Editing Virology (section)

==Molecular biology==
Molecular virology is the study of viruses at the level of nucleic acids and proteins. The methods invented by [[molecular biology|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===
[[File:CsCl density gradient centrifugation.jpg|right
|thumb|Caesium chloride (CsCl) solution and two morphological types of [[rotavirus]]. Following centrifugation at 100&nbsp;g a density gradient forms in the CsCl solution and the virus particles separate according to their densities. The tube is 10&nbsp;cm tall. The viruses are the two "milky" zones close together.<ref name="pmid6290520"/>]]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====
[[Centrifuge]]s 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 [[ultracentrifuge]]s with a top speed of around 100,000&nbsp;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.<ref name="pmid32825599">{{cite journal |vauthors=Zhou Y, McNamara RP, Dittmer DP |title=Purification Methods and the Presence of RNA in Virus Particles and Extracellular Vesicles |journal=Viruses |volume=12 |issue=9 |date=August 2020 |page=917 |pmid=32825599 |pmc=7552034 |doi=10.3390/v12090917 |url=|doi-access=free }}</ref>

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.<ref name="pmid6290520">{{cite journal |vauthors=Beards GM |title=A method for the purification of rotaviruses and adenoviruses from faeces |journal=Journal of Virological Methods |volume=4 |issue=6 |pages=343–52 |date=August 1982 |pmid=6290520 |doi=10.1016/0166-0934(82)90059-3 |url=}}</ref>  Buoyant density centrifugation can also be used to purify the components of viruses such as their nucleic acids or proteins.<ref name="pmid31043560">{{cite journal |vauthors=Su Q, Sena-Esteves M, Gao G |title=Purification of the Recombinant Adenovirus by Cesium Chloride Gradient Centrifugation |journal=Cold Spring Harbor Protocols |volume=2019 |issue=5 |pages= pdb.prot095547|date=May 2019 |pmid=31043560 |doi=10.1101/pdb.prot095547 |s2cid=143423942 |url=}}</ref>

====Electrophoresis====
[[File:Coomassie blue stained gel.png|thumb|right|200px|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 gel]]s. The separated molecules are revealed using stains such as [[Coomassie brilliant blue|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]].<ref name="pmid20127703">{{cite journal |vauthors=Klepárník K, Boček P |title=Electrophoresis today and tomorrow: Helping biologists' dreams come true |journal=BioEssays |volume=32 |issue=3 |pages=218–226 |date=March 2010 |pmid=20127703 |doi=10.1002/bies.200900152 |s2cid=41587013 |url=}}</ref>

===Sequencing of viral genomes===
{{Main|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.<ref name=":0" /> NGS has surpassed traditional Sanger as the most popular approach for generating viral genomes.<ref name=":0">{{cite journal |last1=Castro |first1=Christina |last2=Marine |first2=Rachel |last3=Ramos |first3=Edward |last4=Ng |first4=Terry Fei Fan |title=The effect of variant interference on de novo assembly for viral deep sequencing |journal=BMC Genomics |pages=421 |doi=10.1186/s12864-020-06801-w |date=22 June 2020 |volume=21 |issue=1 |pmid=32571214 |pmc=7306937 |doi-access=free }}</ref> Viral genome sequencing as become a central method in viral [[epidemiology]] and [[virus classification|viral classification]].

===Phylogenetic analysis===
Data from the sequencing of viral genomes can be used to determine evolutionary relationships and this is called [[phylogenetic analysis]].<ref name="pmid30531947">{{cite journal |vauthors=Cui J, Li F, Shi ZL |title=Origin and evolution of pathogenic coronaviruses |journal=Nature Reviews. Microbiology |volume=17 |issue=3 |pages=181–192 |date=March 2019 |pmid=30531947 |pmc=7097006 |doi=10.1038/s41579-018-0118-9 |url=}}</ref> 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]]).<ref>Gorbalenya AE, Lauber C. Phylogeny of Viruses. Reference Module in Biomedical Sciences. 2017;B978-0-12-801238-3.95723-4. doi:10.1016/B978-0-12-801238-3.95723-4</ref>

===Cloning===
{{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.<ref name="pmid32404960">{{cite journal |vauthors=Koch L |title=A platform for RNA virus cloning |journal=Nature Reviews. Genetics |volume=21 |issue=7 |pages=388 |date=July 2020 |pmid=32404960 |pmc=7220607 |doi=10.1038/s41576-020-0246-8 |url=}}</ref> 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.<ref name="pmid32365353">{{cite journal |vauthors=Thi Nhu Thao T, Labroussaa F, Ebert N, V'kovski P, Stalder H, Portmann J, Kelly J, Steiner S, Holwerda M, Kratzel A, Gultom M, Schmied K, Laloli L, Hüsser L, Wider M, Pfaender S, Hirt D, Cippà V, Crespo-Pomar S, Schröder S, Muth D, Niemeyer D, Corman VM, Müller MA, Drosten C, Dijkman R, Jores J, Thiel V |title=Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform |journal=Nature |volume=582 |issue=7813 |pages=561–565 |date=June 2020 |pmid=32365353 |doi=10.1038/s41586-020-2294-9 |bibcode=2020Natur.582..561T |s2cid=213516085 |url=|doi-access=free }}</ref> There are several proven methods for cloning viruses and their components. Small pieces of DNA called [[cloning vector]]s 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.<ref name="pmid31219641">{{cite journal |vauthors=Rosano GL, Morales ES, Ceccarelli EA |title=New tools for recombinant protein production in Escherichia coli: A 5-year update |journal=Protein Science   |volume=28 |issue=8 |pages=1412–1422 |date=August 2019 |pmid=31219641 |pmc=6635841 |doi=10.1002/pro.3668 |url=}}</ref>

===Phage virology===
The viruses that reproduce in bacteria, archaea and fungi are informally called "phages",<ref name="pmid16791793">{{cite journal |vauthors=Pennazio S |title=The origin of phage virology |journal=Rivista di Biologia |volume=99 |issue=1 |pages=103–29 |date=2006 |pmid=16791793 |doi= |url=}}</ref> and the ones that infect bacteria – [[bacteriophages]] – in particular are useful in virology and biology in general.<ref name="pmid29853167">{{cite journal |vauthors=Harada LK, Silva EC, Campos WF, Del Fiol FS, Vila M, Dąbrowska K, Krylov VN, Balcão VM |title=Biotechnological applications of bacteriophages: State of the art |journal=Microbiological Research |volume=212-213 |issue= |pages=38–58 |date=2018 |pmid=29853167 |doi=10.1016/j.micres.2018.04.007 |s2cid=46921731 |url=|doi-access=free |hdl=1822/54758 |hdl-access=free }}</ref> Bacteriophages were some of the first viruses to be discovered, early in the twentieth century,<ref name="pmid31216787">{{cite journal |vauthors=Stone E, Campbell K, Grant I, McAuliffe O |title=Understanding and Exploiting Phage-Host Interactions |journal=Viruses |volume=11 |issue=6 |date=June 2019 |page=567 |pmid=31216787 |pmc=6630733 |doi=10.3390/v11060567 |url=|doi-access=free }}</ref> and because they are relatively easy to grow quickly in laboratories, much of our understanding of viruses originated by studying them.<ref name="pmid31216787"/> 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.<ref name="pmid33504115">{{cite journal |vauthors=Nagano K, Tsutsumi Y |title=Phage Display Technology as a Powerful Platform for Antibody Drug Discovery |journal=Viruses |volume=13 |issue=2 |date=January 2021 |page=178 |pmid=33504115 |pmc=7912188 |doi=10.3390/v13020178 |url=|doi-access=free }}</ref>