Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

ABSTRACT The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed...

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Autores principales:	Elizabeth R. Aguilera, Andrea K. Erickson, Palmy R. Jesudhasan, Christopher M. Robinson, Julie K. Pfeiffer
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	coinfection evolution mutagen poliovirus Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/d9eeee66d3594dfe8d3c8f5f51a8504d
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spelling	oai:doaj.org-article:d9eeee66d3594dfe8d3c8f5f51a8504d2021-11-15T15:50:59ZPlaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies10.1128/mBio.02020-162150-7511https://doaj.org/article/d9eeee66d3594dfe8d3c8f5f51a8504d2017-05-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02020-16https://doaj.org/toc/2150-7511ABSTRACT The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high. IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.Elizabeth R. AguileraAndrea K. EricksonPalmy R. JesudhasanChristopher M. RobinsonJulie K. PfeifferAmerican Society for MicrobiologyarticlecoinfectionevolutionmutagenpoliovirusMicrobiologyQR1-502ENmBio, Vol 8, Iss 2 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	coinfection evolution mutagen poliovirus Microbiology QR1-502
spellingShingle	coinfection evolution mutagen poliovirus Microbiology QR1-502 Elizabeth R. Aguilera Andrea K. Erickson Palmy R. Jesudhasan Christopher M. Robinson Julie K. Pfeiffer Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
description	ABSTRACT The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high. IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations.
format	article
author	Elizabeth R. Aguilera Andrea K. Erickson Palmy R. Jesudhasan Christopher M. Robinson Julie K. Pfeiffer
author_facet	Elizabeth R. Aguilera Andrea K. Erickson Palmy R. Jesudhasan Christopher M. Robinson Julie K. Pfeiffer
author_sort	Elizabeth R. Aguilera
title	Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
title_short	Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
title_full	Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
title_fullStr	Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
title_full_unstemmed	Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies
title_sort	plaques formed by mutagenized viral populations have elevated coinfection frequencies
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/d9eeee66d3594dfe8d3c8f5f51a8504d
work_keys_str_mv	AT elizabethraguilera plaquesformedbymutagenizedviralpopulationshaveelevatedcoinfectionfrequencies AT andreakerickson plaquesformedbymutagenizedviralpopulationshaveelevatedcoinfectionfrequencies AT palmyrjesudhasan plaquesformedbymutagenizedviralpopulationshaveelevatedcoinfectionfrequencies AT christophermrobinson plaquesformedbymutagenizedviralpopulationshaveelevatedcoinfectionfrequencies AT juliekpfeiffer plaquesformedbymutagenizedviralpopulationshaveelevatedcoinfectionfrequencies
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Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

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