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

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.

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

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.

Detection of three independently-generated DS-1-like G9P[8] reassortant rotavirus A strains during the G9P[8] dominance in Vietnam, 2016-2018.

A rapid increase and dominance of G9P[8] Rotavirus A strains occurred in northern Vietnam between 2016 and 2018, during which period there appeared three G9P[8] strains possessing short RNA patterns. To understand how the first-ever G9P[8] strains possessing short RNA patterns were formed, next generation sequencing technology was used to examine the whole genomes of the three strains, i.e., RVA/Human-wt/VNM/RVN16.1024/2016/G9P[8], RVA/Human-wt/VNM/RVN17.0879/2017/G9P[8], and RVA/Human-wt/VNM/RVN18.0197/2018/G9P[8], and those of seven representative G9P[8] strains possessing long RNA patterns. The VP7 genes of the short and long G9P[8] strains were > 99% identical, indicating that the origin was in the co-circulating, dominant, long G9P[8] strains. On the other hand, the VP4 genes likely derived from recently-emerging G1/G3/G8P[8] strains possessing the DS-1 backbone. At the lineage level, however, the backbone genes of any one strain differed from that of the other two in the VP1, VP3 or NSP4 gene. Moreover, even at the nucleotide sequence level of the backbone genes belonging to the same lineage, the identities between the three strains were lower than those expected for the strains deriving from an immediate, common ancestor. Thus, the three strains were likely formed by independent reassortment events in which the VP7 gene of the currently dominant G9P[8] strains was incorporated into co-circulating G1/G3/G8P[8] strains possessing similar yet distinct DS-1-like backbone genes. The observation that all of the three reassortant G9P[8] strains were detected only once among the prevalent, ordinary G9P[8] strains suggests that acquisition of the DS-1-like backbone genes unlikely provided selective advantage over the parental Wa-like G9P[8] strains.

Sub-genotype phylogeny of the non-G, non-P genes of genotype 2 Rotavirus A strains.

Recent increase in the detection of unusual G1P[8], G3P[8], G8P[8], and G9P[4] Rotavirus A (RVA) strains bearing the DS-1-like constellation of the non-G, non-P genes (hereafter referred to as the genotype 2 backbone) requires better understanding of their evolutionary relationship. However, within a genotype, there is lack of a consensus lineage designation framework and a set of common sequences that can serve as references. Phylogenetic analyses were carried out on over 8,500 RVA genotype 2 genes systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. In line with previous designations, using pairwise comparison of cogent nucleotide sequences and stringent bootstrap support, reference lineages were defined. This study proposes a lineage framework and provides a dataset ranging from 34 to 145 sequences for each genotype 2 gene for orderly lineage designation of global genotype 2 genes of RVAs detected in human and animals. The framework identified five to 31 lineages depending on the gene. The least number of lineages (five to seven) were observed in genotypes A2 (NSP1), T2 (NSP3) and H2 (NSP5) which are limited to human RVA whereas the most number of lineages (31) was observed in genotype E2 (NSP4). Sharing of the same lineage constellations of the genotype 2 backbone genes between recently-emerging, unusual G1P[8], G3P[8], G8P[8] and G9P[4] reassortants and many contemporary G2P[4] strains provided strong support to the hypothesis that unusual genotype 2 strains originated primarily from reassortment events in the recent past involving contemporary G2P[4] strains as one parent and ordinary genotype 1 strains or animal RVA strains as the other. The lineage framework with selected reference sequences will help researchers to identify the lineage to which a given genotype 2 strain belongs, and trace the evolutionary history of common and unusual genotype 2 strains in circulation.

Whole genome characterization of feline-like G3P[8] reassortant rotavirus A strains bearing the DS-1-like backbone genes detected in Vietnam, 2016.

While conducting rotavirus gastroenteritis surveillance in Vietnam, two G3P[8] rotavirus A specimens possessing an identical short RNA electropherotype were detected. They were RVA/Human-wt/VNM/0232/2016/G3P[8] and RVA/Human-wt/VNM/0248/2016/G3P[8], and recovered from 9 and 23 months old boys, respectively. The patients developed diarrhoea within one-week interval in March 2016 but in places >100 km apart in northern Vietnam. Whole genome sequencing of the two G3P[8] rotavirus A strains revealed that their genomic RNA sequences were identical across the 11 genome segments, suggesting that they derived from a single clone. The backbone gene constellation was I2-R2-C2-M2-A2-N2-T2-E2-H2. The backbone genes and the VP4 gene had a virtually identical nucleotide sequences with identities ranging from 99.2 to 100% to the corresponding genes of RVA/Human-wt/VNM/1149/2014/G8P[8]; the prototype of recently-emerging bovine-like G8P[8] reassortant strains in Vietnam. On the other hand, the VP7 gene was 98.8% identical with that of RVA/Human-wt/CHN/E2451/2011/G3P[9], and they were clustered together in the lineage represented by RVA/Cat-tc/JPN/FRV-1/1986/G3P[9]. The observations led us to hypothesise that one of the bovine-like G8P[8] strains bearing the DS-1-like backbone genes reassorted with a locally circulating FRV-1-like strain to gain the G3 VP7 gene and to emerge as a thus-far undescribed feline-like G3P[8] reassortant strain. The identification of feline-like G3P[8] strains bearing the DS-1-like backbone genes exemplifies the strength and necessity of the whole genome sequencing approach in monitoring, describing and understanding the evolutionary changes that are occurring in emerging strains and their interactions with co-circulating strains.

Study of Complete Genome Sequences of Rotavirus A Epidemics and Evolution in Japan in 2012-2014.

A comprehensive molecular epidemiological study using next-generation sequencing technology was conducted on 333 rotavirus A (RVA)-positive specimens collected from six sentinel hospitals across Japan over three consecutive seasons (2012-2014). The majority of the RVA isolates were grouped into five genotype constellations: Wa-like G1P[8], DS-1-like G1P[8], G2P[4], G3P[8] and G9P[8]. Phylogenetic analysis showed that the distribution of strains varied by geographical locations and epidemic seasons. The VP7 genes of different G types were estimated to evolve at 7.26 × 10-4-1.04 × 10-3 nucleotide substitutions per site per year. The Bayesian time-scaled tree of VP7 showed that the time to the most recent common ancestor of epidemic strains within a region was 1-3 years, whereas that of the epidemic strains across the country was 2-6 years. This study provided, for the first time, the timeframe during which an epidemic strain spread locally and within the country and baseline information needed to predict how rapidly RVAs spread.

Whole genome characterisation of G11P[25] and G9P[19] rotavirus A strains from adult patients with diarrhoea in Nepal.

Rotavirus A (RVA) causes acute diarrhoea in children and less frequently in adults. However, the knowledge about the genotype distribution of RVA strains circulating in adults is limited particularly in developing countries. This study aimed to characterise the RVA strains detected from adult patients with diarrhoea in Nepal. A total of 47 RVA positive stool samples from adult patients with diarrhoea in Kathmandu, Nepal during 2007-2008 were examined for the G and P genotypes by sequencing. Nearly half (49%) of the samples were genotyped as G9P[8] (n = 23), G1P[8], G2P[4] (n = 5 each), G12P[8] (n = 4), G12P[6] (n = 3), G1P[6] (n = 2), G3P[8] and G9P[6] (n = 1 each). Interestingly, two G11P[25] and one G9P[19] strains detected were further subjected to Illumina MiSeq next generation sequencing to determine their whole genome sequences. The genotype constellations of RVA/Human-wt/NPL/TK2615/2008/G11P[25] and RVA/Human-wt/NPL/TK2620/2008/G11P[25] were I12-R1-C1-M1-A1-N1-T1-E1-H1, whereas that of RVA/Human-wt/NPL/TK1797/2007/G9P[19] was I5-R1-C1-M1-A8-N1-T1-E1-H1. The 11 genes of TK2615 and TK2620 were virtually identical, and they were either porcine-like or unique except the VP2 and NSP1 genes which were of human RVA origin. The two G11P[25] strains were also very similar to KTM368, another G11P[25] isolated from a child in Nepal in 2004. On the other hand, no gene of TK1797 was likely to be of human RVA origin. The observation that porcine-like RVAs were detected from adult patients justifies further studies to explore the role of adults in the interspecies transmission of animal RVA to humans.

Full genotype constellations of six feline Rotavirus A strains isolated in Japan in the 1990s including a rare A15 NSP1 genotype.

Full genome sequencing of six feline Rotavirus A (RVA) strains isolated in Japan in the 1990s revealed three genotype constellations, one of which had a unique constellation of G3-P[3]-I3-R3-C3-M3-A15-N3-T3-E3-H6. Genotype A15, carried by RVA/Cat-tc/JPN/FRV348/1994/G3P[3], is a rare NSP1 genotype, and only one human and one canine RVA strains have thus far been reported to carry this genotype. The other three G3P[3] strains (FRV72, FRV73, and FRV303) possessed a constellation of I3-R3-C2-M3-A9-N2-T3-E3-H6, whereas two G3P[9] strains (FRV317 and FRV384) possessed a constellation of I3-R3-C3-M3-A3-N3-T3-E3-H3.

Porcine-like G3P[6] and G4P[6] rotavirus A strains detected from children with diarrhoea in Vietnam.

Animal rotavirus A (RVA) strains can infect children and cause diarrhoea. We determined the full genome sequences of one G3P[6] strain (NT0001) and five G4P[6] strains (NT0042, NT0077, NT0205, NT0599, and NT0621) detected from children with diarrhoea in Vietnam in 2007-2008. Strain NT0001 had a genotype constellation of: G3-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H1, strain NT0042: G4-P[6]-I1-R1-C1-M1-A8-N1-T1-E1-H1, strain NT0077: G4-P[6]-I5-R1-C1-M1-A8-N1-T7-E1-H1, and strains NT0205, NT0599, and NT0621: G4-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence divergence data and phylogenetic analysis showed that they were different porcine RVA strains that independently and directly crossed the host species barrier to infect children.

Free-Chlorine Disinfection as a Selection Pressure on Norovirus.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.

Detection in Japan of an equine-like G3P[8] reassortant rotavirus A strain that is highly homologous to European strains across all genome segments.

Equine-like G3P[8] rotavirus A strains with DS-1-like backbone genes have emerged since 2013. An equine-like RVA/Human-wt/JPN/15R429/2015/G3P[8] strain possessing I2-R2-C2-M2-A2-N2-T2-E2-H2 was detected in Japan in 2015. Its VP7 gene was ≥ 99.3% identical to those of equine-like G3P[4] strains detected in Japan, and the remaining 10 genes were 98.6-99.8% identical to G1P[8] double-gene reassortants detected in Japan, Thailand and the Philippines. Thus, 15R429 was likely generated through reassortment between the equine-like G3P[4] and G1P[8] reassortant strains. Notably, 15R429 was 98.5-99.8% identical across all 11 genes of the equine-like G3P[8] strains detected in Spain and Hungary in 2015.

Reassortant DS-1-like G1P[4] Rotavirus A strains generated from co-circulating strains in Vietnam, 2012/2013.

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human-wt/VNM/SP127/2013/G1P[4] and RVA/Human-wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2, indicated that they were G1 VP7 mono-reassortants possessing DS-1-like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa-like strain RVA/Human-wt/VNM/SP110/2012/G1P[8] which co-circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS-1-like strains RVA/Human-wt/VNM/SP015/2012/G2P[4] (and SP108) that co-circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore-mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110-like and SP015-like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter-genogroup reassortment between co-circulating G1P[8] and G2P[4] strains within the same place and season.

Effectiveness of rotavirus vaccines against hospitalisations in Japan.

BACKGROUND: In Japan, rotavirus hospitalisation occurs at a rate from 2.8 to 13.7 per 1000 child-years among children age less than 5 years, and it imposes a substantial burden to the healthcare system in the country. While both monovalent (RV1) and pentavalent (RV5) rotavirus vaccines are licensed in Japan, neither has been incorporated in the national infant immunization programme. In this study, we estimated vaccine effectiveness (VE) in Japan. METHODS: This study was conducted in Yuri-Kumiai General Hospital located in a city in the north-western part of Japan. Age-eligible children for rotavirus vaccination were enrolled if they were hospitalized for rotavirus gastroenteritis between September 2013 and August 2016. Rotavirus gastroenteritis was defined by the detection of rotavirus antigen by immunochromatography. "Vaccinated" was defined as infant inoculated with at least one dose of either RV1 or RV5. A conditional logistic regression analysis was performed by modelling the year of birth, year of admission, residence of the children and vaccination status, and by matching the age of cases with that of test-negative controls. The adjusted odds ratio of the vaccinated over unvaccinated was then used to calculate VE in the formula of (1 - adjusted odds ratio) × 100. RESULTS: Out of the 244 patients enrolled, rotavirus antigen was detected in 55 (22.5%) of whom 10 (18.2%) were vaccinated, whereas 94 (49.7%) of 189 test-negative controls were vaccinated. During the study period, the vaccine uptake rate in the controls increased from 36.2% to 61.8%. On the other hand, the vaccination coverage over the three years was 64.2% in Yuri-Honjo city (three quarters of the catchment), and 91.4% in Nikaho city (one quarter of the catchment). The VE was calculated to be 70.4% (95% confidence interval: 36.0-86.4%, P = 0.002). The point estimate of the VE was lower but its 95% confidence interval overlaps those of the efficacies obtained from clinical trials in Japan. CONCLUSION: The rotavirus vaccine was effective in the real-world setting in Japan as in the clinical trials, and the introduction of rotavirus vaccine in the national infant immunization schedule will substantially reduce the number of rotavirus gastroenteritis hospitalisation in Japan.

Whole genome characterisation of a porcine-like human reassortant G26P[19] Rotavirus A strain detected in a child hospitalised for diarrhoea in Nepal, 2007.

A rare G26 Rotavirus A strain RVA/Human-wt/NPL/07N1760/2007/G26P[19] was detected in a child hospitalised for acute diarrhoea in Kathmandu, Nepal. The complete genome of 07N1760 was determined in order to explore its evolutionary history as well as examine its relationship to a Vietnamese strain RVA/Human-wt/VNM/30378/2009/G26P[19], the only G26 strain whose complete genotype constellation is known. The genotype constellation of 07N1760 was G26-P[19]-I12-R1-C1-M1-A8-N1-T1-E1-H1, a unique constellation identical to that of the Vietnamese 30378 except the VP6 gene. Phylogenetic analysis revealed that both strains were unrelated at the lineage level despite their similar genotype constellation. The I12 VP6 gene of 07N1760 was highly divergent from the six currently deposited I12 sequences in the GenBank. Except for its NSP2 gene, the remaining genes of 07N1760 shared lineages with porcine and porcine-like human RVA genes. The NSP2 gene belonged to a human RVA N1 lineage which was distinct from typical porcine and porcine-like human lineages. In conclusion, the Nepali G26P[19] strain 07N1760 was a porcine RVA strain which derived an NSP2 gene from a human Wa-like RVA strain by intra-genotype reassortment probably after transmission to the human host.

Regional Variations in the Incidence of Rotavirus Hospitalization in Children Living in Defined Regions of Akita and Kyoto Prefectures, Japan.

Variable incidence rates of rotavirus gastroenteritis hospitalizations have been reported in Japan. However, it is not known whether the observed regional differences were due to the real difference in the occurrence of severe disease or other causes. This study aimed to determine the incidence rates of rotavirus hospitalization among children aged <5 years in the Yuri district in Akita prefecture and the Nantan district in Kyoto prefecture between March 2012 and February 2013. During this period, rotavirus vaccine uptake rates were equally low in both regions. All specimens were evaluated using the standardized case definition, severity scores, and the same assays. There were 44 rotavirus cases (44%) among 101 acute gastroenteritis-related hospitalizations in the Yuri district with a catchment population of 3,853, and 18 rotavirus cases (47%) among 38 acute gastroenteritis-related hospitalizations in the Nantan district with a catchment population of 5,128. While the severity score at the time of the hospitalizations was 11 in both hospitals, the incidence rates in Akita and Kyoto were 11.7 (95% confidence interval [CI]: 8.5-15.6) and 3.9 (95% CI: 2.1-5.5) per 1,000 child-years, respectively. Thus, there was a real difference in the occurrence rate of severe rotavirus infections between the 2 regions.

Evolution of DS-1-like G1P[8] double-gene reassortant rotavirus A strains causing gastroenteritis in children in Vietnam in 2012/2013.

Rotavirus A (RVA) strains, a leading cause of severe gastroenteritis in children worldwide, commonly possess the Wa or DS-1 genotype constellations. During a hospital-based study conducted in Hanoi, Vietnam, in the 2012-2013 rotavirus season, G1P[8] strains with a virtually identical short RNA migration pattern were detected in 20 (14%) of 141 rotavirus-positive samples. Two representatives of these strains were shown by whole-genome sequencing to be double-gene reassortants possessing the genotype constellation of G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Sequencing and a database search revealed that these Vietnamese G1P[8] double-gene reassortant strains shared an immediate ancestor with a locally circulating G2P[4] strain in all of the inner-capsid and non-structural protein genes, whereas they were more closely related in the VP7 and VP4 genes to a Chinese G1P[8] strain and a Chinese G3P[8] strain, respectively, than to locally circulating G1P[8] strains. Despite the marked similarity between Japanese and Thai G1P[8] double-gene reassortant strains, phylogenetic analysis suggested that the Vietnamese and Japanese/Thai G1P[8] double-gene reassortant strains originated from independent reassortment events. Clinically, children infected with Vietnamese G1P[8] double-gene reassortant strains experienced severe diarrhoea, but it was not more severe than that in children infected with ordinary G1P[8] strains. In conclusion, Vietnamese G1P[8] double-gene reassortant strains originated from a locally circulating G2P[4] strain and caused severe diarrhoea, but there was no evidence of increased virulence.

Molecular epidemiology of Rotavirus A, causing acute gastroenteritis hospitalizations among children in Nha Trang, Vietnam, 2007-2008: Identification of rare G9P[19] and G10P[14] strains.

Rotavirus A (RVA) causes acute diarrhea in children as well as animals. As part of a cross-sectional study of children less than 5 years of age hospitalized for acute diarrhea in Vietnam during a 15-month period (2007-2008), 322 (43.5%) of 741 fecal specimens contained RVA with 92% either G1P[8] or G3P[8]. This study was undertaken to further characterize strains that remained untypeable to complete the G and P genotypes of the 322 rotavirus-positive specimens. While 307 (95.3%) strains possessed the common human RVA genotypes: G1P[8] (45.0%), G2P[4] (2.8%), G3P[8] (46.9%), and G9P[8] (0.6%), sequencing of initially untypeable specimens revealed the presence of two unusual strains designated NT0073 and NT0082 possessing G9P[19] and G10P[14], respectively. The genotype constellation of NT0073 (G9-P[19]-I5-R1-C1-M1-A8-N1-T7-E1-H1) and the phylogenetic trees suggested its origin as a porcine RVA strain causing diarrhea in a 24-month-old girl whereas the genotype constellation of NT0082 (G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3) and the phylogenetic trees suggested its origin as an RVA strain of artiodactyl origin (such as cattle, sheep and goats) causing diarrhea in a 13-month-old boy. This study showed that RVA strains of animal host origin were not necessarily attenuated in humans. A hypothesis may be postulated that P[19] and P[14] VP4 spike proteins helped the virus to replicate in the human intestine but that efficient onward human-to-human spread after crossing the host species barrier may require the virus to obtain some additional features as there was no evidence of widespread transmission with the limited sampling performed over the study period. J. Med. Virol. 89:621-631, 2017. © 2016 Wiley Periodicals, Inc.

Genomic constellation and evolution of Ghanaian G2P[4] rotavirus strains from a global perspective.

Understanding of the genetic diversity and evolution of Rotavirus A (RVA) strains, a common cause of severe diarrhoea in children, needs to be based on the analysis at the whole genome level in the vaccine era. This study sequenced the whole genomes of six representative G2P[4] strains detected in Ghana from 2008 to 2013, and analysed them phylogenetically with a global collection of G2P[4] strains and African non-G2P[4] DS-1-like strains. The genotype constellation of the study strains was G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Strains from the same season were highly identical across the whole genome while strains from different seasons were more divergent from each other. The VP7, VP4, VP2, NSP1, and NSP5 genes belonged to lineage IVa; the VP6, VP1, NSP2, and NSP3 genes belonged to lineage V, and all these genes evolved in the same fashion as the global strains. In the NSP4 gene, lineages V (2008) and X (2009) were replaced by VI (2012/2013) whereas in the VP3 gene, lineage V (2008/2009) was replaced by VII (2012/2013) and these replacements coincided with the vaccine introduction period (2012). The evolutionary rate of the NSP4 gene was 1.2×10-3 substitutions/site/year and was rather comparable to that of the remaining 10 genes. The multiple NSP4 lineages were explained by intra-genotype reassortment with co-circulating African human DS-1-like strains bearing G2[6], G3P[6], G6[6] and G8. There was no explicit evidence of the contribution of animal RVA strains to the genome of the Ghanaian G2P[4] strains. In summary, this study revealed the dynamic evolution of the G2P[4] strains through intra-genotype reassortment events leading to African specific lineages such IX and X in the NSP4 gene. So far, there was no evidence of a recent direct involvement of animal RVA genes in the genome diversity of African G2P[4] strains.

Whole genome characterization of a G6P[5] rotavirus A strain isolated from a stray cat in Japan.

The whole genome of an unusual G6P[5] rotavirus A strain named FRV537, which was isolated from a stray cat in Japan, was characterized to determine its species of origin. The genotype constellation of FRV537 was G6-P[5]-I2-R2-C2-M2-A13-N2- T6-E2-H3. No known feline rotavirus has this genotype constellation; the Japanese equine strain OH-4 is the only known strain that does. While FRV537 shares the same genotype with some feline rotaviruses in all genes except those encoding VP4 and NSP1, none of these genes are closely related to those of known feline rotaviruses. By contrast, G6P[5] is almost exclusively present in bovine rotaviruses. The VP7 and VP4 genes of FRV537 formed a lineage with typical bovine rotaviruses with high bootstrap values. As to the internal capsid and nonstructural gene constellation, three bovine rotavirus strains had a constellation identical to that of FRV537. Moreover, each of the genotypes of FRV537 was found to be a common bovine genotype. In addition to the high nucleotide sequence identities between FRV537 and bovine rotaviruses in each genome segment (≥95%), phylogenetic analysis revealed a close relationship to bovine/artiodactyl rotaviruses. Thus, the molecular and phylogenetic evidence suggests that FRV537 isolated from a stray cat was of bovine rotavirus origin.