Comparison of three neurotropic viruses reveals differences in viral dissemination to the central nervous system

Luethy, Lauren N.; Erickson, Andrea K.; Jesudhasan, Palmy R.; Ikizler, Mine R.; Dermody, Terence S.; Pfeiffer, Julie K.

doi:10.1016/j.virol.2015.09.019

Cited by 33 publications

(29 citation statements)

References 57 publications

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“…We began by using 10 genetically distinct polioviruses that each contain unique silent point mutations and are discriminated by hybridization of reverse transcription-PCR (RT-PCR) products with specific probes. We previously demonstrated that these 10 marked viruses are equally fit (37–39). We mixed equal amounts of the 10 viruses and infected HeLa cells at an extremely low multiplicity of infection (MOI) such that ~2 to 20 plaques would be generated on each plate of 10 6 cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

Aguilera

Erickson

Jesudhasan

et al. 2017

mBio

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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.

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Section: Resultsmentioning

confidence: 99%

Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

Aguilera

Erickson

Jesudhasan

et al. 2017

mBio

Self Cite

View full text Add to dashboard Cite

show abstract

“…We began by using ten genetically distinct polioviruses that each contain unique silent point mutations and are discriminated by hybridization of RT-PCR products with specific probes. We previously demonstrated that these ten marked viruses are equally fit (37–39). We mixed equal amounts of the ten viruses and infected HeLa cells at an extremely low MOI such that ~2-20 plaques would be generated on each plate of 10 6 cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Plaques formed by mutagenized viral populations have elevated co-infection frequencies

Aguilera

Erickson

Jesudhasan

et al. 2017

Preprint

Self Cite

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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, plaque number 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 5-7% 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 co-infection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased co-infection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher co-infection frequencies due to fitness restoring processes such as complementation and recombination. Indeed, we found that co-infection 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 three-fold, since co-infection 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 co-infection may assist plaque formation in situations where the amount of genome damage is high.

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“…Barcoded RNA viral genomes were used to identify bottlenecks in viral diversity, both inside the infected host [14, 15] and during transmission among hosts [16]. Thus, barcoding of viral genomes can be a useful tool in studying bottlenecks during viral replication, even on the single cell level [17].…”

Section: Introductionmentioning

confidence: 99%

Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

Cohen

Kobiler

2016

PLoS Pathog

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Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells.

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Comparison of three neurotropic viruses reveals differences in viral dissemination to the central nervous system

Cited by 33 publications

References 57 publications

Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

Plaques Formed by Mutagenized Viral Populations Have Elevated Coinfection Frequencies

Plaques formed by mutagenized viral populations have elevated co-infection frequencies

Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells

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