Editing Virology (section)

==Quantitation and viral loads==
{{main|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.<ref name="pmid34369836">{{cite journal |vauthors=Lee MJ |title=Quantifying SARS-CoV-2 viral load: current status and future prospects |journal=Expert Review of Molecular Diagnostics |volume=21 |issue=10 |pages=1017–1023 |date=October 2021 |pmid=34369836 |pmc=8425446 |doi=10.1080/14737159.2021.1962709 |url=}}</ref> 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.<ref name =Payne/>

===Infectivity assays===
[[File:Plaque assay macro.jpg|thumb|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.<ref name="pmid29925033">{{cite journal |vauthors=Mistry BA, [[Maria Rita D'Orsogna|D'Orsogna MR]], Chou T |title=The Effects of Statistical Multiplicity of Infection on Virus Quantification and Infectivity Assays |journal=Biophysical Journal |volume=114 |issue=12 |pages=2974–2985 |date=June 2018 |pmid=29925033 |pmc=6026352 |doi=10.1016/j.bpj.2018.05.005 |arxiv=1805.02810 |bibcode=2018BpJ...114.2974M |url=}}</ref> 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.<ref name="pmid15585191">{{cite journal |vauthors=Kashuba C, Hsu C, Krogstad A, Franklin C |title=Small mammal virology |journal=The Veterinary Clinics of North America. Exotic Animal Practice |volume=8 |issue=1 |pages=107–22 |date=January 2005 |pmid=15585191 |pmc=7110861 |doi=10.1016/j.cvex.2004.09.004 |url=}}</ref> 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 value]]s 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 [[Minimal infective dose|ID <sub>50</sub>]].<ref name="pmid21474258">{{cite journal |vauthors=Cutler TD, Wang C, Hoff SJ, Kittawornrat A, Zimmerman JJ |title=Median infectious dose (ID<sub>50</sub>) of porcine reproductive and respiratory syndrome virus isolate MN-184 via aerosol exposure |journal=Veterinary Microbiology |volume=151 |issue=3–4 |pages=229–37 |date=August 2011 |pmid=21474258 |doi=10.1016/j.vetmic.2011.03.003 |url=}}</ref> 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.<ref name="pmid33624268">{{cite journal |vauthors=Moon K, Cho JC |title=Metaviromics coupled with phage-host identification to open the viral "black box" |journal=Journal of Microbiology (Seoul, Korea) |volume=59 |issue=3 |pages=311–323 |date=March 2021 |pmid=33624268 |doi=10.1007/s12275-021-1016-9 |s2cid=232023531 |url=}}</ref>
[[File:Virus Infected Cells.jpg|thumb|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 (TCID<sub>50</sub>) 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/<ref name="pmid28701394">{{cite journal |vauthors=Salgado EN, Upadhyayula S, Harrison SC |title=Single-Particle Detection of Transcription following Rotavirus Entry |journal=Journal of Virology |volume=91 |issue=18 |pages= |date=September 2017 |pmid=28701394 |pmc=5571246 |doi=10.1128/JVI.00651-17 |url=}}</ref>

===Viral load assays===
{{main|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 [[Polymerase chain reaction|PCR]].<ref name="pmid33627730">{{cite journal |vauthors=Yokota I, Hattori T, Shane PY, Konno S, Nagasaka A, Takeyabu K, Fujisawa S, Nishida M, Teshima T |title=Equivalent SARS-CoV-2 viral loads by PCR between nasopharyngeal swab and saliva in symptomatic patients |journal=Scientific Reports |volume=11 |issue=1 |pages=4500 |date=February 2021 |pmid=33627730 |pmc=7904914 |doi=10.1038/s41598-021-84059-2 |bibcode=2021NatSR..11.4500Y |url=}}</ref> Viral load tests are an important in the control of infections by HIV.<ref name="pmid31515967">{{cite journal |vauthors=Nichols BE, Girdwood SJ, Crompton T, Stewart-Isherwood L, Berrie L, Chimhamhiwa D, Moyo C, Kuehnle J, Stevens W, Rosen S |title=Monitoring viral load for the last mile: what will it cost? |journal=Journal of the International AIDS Society |volume=22 |issue=9 |pages=e25337 |date=September 2019 |pmid=31515967 |pmc=6742838 |doi=10.1002/jia2.25337 |url=}}</ref> This versatile method can be used for plant viruses.<ref name="pmid28315718">{{cite journal |vauthors=Shirima RR, Maeda DG, Kanju E, Ceasar G, Tibazarwa FI, Legg JP |title=Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |journal=Journal of Virological Methods |volume=245 |issue= |pages=5–13 |date=July 2017 |pmid=28315718 |pmc=5429390 |doi=10.1016/j.jviromet.2017.03.003 |url=}}</ref><ref name="pmid32765569">{{cite journal |vauthors=Rubio L, Galipienso L, Ferriol I |title=Detection of Plant Viruses and Disease Management: Relevance of Genetic Diversity and Evolution |journal=Frontiers in Plant Science |volume=11 |issue= |pages=1092 |date=2020 |pmid=32765569 |pmc=7380168 |doi=10.3389/fpls.2020.01092 |url=|doi-access=free }}</ref>