Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Kadoya, Syun suke; Sano, Daisuke

doi:10.3791/58821

Cited by 4 publications

(10 citation statements)

References 6 publications

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“…The infectious titer (PFU/mL) of each passage was measured by plaque assay. According to our previous report (16), serially diluted RRV suspensions were treated with 1 μg/μL trypsin from a porcine pancreas. Then, the RRV suspensions were inoculated onto confluent MA104 cells in the 6-well plate, and the plate was incubated at 37 °C with 5 % CO 2 for 90 min.…”

Section: Methodsmentioning

confidence: 99%

“…A cell-binding assay was conducted according to the steps outlined in previous reports (16, 17). MA104 cells were incubated on the 24 well-plate for 2 or 3 days, and then the cell culture medium was removed and washed twice with tris-buffered saline (TBS).…”

Section: Methodsmentioning

confidence: 99%

“…Following the recommendations from our previous report (16), MA104 cells were inoculated on the T25 flask for 2 - 3 days, and then the cell culture medium was removed and washed twice with Dulbecco’s PBS. The 400 μL of suspensions containing RRV (MOI of 0.01) were inoculated to the flasks individually.…”

Section: Methodsmentioning

confidence: 99%

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Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

Kadoya

Urayama

Nunoura

et al. 2019

Preprint

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RNA viruses form a dynamic distribution of mutant swarm (termed “quasispecies”) due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission ex-emplifies a bottleneck effect in that only part of a viral population can reach the next susceptible hosts. In the present study, the rhesus rotavirus (RRV) strain of Rotavirus A was serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001 in duplicate (the 1st and 2nd lineages), and three phenotypes (infectious titer, cell binding ability and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and non-synonymous substitutions on rotaviral genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate that a stronger bottleneck effect can create more sequence spaces for minor mutants originally existing in a hidden layer of mutant swarm.IMPORTANCEIn this study, we investigated a bottleneck effect on an RRV population, which may drastically impact a viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified a human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck could create a new sequence space in a population for minor mutants originally existing in a hidden layer of a mutant swarm of the double-stranded RNA virus. The results of this study suggest that the genetic drift caused by a bottleneck in a human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected by the molecular epidemiology using next generation sequencing in which the viral genetic diversity within a viral population is investigated.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

Kadoya

Urayama

Nunoura

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…The infectious titer (PFU/mL) of each passage was measured in triplicate by plaque assay. According to our previous report (17), serially diluted RRV suspensions were treated with 1 µg/µL trypsin from a porcine pancreas. Then, the RRV suspensions were inoculated onto confluent MA104 cells in the 6-well plate, and the plate was incubated at 37ºC with 5% on September 8, 2020 by guest http://jvi.asm.org/ Downloaded from CO for 90 min.…”

Section: Serial Passagesmentioning

confidence: 99%

“…A cell-binding assay was conducted in triplicate according to the steps outlined in previous reports (17,18). MA104 cells were incubated on the 24 well-plate for 2 or 3 days, and then the cell culture medium was removed and washed twice with tris-buffered saline (TBS).…”

Section: Cell Binding Assaymentioning

confidence: 99%

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

Kadoya

Urayama

Nunoura

et al. 2020

J Virol

Self Cite

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RNA viruses form a dynamic distribution of mutant swarms (termed “quasispecies”) due to the accumulation of mutations in the viral genome. The genetic diversity of a viral population is affected by several factors, including a bottleneck effect. Human-to-human transmission exemplifies a bottleneck effect, in that only part of a viral population can reach the next susceptible hosts. In the present study, two lineages of the rhesus rotavirus (RRV) strain of rotavirus A were serially passaged five times at a multiplicity of infection (MOI) of 0.1 or 0.001, and three phenotypes (infectious titer, cell binding ability, and specific growth rate) were used to evaluate the impact of a bottleneck effect on the RRV population. The specific growth rate values of lineages passaged under the stronger bottleneck (MOI of 0.001) were higher after five passages. The nucleotide diversity also increased, which indicated that the mutant swarms of the lineages under the stronger bottleneck effect were expanded through the serial passages. The random distribution of synonymous and nonsynonymous substitutions on rotavirus genome segments indicated that almost all mutations were selectively neutral. Simple simulations revealed that the presence of minor mutants could influence the specific growth rate of a population in a mutant frequency-dependent manner. These results indicate a stronger bottleneck effect can create more sequence spaces for minor sequences. IMPORTANCE In this study, we investigated a bottleneck effect on an RRV population that may drastically affect the viral population structure. RRV populations were serially passaged under two levels of a bottleneck effect, which exemplified human-to-human transmission. As a result, the genetic diversity and specific growth rate of RRV populations increased under the stronger bottleneck effect, which implied that a bottleneck created a new space in a population for minor mutants originally existing in a hidden layer, which includes minor mutations that cannot be distinguished from a sequencing error. The results of this study suggest that the genetic drift caused by a bottleneck in human-to-human transmission explains the random appearance of new genetic lineages causing viral outbreaks, which can be expected according to molecular epidemiology using next-generation sequencing in which the viral genetic diversity within a viral population is investigated.

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The Intrapopulation Genetic Diversity of RNA Virus May Influence the Sensitivity of Chlorine Disinfection

et al. 2022

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RNA virus populations are not clonal; rather, they comprise a mutant swarm in which sequences are slightly different from the master sequence. Genetic diversity within a population (intrapopulation genetic diversity) is critical for RNA viruses to survive under environmental stresses. Disinfection has become an important practice in the control of pathogenic viruses; however, the impact of intrapopulation genetic diversity on the sensitivity of disinfection, defined as –log10 (postdisinfected infectious titer/predisinfected titer), has not been elucidated. In this study, we serially passaged populations of rhesus rotavirus. We demonstrated that populations with reduced chlorine sensitivity emerged at random and independently of chlorine exposure. Sequencing analysis revealed that compared with sensitive populations, less-sensitive ones had higher non-synonymous genetic diversity of the outer capsid protein gene, suggesting that changes in the amino acid sequences of the outer capsid protein were the main factors influencing chlorine sensitivity. No common mutations were found among less-sensitive populations, indicating that rather than specific mutations, the diversity of the outer capsid protein itself was associated with the disinfection sensitivity and that the disinfection sensitivity changed stochastically. Simulation results suggest that the disinfection sensitivity of a genetically diverse population is destabilized if cooperative viral clusters including multiple sequences are formed. These results advocate that any prevention measures leading to low intrapopulation genetic diversity are important to prevent the spread and evolution of pathogenic RNA viruses in society.

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Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Cited by 4 publications

References 6 publications

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

Bottleneck Size-Dependent Changes in the Genetic Diversity and Specific Growth Rate of a Rotavirus A Strain

The Intrapopulation Genetic Diversity of RNA Virus May Influence the Sensitivity of Chlorine Disinfection

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