to convert Ct values to absolute quantities, enabling precise monitoring but requiring calibration for accuracy across assays. Droplet digital PCR (ddPCR) offers an alternative for absolute quantification of viral loads without the need for standard curves, partitioning the sample into thousands of droplets for parallel PCR reactions and counting positive droplets via Poisson statistics. This method provides direct copy number estimates per microliter, improving precision in low-load scenarios and reducing variability from amplification efficiencies. ddPCR has been particularly valuable for viruses like and , where it detects subtle changes in viral genomes that qPCR might overlook due to its reliance on relative thresholds. Viral loads are typically reported in units of copies per milliliter (copies/mL) for blood plasma or serum, reflecting the concentration of detectable nucleic acids. In untreated infections, viral loads often exceed 10^5 copies/mL, correlating with rapid disease progression and high transmission risk. For HCV, clinical thresholds define treatment success; sustained virologic response (SVR), indicating cure, is achieved when viral load becomes undetectable 12 weeks post-therapy, with typical assay limits of detection around 15-50 international units per milliliter (IU/mL). A key limitation of PCR-based viral load assays is their inability to differentiate between infectious virions and defective or non-infectious particles, as they amplify any intact genetic material, including remnants from cleared infections. This can lead to overestimation of active viral burden, necessitating complementary tests for viability in certain contexts.

Pathogenesis

Host-Virus Interactions

Host-virus interactions represent the dynamic molecular interface where viruses engage with host cellular machinery to facilitate infection, replication, and persistence, while hosts deploy innate defenses to detect and counter viral threats. At the cellular level, these interactions encompass recognition of viral components by host pattern recognition receptors (PRRs), viral strategies to subvert host signaling pathways, modulation of programmed cell death, establishment of latent states, and ongoing evolutionary pressures that shape receptor utilization. These processes highlight an intricate balance between viral exploitation and host resistance, often determining the outcome of infection. Pattern recognition by host PRRs initiates the antiviral response, with Toll-like receptors (TLRs) playing a central role in detecting viral nucleic acids. For instance, endosomal TLR3 recognizes double-stranded RNA produced during , while TLR7 and TLR8 detect single-stranded viral RNA, leading to activation of signaling cascades that culminate in type I (IFN) production. This IFN response induces an antiviral state in infected and neighboring cells by upregulating interferon-stimulated genes (ISGs) that inhibit . Cytosolic PRRs such as RIG-I and further complement TLRs by sensing viral RNA in the cytoplasm, amplifying the signaling through and pathways. Viruses have evolved sophisticated mechanisms to modulate host responses, thereby evading innate immunity. In , the accessory protein ORF6 inhibits IFN signaling by interacting with the nuclear pore complex components Nup98 and Rae1, blocking the nuclear import of and STAT2 transcription factors essential for IFN-stimulated . Similarly, other viral proteins, such as NS1, sequester double-stranded to prevent PRR activation, while ICP0 disrupts signaling. These inhibitors allow viruses to dampen the early antiviral state, promoting efficient replication before adaptive immunity engages. Viruses differentially regulate host cell apoptosis to optimize their lifecycle, with some promoting it to facilitate spread and others inhibiting it for persistence. HIV-1 induces pro-apoptotic effects in infected CD4+ T cells through its Vpr protein, which activates caspase-3/7 and mitochondrial pathways, contributing to T cell depletion. In contrast, adenoviruses employ anti-apoptotic proteins like E1B-19K, a Bcl-2 homolog that binds and inhibits pro-apoptotic Bax and Bak, preventing cytochrome c release and caspase activation to sustain the infected cell for progeny production. The E3 region proteins, including 14.7K and RID complex, further block death receptor-mediated apoptosis by internalizing Fas and TRAIL receptors. This strategic control of apoptosis underscores viral adaptation to host cell fate decisions. Latency enables certain viruses to evade immune detection and persist long-term, often through episomal maintenance of the viral genome. Herpesviruses, such as Epstein-Barr virus (EBV) and (HSV), establish latency by circularizing their DNA into that remain extrachromosomal in the host nucleus, avoiding integration to prevent host genome disruption. Proteins like EBV's EBNA1 bind to viral origins of replication (oriP) to ensure episome segregation during host , while latency-associated transcripts in HSV suppress lytic . This episomal state allows periodic reactivation triggered by stress signals, balancing persistence with transmission. Co-evolution between viruses and hosts manifests as an that influences receptor usage for viral entry and host defense. Viruses select for host receptors that provide efficient attachment, but hosts counter by evolving polymorphisms in these receptors to reduce susceptibility, as seen in the CCR5 delta32 mutation conferring resistance. Positive selection pressures on viral receptor genes across mammals indicate recurrent adaptation to host countermeasures, with viruses in turn developing variants to exploit new receptors. This reciprocal evolution drives diversity in receptor-ligand interactions, shaping viral and host specificity over time.

Disease Mechanisms

Viral infections lead to clinical manifestations through diverse mechanisms that disrupt host cellular functions, trigger immune responses, or persist over time, resulting in acute symptoms, chronic conditions, or population-level outbreaks. These processes range from direct damage to infected cells to indirect effects mediated by the , often culminating in tissue pathology and systemic illness. Understanding these pathways is crucial for elucidating disease progression in virology. Direct occurs when viruses replicate within host cells, causing structural damage and eventual , which contributes to tissue dysfunction and acute symptoms. For instance, induces cell lysis by hijacking cellular machinery to form replication complexes on membranous structures, leading to membrane rearrangement and release of progeny virions that destroy motor neurons, resulting in . Similarly, human papillomavirus (HPV) oncogenes, particularly E6 and E7, drive cellular transformation by inactivating tumor suppressors like and Rb, promoting uncontrolled proliferation and progression to malignancies such as . These mechanisms highlight how viral proteins can directly alter host cell fate without immune involvement. Immune-mediated damage arises when the host's response to viral infection exacerbates pathology, often through excessive or misguided . storms, characterized by hypersecretion of proinflammatory cytokines like IL-6 and TNF-α, were a key factor in the lethality of the 1918 pandemic, where the triggered massive immune activation in young adults, leading to lung edema and . Post-viral , such as in Guillain-Barré syndrome (GBS), involves molecular mimicry where immune responses to viral antigens cross-react with peripheral nerve components, causing demyelination and acute flaccid paralysis following infections like or . These processes underscore the dual role of immunity in viral clearance versus amplification of disease. Chronic viral effects stem from persistent or latent infections that evade clearance, leading to long-term organ damage. Hepatitis B virus (HBV) persistence is primarily due to the maintenance of covalently closed circular DNA (cccDNA) in hepatocytes, sustaining low-level replication and chronic inflammation that progresses to fibrosis and cirrhosis over decades. Viral DNA integration into the host genome can also occur, increasing hepatocellular carcinoma risk. In contrast, varicella-zoster virus (VZV) establishes latency in sensory ganglia neurons after primary chickenpox infection, with reactivation—often triggered by waning immunity—causing herpes zoster (shingles) through viral replication in dermatomes, resulting in painful vesicular rash and potential postherpetic neuralgia. These examples illustrate how viruses exploit host tolerance for lifelong carriage and recurrent pathology. Zoonotic spillovers represent a critical mechanism for emerging viral diseases, where viruses cross species barriers from animal reservoirs to humans, initiating epidemics. HIV originated from multiple cross-species transmissions of simian immunodeficiency virus (SIV) from chimpanzees to humans in early 20th-century , likely via hunting, leading to the global AIDS through adaptation and human-to-human spread. Similarly, , the cause of , spilled over from bats—natural reservoirs of sarbecoviruses—possibly via an intermediate host at a wildlife market in , , in late 2019, resulting in a with over 700 million cases worldwide. Such events emphasize the role of ecological interfaces in viral emergence. Emerging threats in viral disease mechanisms include antiviral resistance and climate-driven spread, which amplify outbreak potential. Resistance arises through viral mutations under drug selective pressure, such as in SARS-CoV-2 where variants evade protease inhibitors by altering target proteins, reducing treatment efficacy and prolonging transmission as observed in 2024-2025 surveillance data. Climate change projections indicate accelerated viral spillovers in the coming decades, with warming expanding vector habitats and altering wildlife-human interfaces, potentially increasing the odds of bat-to-mammal viral transmission spillovers more than 400-fold by 2070 in parts of biodiversity hotspots like Southeast Asia. These factors pose ongoing challenges to virological control.

Applications

Vaccines and Antivirals

Vaccines represent a of virology in preventing viral infections by stimulating the host to produce protective antibodies and memory cells without causing . These prophylactic agents target specific viruses and have evolved from early empirical approaches to sophisticated molecular designs, significantly reducing the global burden of diseases like , , and COVID-19. Antivirals, in contrast, provide therapeutic intervention by inhibiting in infected individuals, often used in combination therapies to combat chronic infections such as or acute outbreaks like . Together, vaccines and antivirals exemplify virology's application in , though challenges like viral mutation and delivery barriers persist. Live-attenuated vaccines, which use weakened forms of the virus to mimic natural infection, induce robust and long-lasting immunity. The measles-mumps-rubella (MMR) vaccine, introduced in 1971, exemplifies this approach by conferring lifelong protection against three viruses through a single administration, with efficacy rates exceeding 97% after two doses. Inactivated vaccines, employing killed virus particles, offer safety for immunocompromised individuals but may require boosters for sustained immunity. The Salk polio vaccine, licensed in 1955, prevented paralytic poliomyelitis by inactivating with , dramatically reducing U.S. cases from over 15,000 annually to near zero within years. Advancements in technology include mRNA platforms, which deliver genetic instructions for viral spike proteins to host cells, triggering antibody production without live virus. The Pfizer-BioNTech (BNT162b2), authorized in 2020, demonstrated 95% efficacy against symptomatic infection in phase 3 trials involving over 44,000 participants. vaccines use modified non-replicating viruses to deliver viral genes, eliciting strong cellular and humoral responses. The for , prequalified by WHO in 2019, showed 97.5% efficacy in a ring vaccination trial during the 2018-2020 outbreaks, preventing further spread in contact groups. Antiviral drugs target specific stages of the viral life cycle to halt replication. analogs mimic building blocks of viral or , causing chain termination during synthesis. Acyclovir, approved in 1982, treats (HSV) infections by selectively inhibiting viral after by viral , reducing lesion duration by 1-2 days in clinical studies. inhibitors disrupt the cleavage of viral polyproteins essential for maturation. In treatment, highly active antiretroviral therapy (HAART) incorporating like , introduced in 1995, suppresses viral loads to undetectable levels in over 90% of adherent patients, transforming into a manageable . RNA-dependent RNA polymerase (RdRp) inhibitors block viral genome replication. , an analog, was granted emergency FDA authorization in 2020 for , shortening recovery time by 5 days in hospitalized patients with oxygen needs, as shown in the ACTT-1 trial. Combination regimens, such as those for , enhance efficacy by targeting multiple viral enzymes, minimizing resistance development. Developing effective vaccines and antivirals faces challenges from , including antigenic drift and shift in , necessitating annual vaccine updates by the WHO to match circulating strains based on global surveillance. For mRNA vaccines, lipid nanoparticle delivery addresses RNA instability, but cold-chain requirements complicate global distribution, as evidenced by logistical hurdles during the 2020-2021 rollout. Achieving , where protects unvaccinated individuals by reducing transmission, requires high coverage thresholds; for , with its (R0) of 12-18, 95% population immunity is needed to prevent outbreaks. The global impact of these interventions is profound: was declared eradicated in 1980 through a WHO-led campaign, eliminating over 300 million cases in the alone. remains nearly eradicated, with wild cases dropping 99.9% since 1988 to fewer than 100 annually by 2023, driven by oral and inactivated vaccines in GPEI initiatives.

Therapeutic Uses

Bacteriophage therapy, utilizing lytic bacteriophages to target and destroy bacterial pathogens, represents a specialized therapeutic application of virology. The discovery of bacteriophages is credited to Frederick Twort in 1915, who observed viral agents lysing bacterial cultures, and Félix d'Hérelle in 1917, who further characterized them and proposed their use against bacterial infections. Early applications in the early 20th century targeted dysentery and cholera, but interest waned in Western countries with the rise of antibiotics; however, phage therapy persisted in Eastern Europe and has seen resurgence in the 21st century amid antibiotic resistance. Modern trials in the 2020s have focused on multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), with clinical case reports and phase I/II studies demonstrating efficacy in treating chronic infections like diabetic foot ulcers and ventilator-associated pneumonia. For instance, a 2023 review highlighted successful compassionate use cases where phages reduced MRSA bacterial loads without adverse effects. Oncolytic viruses, engineered to selectively replicate in and lyse cancer cells while sparing healthy tissue, offer another key therapeutic avenue in virology. These viruses often derive from herpesviruses, adenoviruses, or poxviruses modified to express immunostimulatory genes, enhancing anti-tumor immunity. A prominent example is (T-VEC), a genetically modified type 1 approved by the U.S. (FDA) in 2015 for unresectable cutaneous, subcutaneous, and nodal lesions in patients with advanced recurrent after initial . Clinical trials showed T-VEC improved durable response rates compared to granulocyte-macrophage colony-stimulating factor alone, with intralesional injection leading to tumor regression in about 26% of patients. Ongoing research explores combinations with checkpoint inhibitors to broaden applicability to other solid tumors. Adeno-associated viruses (AAVs) serve as critical , delivering functional genes to treat genetic disorders caused by viral or non-viral mutations. AAVs are favored for their low , ability to transduce non-dividing cells, and long-term without integration into the host . The FDA approved (Luxturna) in 2017, an AAV2-based therapy for biallelic mutation-associated retinal , a form of inherited blindness. Administered via subretinal injection, Luxturna restores the , improving and mobility in low-light conditions for treated patients, marking the first FDA-approved for an inherited disease. Subsequent AAV applications target conditions like and hemophilia, with over 100 clinical trials underway by 2025. Phage therapy holds distinct advantages over traditional antibiotics, including high specificity for target , which minimizes disruption to the host , and at sites for self-dosing. Unlike antibiotics, phages do not broadly select for resistance in non-target and can evolve rapidly to counter bacterial resistance mechanisms, reducing the likelihood of widespread resistance buildup. Regulatory frameworks support these applications through compassionate use programs; in the , phages can be prepared magistraly under Article 5 of Directive 2001/83/EC for individualized treatment of life-threatening infections, bypassing full . However, challenges persist in , including purification to remove bacterial endotoxins and scaling production for broader clinical use, which require GMP-compliant facilities and phage banking to ensure rapid matching to patient isolates. These hurdles underscore the need for harmonized international guidelines to facilitate wider adoption.

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