# Miray Bombom : "Methods Used in Virology"

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# Contents :
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1. Culturing wild virus isolates
2. Enumeration of viruses
3. Measuring infectious virus titres and measuring physical virus titres
4. Detecting virus in a sample

4.1.ELISA
4.2.PCR and bDNA assays
4.3.Detection of virus-specific antibodies (serology, applicable only to animal viruses)

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>*What is the first time we come into touch with a virus from a scientific point of view ? How did we found about it ?*

>""***PATHOGENESİS*** ""

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src="https://i.imgur.com/pohRYeK.png
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>The technology that supports viral research extends the whole field of contemporary biology.

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Viruses are too tiny to observed without electron microscopy (EM),which requires concentrations in excess of 10*11 particles per ml, or even more if a virus has a distinguishing shape, as well as costly equipment and a highly competent operator.**That is why some methods are required in virus studies.**

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# Culturing Wild Virus Isolates

* **Growth in vitro of wild virus isolates is unreliable and imposes strong selection on the virus to change.**

* **It is difficult to study a virus that does not grow to high titre in cultured cells or in a convenient whole organism model.**

High concentrations are rarely achieved in a specimen and, even if it is possible to identify a new virus in appropriate samples directly,require that it be cultured in an amenable laboratory system.Such culturing is important in part because the virus will need to be amplified to produce sufficient to study, but also because most of the important knowledge about a virus will come from studying its effects in infected cells or an infected organism.

For viruses of host species other than humans, a certain amount may be achievable using the intact host organism if that is amenable to laboratory work but, even here, there are serious limitations on experimentation and work in cell cultures or organ cultures is much preferable; for viruses of humans, such culture experiments are crucial because experimental infection of humans would not be ethical.

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src="https://i.imgur.com/vFNfuBM.png" width="750" height="500" />

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# Enumaration of Viruses
Perhaps the most important techniques in virology are those that allow the enumeration of viruses. Without accurate quantitation of virus to use in an experiment it is impossible to carry these out under reproducible conditions.

How do we quantify the amount of virus we have in a sample or in a stock that has been grown in a laboratory?
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Almost all methods to measure the amount of virus will actually determine its concentration in the sample.

The virus concentration is often referred to as the virus titre, expressed in virus units per ml of sample. There are two different measures of virus titre that are useful: a physical particle count,and an infectivity.You might imagine that these two numbers would be very similar for any given virus sample, but in fact the ratio of physical to infectious particles is frequently high, with values of between 10 and 100 being common in well-studied animal viruses.

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Why should the physical and infectious titres of a sample differ so dramatically?
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In part, it reflects inefficiencies in assays of infectivity, i.e. not all particles that could potentially score as infectious in the assay actually do so on any single occasion. Reasons for this include random chance, and also the effects of intracellular resistance mechanisms that act against an incoming infectious particle, but it is also the case that great numbers of defective particles are produced in many virus infections.

Such particles normally contain an incomplete or mutated copy of the virus genetic material, such that one or more products essential to infection cannot be successfully encoded. Under conditions where a cell is only infected by a single particle, the inability to make any one of the crucial viral proteins would mean no productive infection would result.

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# Measuring Infectious Virus Titres
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In 1952, Renato Dulbecco (1914–2012) published his landmark paper that established the plaque assay as a means of counting viable animal viruses. Adapting a concept already established for measuring bacteriophage infectivity, he used Western Equine Encephalitis virus to produce plaques in monolayers of chick embryo fibroblasts and showed a linear relationship between virus dilution and measured plaque number.
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The classic method for determining infectious virus titres is the plaque assay, which can be used, with appropriate hosts, to measure bacteriophage, animal virus or plant virus concentrations

To perform the assay for a mammalian virus, a sample is serially diluted and a known volume of each dilution (often termed an inoculum) is then applied to infect confluent monolayers of susceptible cells. At lower dilution factors, i.e. more concentrated virus, many if not all cells in the culture will be infected; such dilutions are not informative.

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An alternative method for determining infectious titre, which again relies on the ability of a virus to cause cpe, is the **T**issue **C**ulture **I**nfectious **D**ose **50%** **(TCID50)** assay.
As before, a virus sample is serially diluted and replicate aliquots of each dilution are applied to susceptible monolayers of cells. Each of the serial dilutions is applied to several equivalent cultures and spread of progeny virus from one part of the monolayer to another is allowed through the use of liquid culture medium.
After a fixed time appropriate to a particular virus/cell combination such that two or three cycles of infection can occur, the cells are fixed and stained and gross cytopathic effect in each cultures is assessed to determine the dilution that gives cpe in 50% of equivalent cultures. At this dilution, the volume used to infect each culture contains, by definition, 1 TCID50 unit of virus.

The titre of the stock, in TCID50 units/ml, can then be calculated. This is an example of an end-point dilution assay.
>A more modern development is the **fluorescent focus assay**. The essential principles are the same as for a plaque assay but, rather than allowing several cycles of infectious spread from the cells initially infected within the assay culture, the cells are fixed during the first cycle (killed in situ) at a time when they would be full of viral proteins if infected. From the fluorescent cell count, a titre can be determined in fluorescent focus units ( ffu) per ml in the same way as for a plaque assay. It is also possible to count the infected fluorescent cells by flow cytometry.
>Importantly, this approach to finding out how much infectious virus is present can be applied to viruses that do not produce cpe in their host cells as well as to cytopathic viruses. The methodology of fluorescent antibody staining is more generally termed immunofluorescence and when applied at higher magnifications it has important applications in the study of virus infections.

*Figure : TCID 50 Test*

*Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection,Stephen L. Wechman,Xiao-Mei Rao et . al,2016*

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# Measuring Physical Virus Titres

<img
src="https://i.imgur.com/91WF5ry.png" width="7500" height="250" />
*Haemagglutination assay. Here an influenza virus is serially diluted from left to right in wells in a plastic plate. Red blood cells (RBCs) are then added to 0.5% v/v and mixed with each dilution of virus. Where there is little or no virus, RBCs settle to a button (from 1/128) indistinguishable from what is seen when no virus is added (row 3). Where sufficient virus is present (up to 1/64 dilution), the RBCs agglutinate and settle in a diffuse pattern.*

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Many viruses have the ability to bind to the surface of red blood cells (RBC) and cross-link them into a clump; in this state the RBCs are said to be agglutinated, hence this virus property is described as haemagglutination (HA). This function is a property of proteins on the surface of the virus particles, which often are also the proteins responsible for attachment to cells during the first steps of an infection; it is for this reason, for example, that the attachment protein of influenza viruses is known as the haemagglutinin. However, HA activity in a virus is not the same as infectivity and, generally, a virus can still agglutinate RBCs when its infectivity is inactivated.

For each virus, HA activity will only be observed with RBCs from specific species. Viruses whose particles have HA activity can be quantified using this property .

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# Detection Virus in a Sample

>Detection of known viruses in clinical or other samples is the area of viral diagnosis. Since direct detection of virus particles is difficult, diagnostic techniques focus on the detection of specific viral components, or inferring the presence of a virus indirectly through the detection of virus-specific antibodies.
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Detection of viral protein: ELISA
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All methods for detecting specific viral proteins in samples rely on the use of specific antibodies. The most widely-used method for viral protein detection is a configuration of ELISA that is otherwise known as a sandwich assay.

Here is a video about the **ELISA** (**E**nzyme **L**inked **I**mmuno **S**orbent **A**ssay) working principle :

{%youtube oyiz2lci4dY%}
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* The amount of compound corresponds to the substrate converted to colored product by the enzyme-linked antibody.
* ELISA has replaced RIA(Radıo Immunoassay)
* Successfully diagnose HIV,rotavirus,syphilis,zika virus
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# Types of ELISA

1. Direct : ELISA reacts with substrate to produce colored product.
2. Indirect : Primary antibody is added.ELISA binds primary antibody reacts with substrate to produce colored product.
3. Sandwich : Epitope of compound binds to the bound antibody.
Primary antibody binds another epitope of compound.[SANDWICH]
Enzyme linked secondary antibody binds primary antibody and coverts substrate to produced colored product.
(https://www.youtube.com/watch?v=JFXVPOym3fs)
1. Competitive
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*Figure : ELISA Types*
*(Suleyman Aydin,A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA,Peptides,2015,)*

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<img
src="https://i.imgur.com/eyaCQlC.png" width="500" hght="250" />

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# Detection of viral nucleic acid: PCR and bDNA Assays
The polymerase chain reaction (PCR), which amplifies a specific DNA segment from a complex mixture of DNA molecules, was devised in 1985 by Kary Mullis. This general technique can be applied to detect viral DNA genomes in a sample among many other uses and it can be extended to detect viral RNA genomes by inclusion of a reverse transcription step that uses a retroviral enzyme (Section 9.3) to convert RNA into DNA.

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<img
src="https://i.imgur.com/07L1anB.png" width="400" ight="500" />
PCR amplification of a target DNA sequence. Thick brown lines: template DNA; red lines: first cycle amplification product; purple lines: second and subsequent cycle product. Turquoise and grey block arrows represent specific left and right primers respectively. Base pairing of these primers to the template DNA defines the PCR amplicon

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>An alternative to PCR for the rapid and quantitative detection of viral nucleic acid sequences is a branched DNA assay

<img
src="https://i.imgur.com/4kaZpyc.png" width="400" ight="500" />

*Branched DNA assay. A generic capture probe (1) is bound to the wells of a microtitre plate. The well is given specificity by binding a capture extender (2). This oligonucleotide has two regions, one able to base pair with the capture probe and the other with specificity for the desired target nucleic acid (3). Bound target is detected by base pairing with a label extender (4), which also has two regions, one specific for a different region of the target and the other that can base pair to the pre-amplifier (5). The pre-amplifier oligo has repeated sequence elements and so can bind multiple copies of the amplifier oligonucleotide that in turn has multiple enzymes coupled to it (6).*

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<img
src="https://i.imgur.com/TOorl2M.png" width="800" ght="500" />

*Virus neutralization test:Virus A loses its infectivity after combining with A-specific antibody (it is neutralized). A-specific antibody does not bind to virus B, so infectivity of virus B is unaffected. The complete test also requires the reciprocal reactions.*

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Here is the video about the qPCR,working mechanism and PCR curve :
{%youtube 1kvy17ugI4w%}

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# Detection of virus-specific antibodies (serology, applicable only to animal viruses)

The presence in a serum sample of antibodies reactive against a virus was traditionally detected in an antibody neutralization test . A test sample of infectious virus was incubated with the serum and the infectivity remaining was then tested on a susceptible cell culture in comparison with controls. Another property of many viruses that can be blocked by antibody and hence used to detect the presence of specific antibody is haemagglutination .

By far the most versatile and widely applied technique for specific antibody detection is the antibody-capture ELISA. This uses exactly the same principles as the assay for viral antigen just described but reconfigured to detect antibodies. Test antigen (either whole virus or protein subunits produced by recombinant DNA techniques) is immobilized on the surface of the wells, and samples that possibly contain antibody are then added in varying dilutions and allowed to bind. After washing away unbound material, any bound Ig is detected using an enzyme-conjugated secondary antibody as before. One problem with using the presence of specific antibody as a test for the presence of a virus is that IgG remains as an aspect of immune memory long after the virus that elicited it has gone from the system. However, an antibody-capture ELISA can be specifically configured to detect only specific IgM molecules. Since IgM is only produced during the active primary response to an antigen, its presence is indicative of a current infection.

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<img
src="https://i.imgur.com/QAekbPN.png" width="350" ght="500" />
*Antibody capture ELISA. Microtitre plate wells are pre-coated with virus or viral antigen (1). Test samples, possibly containing specific antibody for that virus, are added to the wells in serial dilutions (2). After allowing time to bind, unbound Ig is washed away and any bound antibody detected by addition of a secondary antibody (3) to which an enzyme is coupled (4). A positive result is revealed by the enzyme converting its substrate to a coloured product.*

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