History of virology
The history of virology — the scientific study of viruses and the infections they cause – began in the closing years of the 19th century. Although Louis Pasteur and Edward Jenner developed the first vaccines to protect against viral infections, they did not know that viruses existed; the first evidence of the existence of viruses came from experiments with filters that had pores small enough to retain bacteria. In 1892, Dmitry Ivanovsky used one of these filters to show that sap from a diseased tobacco plant remained infectious to healthy tobacco plants despite having been filtered. Martinus Beijerinck called the filtered, infectious substance a "virus" and this discovery is considered to be the beginning of virology; the subsequent discovery and partial characterization of bacteriophages by Frederick Twort and Félix d'Herelle further catalyzed the field, by the early 20th century many viruses had been discovered. In 1926, Thomas Milton Rivers defined viruses as obligate parasites. Viruses were demonstrated to be particles, rather than a fluid, by Wendell Meredith Stanley, the invention of the electron microscope in 1931 allowed their complex structures to be visualised.
Despite his other successes, Louis Pasteur was unable to find a causative agent for rabies and speculated about a pathogen too small to be detected using a microscope. In 1884, the French microbiologist Charles Chamberland invented a filter – known today as the Chamberland filter – that had pores smaller than bacteria. Thus, he could pass a solution containing bacteria through the filter and remove them from the solution. In 1876, Adolf Mayer, who directed the Agricultural Experimental Station in Wageningen was the first to show that what he called "Tobacco Mosaic Disease" was infectious, he thought that it was caused by either a toxin or a small bacterium. In 1892, the Russian biologist Dmitry Ivanovsky used a Chamberland filter to study what is now known as the tobacco mosaic virus, his experiments showed that crushed leaf extracts from infected tobacco plants remain infectious after filtration. Ivanovsky suggested the infection might be caused by a toxin produced by bacteria, but did not pursue the idea.
In 1898, the Dutch microbiologist Martinus Beijerinck, a microbiology teacher at the Agricultural School in Wageningen repeated experiments by Adolf Mayer and became convinced that filtrate contained a new form of infectious agent. He observed that the agent multiplied only in cells that were dividing and he called it a contagium vivum fluidum and re-introduced the word virus. Beijerinck maintained that viruses were liquid in nature, a theory discredited by the American biochemist and virologist Wendell Meredith Stanley, who proved that they were in fact, particles. In the same year Friedrich Loeffler and Paul Frosch passed the first animal virus through a similar filter and discovered the cause of foot-and-mouth disease. In 1881, Carlos Finlay, a Cuban physician, first conducted and published research that indicated that mosquitoes were carrying the cause of yellow fever, a theory proved in 1900 by commission headed by Walter Reed. During 1901 and 1902, William Crawford Gorgas organised the destruction of the mosquitoes' breeding habitats in Cuba, which reduced the prevalence of the disease.
Gorgas organised the elimination of the mosquitoes from Panama, which allowed the Panama Canal to be opened in 1914. The virus was isolated by Max Theiler in 1932 who went on to develop a successful vaccine. By 1928 enough was known about viruses to enable the publication of Filterable Viruses, a collection of essays covering all known viruses edited by Thomas Milton Rivers. Rivers, a survivor of typhoid fever contracted at the age of twelve, went on to have a distinguished career in virology. In 1926, he was invited to speak at a meeting organised by the Society of American Bacteriology where he said for the first time, "Viruses appear to be obligate parasites in the sense that their reproduction is dependent on living cells."From the 1950s to the 1960s, Chester M. Southam, a prominent virologist, injected malignant HeLa cells into cancer patients, healthy individuals, prison inmates from the Ohio Penitentiary in order to observe if cancer could be transmitted, he was examining if one could become immune to cancer by developing an acquired immune response in hopes of creating a vaccine for cancer.
The notion that viruses were particles was not considered unnatural and fitted in nicely with the germ theory. It is assumed that Dr. J. Buist of Edinburgh was the first person to see virus particles in 1886, when he reported seeing "micrococci" in vaccine lymph, though he had observed clumps of vaccinia. In the years that followed, as optical microscopes were improved "inclusion bodies" were seen in many virus-infected cells, but these aggregates of virus particles were still too small to reveal any detailed structure, it was not until the invention of the electron microscope in 1931 by the German engineers Ernst Ruska and Max Knoll, that virus particles bacteriophages, were shown to have complex structures. The sizes of viruses determined using this new microscope fitted in well with those estimated by filtration experiments. Viruses were expected to be small; some were only a little smaller than the smallest known bacteria, the smaller viruses were of similar sizes to complex organic molecules.
In 1935, Wendell Stanley examined the tobacco mosaic virus and found it was made of protein. In 1939, Stanley and Max Lauffer separated the virus into protein and nucleic acid, shown by Stanl
Social history of viruses
The social history of viruses describes the influence of viruses and viral infections on human history. Epidemics caused by viruses began when human behaviour changed during the Neolithic period, around 12,000 years ago, when humans developed more densely populated agricultural communities; this allowed viruses to spread and subsequently to become endemic. Viruses of plants and livestock increased, as humans became dependent on agriculture and farming, diseases such as potyviruses of potatoes and rinderpest of cattle had devastating consequences. Smallpox and measles viruses are among the oldest. Having evolved from viruses that infected other animals, they first appeared in humans in Europe and North Africa thousands of years ago; the viruses were carried to the New World by Europeans during the time of the Spanish Conquests, but the indigenous people had no natural resistance to the viruses and millions of them died during epidemics. Influenza pandemics have been recorded since 1580, they have occurred with increasing frequency in subsequent centuries.
The pandemic of 1918–19, in which 40–50 million died in less than a year, was one of the most devastating in history. Louis Pasteur and Edward Jenner were the first to develop vaccines to protect against viral infections; the nature of viruses remained unknown until the invention of the electron microscope in the 1930s, when the science of virology gained momentum. In the 20th century many diseases both old and new were found to be caused by viruses. There were epidemics of poliomyelitis that were only controlled following the development of a vaccine in the 1950s. HIV is one of the most pathogenic new viruses to have emerged in centuries. Although scientific interest in them arose because of the diseases they cause, most viruses are beneficial, they drive evolution by transferring genes across species, play important roles in ecosystems and are essential to life. Over the past 50,000–100,000 years, as modern humans increased in numbers and dispersed throughout the world, new infectious diseases emerged, including those caused by viruses.
Earlier, humans lived in small, isolated communities, most epidemic diseases did not exist. Smallpox, the most lethal and devastating viral infection in history, first emerged among agricultural communities in India about 11,000 years ago; the virus, which only infected humans descended from the poxviruses of rodents. Humans came into contact with these rodents, some people became infected by the viruses they carried; when viruses cross this so-called "species barrier", their effects can be severe, humans may have had little natural resistance. Contemporary humans lived in small communities, those who succumbed to infection either died or developed immunity; this acquired immunity is only passed down to offspring temporarily, by antibodies in breast milk and other antibodies that cross the placenta from the mother's blood to the unborn child's. Therefore, sporadic outbreaks occurred in each generation. In about 9000 BC, when many people began to settle on the fertile flood plains of the River Nile, the population became dense enough for the virus to maintain a constant presence because of the high concentration of susceptible people.
Other epidemics of viral diseases that depend on large concentrations of people, such as mumps and polio first occurred at this time. The Neolithic age, which began in the Middle East in about 9500 BC, was a time when humans became farmers; this agricultural revolution embraced the development of monoculture and presented an opportunity for the rapid spread of several species of plant viruses. The divergence and spread of sobemoviruses – southern bean mosaic virus – date from this time; the spread of the potyviruses of potatoes, other fruits and vegetables, began about 6,600 years ago. About 10,000 years ago the humans who inhabited the lands around the Mediterranean basin began to domesticate wild animals. Pigs, goats, horses, camels and dogs were all kept and bred in captivity; these animals would have brought their viruses with them. The transmission of viruses from animals to humans can occur, but such zoonotic infections are rare and subsequent human-to-human transmission of animal viruses is rarer, although there are notable exceptions such as influenza.
Most viruses would have posed no threat to humans. The rare epidemics of viral diseases originating in animals would have been short-lived because the viruses were not adapted to humans and the human populations were too small to maintain the chains of infection. Other, more ancient, viruses have been less of a threat. Herpes viruses first infected the ancestors of modern humans over 80 million years ago. Humans have developed a tolerance to these viruses, most are infected with at least one species. Records of these milder virus infections are rare, but it is that early hominids suffered from colds and diarrhoea caused by viruses just as humans do today. More evolved viruses cause epidemics and pandemics – and it is these that history records; the influenza virus is one that seems to have crossed the species barrier from pigs to ducks and water fowl and hence to humans. It is possible that a fatal plague in the Middle East at the time of the late 18th Dynasty was associated with this transmission at Amarna.
Among the earliest records of a viral infection is an Egyptian stele thought to depict an Egyptian priest from the 18th Dynasty with a foot drop deformity characteristic of a poliovirus infection. The mummy of Siptah – a ruler during the 19th Dynasty – shows signs of poliomyelitis, that of Ramesses V and some other Egyptian mummies buried over 3000 years ago show evidence of smallpox. Ther
A giant virus is a large virus, some of which are larger than typical bacteria. They are giant nucleocytoplasmic large DNA viruses that have large genomes compared to other viruses and contain many unique genes not found in other life forms. While the exact criteria as defined in the scientific literature vary, giant viruses are described as viruses having large, pseudo-icosahedral capsids that may be surrounded by a thick layer of filamentous protein fibers; the viruses' large, double-stranded DNA genomes encode a large contingent of genes. While few giant viruses have been characterized in detail, the most notable examples are the phylogenetically related megavirus and mimivirus — belonging to the Megaviridae and Mimiviridae families — due to their having the largest capsid diameters of all known viruses. Viral replication in giant viruses occurs within large circular virus factories located within the cytoplasm of the infected host cell; this is similar to the replication mechanism used by Poxviridae, though whether this mechanism is employed by all giant viruses or only mimivirus and the related mamavirus has yet to be determined.
These virion replication factories are themselves subject to infection by the virophage satellite viruses, which inhibit or impair the reproductive capabilities of the complementary virus. The genomes of giant viruses are the largest known for viruses, contain genes that encode for important elements of translation machinery, a characteristic, believed to be indicative of cellular organisms; these genes include multiple genes encoding a number of aminoacyl tRNA synthetases, enzymes that catalyze the esterification of specific amino acids or their precursors to their corresponding cognate tRNAs to form an aminoacyl tRNA, used during translation. The presence of four aminoacyl tRNA synthetase encoding genes in mimivirus and mamavirus genomes, both species within the Mimiviridae family, as well as the discovery of seven aminoacyl tRNA synthetase genes, including the four genes present in Mimiviridae, in the megavirus genome provide evidence for a possible scenario in which these large DNA viruses evolved from a shared ancestral cellular genome by means of genome reduction.
Their discovery and subsequent characterization has triggered some debate concerning the evolutionary origins of giant viruses. The two main hypotheses for their origin are that either they evolved from small viruses, picking up DNA from host organisms, or that they evolved from complicated organisms into the current form, not self-sufficient for reproduction. What sort of complicated organism giant viruses might have diverged from is a topic of debate. One proposal is that the origin point represents a fourth domain of life, but this is not universally accepted. Table 1 - Largest giant viruses with complete sequenced genomesThe whole list is in the Giant Virus Toplist created by the Giant Virus Finder software. Table 2 - Specific common features among giant viruses1Mutator S and its homologs are a family of DNA mismatch repair proteins involved in the mismatch repair system that acts to correct point mutations or small insertion/deletion loops produced during DNA replication, increasing the fidelity of replication.
2A stargate is a five-pronged star structure present on the viral capsid forming the portal through which the internal core of the particle is delivered to the host's cytoplasm. Nucleocytoplasmic large DNA viruses Pandoravirus Klosneuvirus Cafeteria roenbergensis virus
Plant viruses are viruses that affect plants. Like all other viruses, plant viruses are obligate intracellular parasites that do not have the molecular machinery to replicate without a host. Plant viruses can be pathogenic to higher plants. Most plant viruses are rod-shaped, with protein discs forming a tube surrounding the viral genome, they have an envelope. The great majority have an RNA genome, small and single stranded, but some viruses have double-stranded RNA, ssDNA or dsDNA genomes. Although plant viruses are not as well understood as their animal counterparts, one plant virus has become iconic: tobacco mosaic virus, the first virus to be discovered; this and other viruses cause an estimated U. S$60 billion loss in crop yields worldwide each year. Plant viruses are grouped into 49 families. However, these figures relate only to cultivated plants, which represent only a tiny fraction of the total number of plant species. Viruses in wild plants have been little studied, but the interactions between wild plants and their viruses do not appear to cause disease in the host plants.
To transmit from one plant to another and from one plant cell to another, plant viruses must use strategies that are different from animal viruses. Plants do not move, so plant-to-plant transmission involves vectors. Plant cells are surrounded by solid cell walls, therefore transport through plasmodesmata is the preferred path for virions to move between plant cells. Plants have specialized mechanisms for transporting mRNAs through plasmodesmata, these mechanisms are thought to be used by RNA viruses to spread from one cell to another. Plant defenses against viral infection include, among other measures, the use of siRNA in response to dsRNA. Most plant viruses encode a protein to suppress this response. Plants reduce transport through plasmodesmata in response to injury; the discovery of plant viruses causing disease is accredited to A. Mayer working in the Netherlands demonstrated that the sap of mosaic obtained from tobacco leaves developed mosaic symptom when injected in healthy plants; however the infection of the sap was destroyed.
He thought. However, after larger inoculation with a large number of bacteria, he failed to develop a mosaic symptom. In 1898, Martinus Beijerinck, a Professor of Microbiology at the Technical University the Netherlands, put forth his concepts that viruses were small and determined that the "mosaic disease" remained infectious when passed through a Chamberland filter-candle; this was in contrast to bacteria microorganisms. Beijerinck referred to the infectious filtrate as a "contagium vivum fluidum", thus the coinage of the modern term "virus". After the initial discovery of the ‘viral concept’ there was need to classify any other known viral diseases based on the mode of transmission though microscopic observation proved fruitless. In 1939 Holmes published a classification list of 129 plant viruses; this was expanded and in 1999 there were 977 recognized, some provisional, plant virus species. The purification of TMV was first performed by Wendell Stanley, who published his findings in 1935, although he did not determine that the RNA was the infectious material.
However, he received the Nobel Prize in Chemistry in 1946. In the 1950s a discovery by two labs proved that the purified RNA of the TMV was infectious which reinforced the argument; the RNA carries genetic information to code for the production of new infectious particles. More virus research has been focused on understanding the genetics and molecular biology of plant virus genomes, with a particular interest in determining how the virus can replicate and infect plants. Understanding the virus genetics and protein functions has been used to explore the potential for commercial use by biotechnology companies. In particular, viral-derived sequences have been used to provide an understanding of novel forms of resistance; the recent boom in technology allowing humans to manipulate plant viruses may provide new strategies for production of value-added proteins in plants. Viruses are small and can only be observed under an electron microscope; the structure of a virus is given by its coat of proteins.
Assembly of viral particles takes place spontaneously. Over 50% of known plant viruses are rod-shaped; the length of the particle is dependent on the genome but it is between 300–500 nm with a diameter of 15–20 nm. Protein subunits can be placed around the circumference of a circle to form a disc. In the presence of the viral genome, the discs are stacked a tube is created with room for the nucleic acid genome in the middle; the second most common structure amongst plant viruses are isometric particles. They are 25–50 nm in diameter. In cases when there is only a single coat protein, the basic structure consists of 60 T subunits, where T is an integer; some viruses may have 2 coat proteins. There are three genera of Geminiviridae that consist of particles that are like two isometric particles stuck together. A small number of plant viruses have, in addition to their coat proteins, a lipid envelope; this is derived from the plant cell membrane as the virus particle buds off from the cell. Viruses can be spread by direct transfer of sap by contact of a wounded plant with a healthy one.
Such contact may occur during agricultural practices, as by damage caused by tools or hands, or as by an animal feeding on the plant. TM
Microbiology is the study of microorganisms, those being unicellular, multicellular, or acellular. Microbiology encompasses numerous sub-disciplines including virology, parasitology and bacteriology. Eukaryotic microorganisms possess membrane-bound cell organelles and include fungi and protists, whereas prokaryotic organisms—all of which are microorganisms—are conventionally classified as lacking membrane-bound organelles and include Bacteria and Archaea. Microbiologists traditionally relied on culture and microscopy. However, less than 1% of the microorganisms present in common environments can be cultured in isolation using current means. Microbiologists rely on molecular biology tools such as DNA sequence based identification, for example 16s rRNA gene sequence used for bacteria identification. Viruses have been variably classified as organisms, as they have been considered either as simple microorganisms or complex molecules. Prions, never considered as microorganisms, have been investigated by virologists, however, as the clinical effects traced to them were presumed due to chronic viral infections, virologists took search—discovering "infectious proteins".
The existence of microorganisms was predicted many centuries before they were first observed, for example by the Jains in India and by Marcus Terentius Varro in ancient Rome. The first recorded microscope observation was of the fruiting bodies of moulds, by Robert Hooke in 1666, but the Jesuit priest Athanasius Kircher was the first to see microbes, which he mentioned observing in milk and putrid material in 1658. Antonie van Leeuwenhoek is considered a father of microbiology as he observed and experimented with microscopic organisms in 1676, using simple microscopes of his own design. Scientific microbiology developed in the 19th century through the work of Louis Pasteur and in medical microbiology Robert Koch; the existence of microorganisms was hypothesized for many centuries before their actual discovery. The existence of unseen microbiological life was postulated by Jainism, based on Mahavira’s teachings as early as 6th century BCE. Paul Dundas notes that Mahavira asserted the existence of unseen microbiological creatures living in earth, water and fire.
Jain scriptures describe nigodas which are sub-microscopic creatures living in large clusters and having a short life, said to pervade every part of the universe in tissues of plants and flesh of animals. The Roman Marcus Terentius Varro made references to microbes when he warned against locating a homestead in the vicinity of swamps "because there are bred certain minute creatures which cannot be seen by the eyes, which float in the air and enter the body through the mouth and nose and thereby cause serious diseases."In the golden age of Islamic civilization, Iranian scientists hypothesized the existence of microorganisms, such as Avicenna in his book The Canon of Medicine, Ibn Zuhr who discovered scabies mites, Al-Razi who gave the earliest known description of smallpox in his book The Virtuous Life. In 1546, Girolamo Fracastoro proposed that epidemic diseases were caused by transferable seedlike entities that could transmit infection by direct or indirect contact, or vehicle transmission.
In 1676, Antonie van Leeuwenhoek, who lived most of his life in Delft, observed bacteria and other microorganisms using a single-lens microscope of his own design. He is considered a father of microbiology as he pioneered the use of simple single-lensed microscopes of his own design. While Van Leeuwenhoek is cited as the first to observe microbes, Robert Hooke made his first recorded microscopic observation, of the fruiting bodies of moulds, in 1665, it has, been suggested that a Jesuit priest called Athanasius Kircher was the first to observe microorganisms. Kircher was among the first to design magic lanterns for projection purposes, so he must have been well acquainted with the properties of lenses, he wrote "Concerning the wonderful structure of things in nature, investigated by Microscope" in 1646, stating "who would believe that vinegar and milk abound with an innumerable multitude of worms." He noted that putrid material is full of innumerable creeping animalcules. He published his Scrutinium Pestis in 1658, stating that the disease was caused by microbes, though what he saw was most red or white blood cells rather than the plague agent itself.
The field of bacteriology was founded in the 19th century by Ferdinand Cohn, a botanist whose studies on algae and photosynthetic bacteria led him to describe several bacteria including Bacillus and Beggiatoa. Cohn was the first to formulate a scheme for the taxonomic classification of bacteria, to discover endospores. Louis Pasteur and Robert Koch were contemporaries of Cohn, are considered to be the father of microbiology and medical microbiology, respectively. Pasteur is most famous for his series of experiments designed to disprove the widely held theory of spontaneous generation, thereby solidifying microbiology's identity as a biological science. One of his students, Adrien Certes, is considered the founder of marine microbiology. Pasteur designed methods for food preservation and vaccines against several diseases such as anthrax, fowl cholera and rabies. Koch is best known for his contributions to the germ theory of disease, proving that specific diseases were caused by specific pathogenic microorganisms.
He developed a series of criteria. Koch was one of the first scientists to focus on the i
A pandemic is an epidemic of disease that has spread across a large region. This may include noncommunicable diseases. A widespread endemic disease, stable in terms of how many people are getting sick from it is not a pandemic. Further, flu pandemics exclude recurrences of seasonal flu. Throughout history, there have been a number such as smallpox and tuberculosis. One of the most devastating pandemics was the Black Death, which killed over 75 million people in 1350; the most recent pandemics include the HIV pandemic as well as the 2009 H1N1 pandemics. A pandemic is an epidemic occurring on a scale which crosses international boundaries affecting a large number of people. Pandemics can occur in important agricultural organisms or in other organisms; the World Health Organization has a six-stage classification that describes the process by which a novel influenza virus moves from the first few infections in humans through to a pandemic. This starts with the virus infecting animals, with a few cases where animals infect people moves through the stage where the virus begins to spread directly between people, ends with a pandemic when infections from the new virus have spread worldwide and it will be out of control until we stop it.
A disease or condition is not a pandemic because it is widespread or kills many people. For instance, cancer is responsible for many deaths but is not considered a pandemic because the disease is not infectious or contagious. In a virtual press conference in May 2009 on the influenza pandemic, Dr Keiji Fukuda, Assistant Director-General ad interim for Health Security and Environment, WHO said "An easy way to think about pandemic … is to say: a pandemic is a global outbreak. You might ask yourself:'What is a global outbreak'? Global outbreak means that we see both spread of the agent … and we see disease activities in addition to the spread of the virus."In planning for a possible influenza pandemic, the WHO published a document on pandemic preparedness guidance in 1999, revised in 2005 and in February 2009, defining phases and appropriate actions for each phase in an aide memoir entitled WHO pandemic phase descriptions and main actions by phase. The 2009 revision, including definitions of a pandemic and the phases leading to its declaration, were finalized in February 2009.
The pandemic H1N1 2009 virus mentioned in the document. All versions of this document refer to influenza; the phases are defined by the spread of the disease. HIV originated in Africa, spread to the United States via Haiti between 1966 and 1972. AIDS is a pandemic, with infection rates as high as 25% in southern and eastern Africa. In 2006, the HIV prevalence rate among pregnant women in South Africa was 29.1%. Effective education about safer sexual practices and bloodborne infection precautions training have helped to slow down infection rates in several African countries sponsoring national education programs. Infection rates are rising again in Asia and the Americas; the AIDS death toll in Africa may reach 90–100 million by 2025. There have been a number of significant pandemics recorded in human history zoonoses which came about with the domestication of animals, such as influenza and tuberculosis. There have been a number of significant epidemics that deserve mention above the "mere" destruction of cities: Plague of Athens, 430 BC.
Typhoid fever killed a quarter of the Athenian troops, a quarter of the population over four years. This disease fatally weakened the dominance of Athens, but the sheer virulence of the disease prevented its wider spread; the exact cause of the plague was unknown for many years. In January 2006, researchers from the University of Athens analyzed teeth recovered from a mass grave underneath the city, confirmed the presence of bacteria responsible for typhoid. Antonine Plague, 165–180 AD. Smallpox brought to the Italian peninsula by soldiers returning from the Near East. At the height of a second outbreak, the Plague of Cyprian, which may have been the same disease, 5,000 people a day were said to be dying in Rome. Plague of Justinian, from 541 to 750, was the first recorded outbreak of the bubonic plague, it started in Egypt, reached Constantinople the following spring, killing 10,000 a day at its height, 40% of the city's inhabitants. The plague went on to eliminate a quarter to a half of the human population that it struck throughout the known world.
It caused Europe's population to drop by around 50% between 550 and 700. Black Death, from 1331 to 1353; the total number of deaths worldwide is estimated at 75 million people. Eight hundred years after the last outbreak, the plague returned to Europe. Starting in Asia, the disease reached Mediterranean and western Europe in 1348, killed an estimated 20 to 30 million Europeans in six years, it was the first of a cycle of European plague epidemics. There were more than 100 plague epidemics in Europe in this period; the disease recurred in England every two to five years from 1361 to 1480. By the 1370s
Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection optimization and adaptation of methods to cell culture; this page discusses various techniques used to quantify viruses in liquid samples. These methods are separated into traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are slow and labor-intensive.
Modern methods are new commercially available products and kits that reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here. Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral plaque assays determine the number of plaque forming units in a virus sample, one measure of virus quantity; this assay is based on a microbiological method conducted in multi-well plates. A confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar or carboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed; the virus infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated.
The infected cell area will create a plaque which can be seen visually or with an optical microscope. Plaque formation can take 3–14 days, depending on the virus being analyzed. Plaques are counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume; the pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle. The focus forming assay 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 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 brief incubation period under a semisolid overlay medium that restricts the spread of infectious virus, creating localized clusters of infected cells. Plates are subsequently probed with fluorescently labeled antibodies against a viral antigen, fluorescence microscopy is used to count and quantify the number of foci; the FFA method yields results in less time than plaque or TCID50 assays, but it can be more expensive in terms of required reagents and equipment. Assay completion time is dependent on the size of area that the user is counting. A larger area can provide a more accurate representation of the sample. Results of the FFA are expressed as focus forming units per milliliter, or FFU/mL. 50% Tissue culture Infective Dose is the measure of infectious virus titer. This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells.
This assay may be more common in clinical research applications where the lethal dose of virus must be determined or if the virus does not form plaques. When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death is manually observed and recorded for each virus dilution, results are used to mathematically calculate a TCID50 result. Due to distinct differences in assay methods and principles, TCID50 and pfu/mL or other infectivity assay results are not equivalent; this method can take up to a week due to cell infectivity time. Two methods used to calculate TCID50 are: Spearman-Karber Reed-Muench methodThe theoretical relationship between TCID50 and PFU is 0.69 PFU = 1 TCID50 based on the Poisson distribution, a probability distribution which describes how many random events occurring at a known average rate are to occur in a fixed space. However it must be emphasized that in practice, this relationship may not hold for the same virus + cell combination, as the two types of assay are set up differently and virus infectivity is sensitive to vario