Editing Serial passage

{{short description|Growing bacteria or viruses in iterations}}
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'''Serial passage''' is the process of growing [[bacteria]] or a [[virus]] in iterations.  For instance, a virus may be grown in one environment, and then a portion of that virus population can be removed and put into a new environment.  This process is repeated with as many stages as desired, and then the final product is studied, often in comparison with the original virus.

This sort of facilitated transmission is often conducted in a laboratory setting, because it is of scientific interest to observe how the virus or bacterium that is being passed [[evolution|evolves]] over the course of the experiment. In particular, serial passage can be quite useful in studies that seek to alter the [[virulence]] of a virus or other [[pathogen]]. One consequence of this is that serial passage can be useful in creating [[vaccine]]s, since scientists can apply serial passage and create a strain of a pathogen that has low virulence, yet has comparable [[immunogenicity]] to the original strain.<ref>{{Cite web |title=Serial passage |url=http://www.biology-online.org/dictionary/Serial_passage |url-status=dead |archive-url=https://web.archive.org/web/20160304090140/http://www.biology-online.org/dictionary/Serial_passage |archive-date=4 March 2016 |access-date=16 April 2014 |website=Biology online}}</ref> This can also create strains that are more transmissible in addition to lower virulence, as demonstrated by A/[[H5N1]] passage in ferrets.<ref name="pmid22723413">{{Cite journal |last=Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ |display-authors=etal |year=2012 |title=Airborne transmission of influenza A/H5N1 virus between ferrets. |journal=Science |volume=336 |issue=6088 |pages=1534–41 |bibcode=2012Sci...336.1534H |doi=10.1126/science.1213362 |pmc=4810786 |pmid=22723413}}</ref>

==Mechanism==
Serial passage can either be performed [[in vitro]] or [[in vivo]].  In the in vitro method, a virus or a strain of bacteria will be isolated and allowed to grow for a certain time.  After the sample has grown for that time, part of it will be transferred to a new environment and allowed to grow for the same period.<ref name="pmid21305003">{{Cite journal |vauthors=Chapuis É, Pagès S, Emelianoff V, Givaudan A, Ferdy JB |date=January 2011 |title=Virulence and pathogen multiplication: a serial passage experiment in the hypervirulent bacterial insect-pathogen Xenorhabdus nematophila |journal=PLOS ONE |volume=6 |issue=1 |pages=e15872 |bibcode=2011PLoSO...615872C |doi=10.1371/journal.pone.0015872 |pmc=3031541 |pmid=21305003 |doi-access=free}}</ref><ref name="pmid11844774">{{Cite journal |vauthors=Somerville GA, Beres SB, Fitzgerald JR, DeLeo FR, Cole RL, Hoff JS, Musser JM |date=March 2002 |title=In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence |journal=Journal of Bacteriology |volume=184 |issue=5 |pages=1430–7 |doi=10.1128/jb.184.5.1430-1437.2002 |pmc=134861 |pmid=11844774}}</ref> This process will be repeated as many times as desired.

Alternatively, an in vivo experiment can be performed where an animal is infected with a pathogen, and this pathogen allowed time to grow in that host before a sample of it is removed from the host and passed to another host.  This process is repeated for a certain number of hosts; the individual experiment determines this number.

When serial passage is performed either in vitro or in vivo, the virus or bacterium may evolve by [[mutating]] repeatedly. Identifying and studying mutations that occur often reveals information about the virus or bacterium being studied.  Accordingly, after serial passage has been performed it can be valuable to compare the resulting virus or sample of bacteria to the original, noting any mutations that have occurred and their collective effects.  Various significant outcomes may occur.  The virulence of the virus may be changed,<ref name="woo">{{Cite journal |vauthors=Woo HJ, Reifman J |date=January 2014 |title=Quantitative modeling of virus evolutionary dynamics and adaptation in serial passages using empirically inferred fitness landscapes |journal=Journal of Virology |volume=88 |issue=2 |pages=1039–50 |doi=10.1128/JVI.02958-13 |pmc=3911671 |pmid=24198414}}</ref> or a virus could evolve to become adapted to a different host environment than that in which it is typically found.<ref name="woo" /> Relatively few passages are necessary to produce a noticeable change in a virus; for instance, a virus can typically adapt to a new host within ten or so passages.<ref name="woo" />

Because serial passage allows for rapid evolution of a virus to its host, it can be used to study the evolution of antibiotic resistance; specifically, for determining what mutations could lead to the development of antibiotic resistance.<ref>{{Cite journal |vauthors=Martínez JL, Baquero F, Andersson DI |date=October 2011 |title=Beyond serial passages: new methods for predicting the emergence of resistance to novel antibiotics |journal=Current Opinion in Pharmacology |volume=11 |issue=5 |pages=439–45 |doi=10.1016/j.coph.2011.07.005 |pmid=21835695}}</ref>

==History==
The technique of serial passage has been around since the 1800s.  In particular, [[Louis Pasteur]]'s work with the [[rabies vaccine]] in the late 1800s exemplifies this method.<ref name="smith">{{Cite journal |vauthors=Smith KA |date=2012 |title=Louis pasteur, the father of immunology? |journal=Frontiers in Immunology |volume=3 |pages=68 |doi=10.3389/fimmu.2012.00068 |pmc=3342039 |pmid=22566949 |doi-access=free}}</ref>

Pasteur created several vaccines over the course of his lifetime.  His work prior to rabies involved attenuation of pathogens, but not through serial passage. In particular, Pasteur worked with [[cholera]] and found that if he cultured bacteria for long periods of time, he could create an effective vaccine.<ref name="smith" /><ref name="schwartz">{{Cite journal |last=Schwartz |first=M. |date=2001 |title=The life and works of Louis Pasteur |journal=Journal of Applied Microbiology |volume=91 |issue=4 |pages=597–601 |doi=10.1046/j.1365-2672.2001.01495.x |pmid=11576293 |issn=1364-5072}}</ref> Pasteur thought that there was something special about oxygen and this was why he was able to attenuate (create a less virulent version of) the bacteria. Pasteur also tried to apply this method to create a vaccine for [[anthrax]], although with less success.<ref name="smith" />

Next, Pasteur wanted to apply this method to create a vaccine for rabies. However, rabies was, unbeknownst to him, caused by a virus, not a bacterial pathogen like cholera and anthrax, and for that reason rabies could not be cultured in the same way that cholera and anthrax could be.<ref name="schwartz" />  Methods for serial passage for viruses in vitro were not developed until the 1940s, when [[John Enders]], [[Thomas Huckle Weller]], and [[Frederick Robbins]] developed a technique for this.  These three scientists subsequently won the [[Nobel Prize]] for their major advancement.<ref>{{Cite journal |vauthors=McCullers JA |date=September 2007 |title=Evolution, benefits, and shortcomings of vaccine management |journal=Journal of Managed Care Pharmacy |volume=13 |issue=7 Suppl B |pages=S2–6 |doi=10.18553/jmcp.2007.13.s7-b.2a |pmc=10438138 |pmid=17874877 |doi-access=free}}</ref>

To solve this problem, Pasteur worked with the rabies virus in vivo.<ref name="smith" /><ref name="schwartz" /> In particular, he took brain tissue from an infected dog and transplanted it into another dog, repeating this process multiple times, and thus performing serial passage in dogs.<ref name="smith" /> These attempts increased the virulence of the virus.<ref name="smith" /> Then, he realized that he could put dog tissue into a monkey to infect it and then perform serial passage in monkeys.<ref name="smith" /> After completing this process and infecting a dog with the resulting virus, Pasteur realized that the virus was less virulent.<ref name="smith" /> Mostly, Pasteur worked with the rabies virus in rabbits.<ref name="schwartz" /> Ultimately, to create his vaccine for rabies, Pasteur used a simple method that involved drying out tissue. As is described in his notebook:

<blockquote>In a series of flasks in which air is maintained in a dry state…each day one suspends a thickness of fresh rabbit spinal tissue taken from a rabbit dead of rabies. Each day as well, one inoculates under the skin of a dog 1 mL of sterilized bouillion, in which has dispersed a small fragment of one of these desiccated spinal pieces, beginning with a piece most distant in time from when it was worked upon, in order to be sure that it is not at all virulent.<ref name="smith" /></blockquote>

Pasteur mostly used other techniques besides serial passage to create his vaccines. However, the idea of attenuating a virus through serial passage still holds.

==Use in vaccines==
One way to attenuate a virus to a host is by passing the virus in a different species.<ref name="woo" />  The idea is that, as a strain of a virus becomes more adapted to a different species, that strain will become less adapted to the original host, thus decreasing in virulence with respect to the original host.<ref name="woo" /> This is the implicit principle that Louis Pasteur was unknowingly making use of when he passed the rabies virus in monkeys and ended up with a virus that was less dangerous to dogs, for example.<ref name="smith" />

The process of serial passage yields a [[live vaccine]].  There are both advantages and disadvantages to this.  Most notably, live vaccines are sometimes more effective and more long-lasting than inactivated or other types of vaccines.<ref>{{Cite web |last=Hunt |first=Richard |title='Vaccines: past successes and future prospects. |url=https://www.microbiologybook.org/lecture/vaccines.htm |access-date=5 May 2014 |website=Microbiology and Immunology On-line.}}</ref><ref name="types">{{Cite web |title=Types of Vaccines |url=https://www.niaid.nih.gov/research/vaccine-types |access-date=5 May 2014 |website=National Institute of Allergy and Infectious Diseases.}}</ref>  However, just as the virus evolved to become attenuated, it may reverse-evolve in the host, leading to infection.<ref name="types" />

==Experiments==
Researchers have conducted many experiments using serial passage.  Some of the experimental uses for serial passage include changing the virulence of a virus, to study the adaptive evolution or potential evolution of [[zoonotic diseases]] to new hosts, and studying [[antibiotic resistance]].

===Increasing virulence for use in animal modeling===
When developing vaccines for viruses, the emphasis is on attenuating the virus, or decreasing its virulence, in a given host.  Sometimes it is useful to employ serial passage to increase the virulence of a virus.  Usually, when serial passage is performed in a species, the result is a virus that is more virulent to that species.<ref name = woo/>

For example, one study<ref name="locher">{{Cite journal |vauthors=Locher CP, Witt SA, Herndier BG, Abbey NW, Tenner-Racz K, Racz P, Kiviat NB, Murthy KK, Brasky K, Leland M, Levy JA |date=January 2003 |title=Increased virus replication and virulence after serial passage of human immunodeficiency virus type 2 in baboons |journal=Journal of Virology |volume=77 |issue=1 |pages=77–83 |doi=10.1128/jvi.77.1.77-83.2003 |pmc=140565 |pmid=12477812}}</ref> used serial passage in baboons to create a strain of HIV-2 that is particularly virulent to baboons.  Typical strains of HIV-2 only infect baboons slowly.<ref name=locher/>  This specificity makes it challenging for scientists to use HIV-2 in animal models of HIV-1, because the animals in the model will only show symptoms slowly. The more virulent strain of HIV-2 could be practical for use in animal models, however.<ref name=locher/>

Another study by Kanta Subbaro involved a serial passage experiment in which mice were infected with [[SARS]].<ref name="zimmer">{{Cite book |last=Zimmer |first=Carl |title=The Tangled Bank |date=2013 |publisher=Robert and Co. Publishers |edition=second |pages=399–427 |chapter=Chapter 15}}</ref>  SARS usually does not make mice particularly sick, however, after the virus had undergone serial passage in the mice, it had become lethal.<ref name="zimmer" />

Changing the virulence of SARS in this way was important, because without a virulent form of SARS to infect laboratory animals, scientists would have been unable to test the effects of SARS in an animal model.<ref name="zimmer" />

More generally, this experiment also reflects a general medical principle: The virulence of a virus is mediated by the difficulty of its transmission.

Generally, if a virus kills its host too quickly, the host will not have a chance to come in contact with other hosts and transmit the virus before dying.  In serial passage, when a virus was being transmitted from host to host regardless of its virulence, such as Subbaro's experiment, the viruses that grow the fastest (and are therefore the most virulent) are selected for.<ref name="zimmer" />

This principle has public health implications, because it suggests that, in very densely populated or overcrowded areas, such as slums or [[Pest house|group quarantine facilities]], natural selection may favour more virulent viruses.

This also helps explain why good hygiene is so important. Good hygiene selects against highly virulent viruses by lowering the ability of pathogens to transmit.<ref name="zimmer" />

Serial passaging has been used to produce mouse-adapted SARS-CoV-2.<ref name="pandey">{{Cite journal |last1=Pandey |first1=Kabita |last2=Acharya |first2=Arpan |year=2021 |title=Animal models for SARS-CoV-2 research: A comprehensive literature review |journal=Transboundary and Emerging Diseases |publication-date=October 2020 |volume=68 |issue=4 |pages=1868–1885 |doi=10.1111/tbed.13907 |pmc=8085186 |pmid=33128861}}</ref><ref>{{cite journal|vauthors=Gu H, Chen Q, Yang G, He L, Fan H, Deng YQ, Wang Y, Teng Y, Zhao Z, Cui Y, Li Y, Li XF, Li J, Zhang NN, Yang X, Chen S, Guo Y, Zhao G, Wang X, Luo DY, Wang H, Yang X, Li Y, Han G, He Y, Zhou X, Geng S, Sheng X, Jiang S, Sun S, Qin CF, Zhou Y|title=Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy|journal=Science|volume=369|issue=6511|pages=1603–1607|date=25 September 2020|pmid=32732280|pmc=7574913|doi=10.1126/science.abc4730}}</ref>

===Influenza===
The [[H5N1]] virus is a particularly lethal strain of [[influenza]]. Currently, it can infect humans, but it is not [[contagious disease|contagious]] between humans. Still, over 600 people worldwide are known to have died from animal-transmitted H5N1 virus,<ref name="zimmer" /> so the transmissibility of the virus is of major concern to scientists.

Several serial passage experiments have been conducted to determine the feasibility of the virus becoming transmissible in humans. In particular, Ron Fouchier and his colleagues did a 10-step serial passage experiment in ferrets.<ref name="zimmer" /> In doing so, they created a strain of influenza that not only infected ferrets, but was transmissible between ferrets.<ref name="zimmer" /> Notably, this strain was very similar to the original strain with which they had infected the first ferret—in other words, only a few mutations were necessary for the virus to become transmissible between ferrets.<ref name="zimmer" /><ref name="puiu">{{Cite web |date=3 May 2012 |title=H5N1 controversial paper shows that bird flu is only a genetic mutation away from mammal flu. |url=http://www.zmescience.com/research/h5n1-bird-flu-paper-genetic-mutation-human-transmission-05032/ |access-date=26 April 2014 |website=ZME Science |vauthors=Puiu T}}</ref>  Similarly, researcher Yoshihiro Kawaoka found that a single mutation is necessary to make the virus transmissible in ferrets.<ref name="puiu" />

Both Fouchier's research and Kawaoka's research were initially censored, per implications for [[bioterrorism]].<ref name="puiu" /> The research was later published, but remained controversial.<ref name="puiu" />

Serial passage is an artificial technique that is used in a laboratory setting, rather than a natural process.  Accordingly, the likelihood that the H5N1 virus would actually mutate to become transmissible in humans is unknown; however, researcher Derek Smith created an evolutionary model to show that this is possible.<ref name="zimmer" />

===Understanding how viruses jump between species===
Another use of serial passage is in understanding how pathogens adapt to new species.  By introducing a pathogen into a new host species and performing serial passage, scientists can observe as the pathogen adapts to its new host and pinpoint the mutations that allow for this adaptation.<ref name="woo" />

== Mathematical modeling ==
Work by Stewart and Levin <ref name="stewart1973partitioning">{{cite journal |vauthors=Stewart, Frank M, Levin, Bruce R |title=Partitioning of Resources and the Outcome of Interspecific Competition: A Model and Some General Considerations |journal=The American Naturalist |volume=107 |issue=954 |pages=171-198 |date=1973 |pmid=|pmc=10425282 |doi=10.3389/fbioe.2023.1210392 |doi-access=free |url=}}</ref> delineated fundamental differences between continuous and serial passage ecosystems and work by Hal Smith explored the applicability of the competitive exclusion principle to serial passage.<ref name="Smith2011Serial">{{cite journal |vauthors= Hal L, Smith | title= Bacterial competition in serial transfer culture |journal=Mathematical Biosciences |volume=229 |issue=2 |pages=149-159 |date=2011 |pmid=21163273|pmc= |doi=10.1016/j.mbs.2010.12.001 |doi-access=free |url=}}</ref> Later, Erez and Lopez et al. <ref name="eLife">{{cite journal |vauthors=Erez A, Lopez JG, Weiner BG, Meir Y, Wingreen NS |title=Nutrient levels and trade-offs control diversity in a serial dilution ecosystem |journal=eLife |volume=9 |issue= |pages= |date=September 2020 |pmid=32915132 |pmc=7486120 |doi=10.7554/eLife.57790 |doi-access=free |url=}}</ref> predicted that serial passage ecosystems are strongly structured by an [[Early-bird effect]] in which otherwise poor competitors can leverage early advantage to outcompete other organisms. Experimental work has demonstrated the existence of this effect empirically.<ref name="Aranda-Díaz">{{cite journal |vauthors=Aranda-Díaz A, Willis L, Nguyen TH, Ho PY, Vila J, Thomsen T, Chavez T, Yan R, Yu FB, Neff N, Sanchez A, Estrela S, Huang KC |title=Assembly of stool-derived bacterial communities follows "early-bird" resource utilization dynamics |journal=Cell Systems |volume= |issue= |pages= |date=April 2025 |pmid=40157357 |pmc= |doi=10.1016/j.cels.2025.101240 |url=}}</ref><ref name="Kim">{{cite journal |vauthors=Kim Y, Flinkstrom Z, Candry P, Winkler MH, Myung J |title=Resource availability governs polyhydroxyalkanoate (PHA) accumulation and diversity of methanotrophic enrichments from wetlands |journal=Front Bioeng Biotechnol |volume=11 |issue= |pages=1210392 |date=2023 |pmid=37588137 |pmc=10425282 |doi=10.3389/fbioe.2023.1210392 |doi-access=free |url=}}</ref>

== See also ==
* [[E. coli long-term evolution experiment]]
* [[Experimental evolution]]
* [[Early-bird effect]]

== References ==
{{reflist}}
{{Use dmy dates|date=November 2020}}

[[Category:Virology]]
[[Category:Microbial population biology]]