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.

Vaccine Effectiveness against DS-1-Like Rotavirus Strains in Infants with Acute Gastroenteritis, Malawi, 2013-2015.

Atypical DS-1-like G1P[8] rotaviruses emerged in 2013 in Malawi after rotavirus vaccine introduction. Vaccine effectiveness among infants hospitalized with acute DS-1-like G1P[8] rotavirus gastroenteritis was 85.6% (95% CI 34.4%-96.8%). These findings suggest that vaccine provides protection against these strains despite their emergence coinciding with vaccine introduction.

Emergence of Double- and Triple-Gene Reassortant G1P[8] Rotaviruses Possessing a DS-1-Like Backbone after Rotavirus Vaccine Introduction in Malawi.

To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (n = 32) possessed a Wa-like genetic constellation, whereas postvaccine G1P[8] strains (n = 18) had a DS-1-like constellation. Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10-4 to 4.1 × 10-3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine escape mutants. While multiple African countries have introduced a rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the postvaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998 to 2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting a limited effect on the recognition of G1-specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation.

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.

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.

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.

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.

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.

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.

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.

Population Impact and Effectiveness of Monovalent Rotavirus Vaccination in Urban Malawian Children 3 Years After Vaccine Introduction: Ecological and Case-Control Analyses.

BACKGROUND: Rotavirus vaccines have been introduced in many low-income African countries including Malawi in 2012. Despite early evidence of vaccine impact, determining persistence of protection beyond infancy, the utility of the vaccine against specific rotavirus genotypes, and effectiveness in vulnerable subgroups is important. METHODS: We compared rotavirus prevalence in diarrheal stool and hospitalization incidence before and following rotavirus vaccine introduction in Malawi. Using case-control analysis, we derived vaccine effectiveness (VE) in the second year of life and for human immunodeficiency virus (HIV)-exposed and stunted children. RESULTS: Rotavirus prevalence declined concurrent with increasing vaccine coverage, and in 2015 was 24% compared with prevaccine mean baseline in 1997-2011 of 32%. Since vaccine introduction, population rotavirus hospitalization incidence declined in infants by 54.2% (95% confidence interval [CI], 32.8-68.8), but did not fall in older children. Comparing 241 rotavirus cases with 692 test-negative controls, VE was 70.6% (95% CI, 33.6%-87.0%) and 31.7% (95% CI, -140.6% to 80.6%) in the first and second year of life, respectively, whereas mean age of rotavirus cases increased from 9.3 to 11.8 months. Despite higher VE against G1P[8] than against other genotypes, no resurgence of nonvaccine genotypes has occurred. VE did not differ significantly by nutritional status (78.1% [95% CI, 5.6%-94.9%] in 257 well-nourished and 27.8% [95% CI, -99.5% to 73.9%] in 205 stunted children;P= .12), or by HIV exposure (60.5% [95% CI, 13.3%-82.0%] in 745 HIV-unexposed and 42.2% [95% CI, -106.9% to 83.8%] in 174 exposed children;P= .91). CONCLUSIONS: Rotavirus vaccination in Malawi has resulted in reductions in disease burden in infants <12 months, but not in older children. Despite differences in genotype-specific VE, no genotype has emerged to suggest vaccine escape. VE was not demonstrably affected by HIV exposure or stunting.

Evolution of a G6P[6] rotavirus strain isolated from a child with acute gastroenteritis in Ghana, 2012.

Unusual human G6P[6] rotavirus A (RVA) strains have been reported sporadically in Europe and Africa, but how they evolved was not fully understood. The whole genome of a Ghanaian G6P[6] strain designated PML1965 (2012) was analysed to understand how it evolved in Africa and to learn how its G6 VP7 gene was related to that of rotaviruses of human and artiodactyl origin. The genotype constellation of RVA/Human-wt/GHA/PML1965/2012/G6P[6] was G6-P-[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. It shared sublineages with G6P[6] strains previously detected in Italy and Africa in all genome segments except the VP6 gene of a few Burkinabe and Cameroonian strains and both the VP6 and NSP4 genes of Guinea Bissau strains. The VP7 gene of the G6P[6] strains appeared to derive from those of human G6P[9] strains, and they were distantly related to the VP7 genes of artiodactyl G6 or human G6P[14] strains. The time of the most recent common ancestor of the VP7 sequences of G6P[6] strains was estimated to be the year 1998. The evolutionary rates of the VP7 genes in bovine and human G6 rotaviruses were 6.93 × 10(-4) and 3.42 × 10(-3) nucleotide substitutions site(-1) year(-1), respectively, suggesting an accelerated adaptive process in the new host. The sequences of the remaining 10 genome segments of PML1965 clustered with those of G2 and G8 human rotaviruses detected in Africa possessing the DS-1-like genetic background. In conclusion, PML1965 evolved from G2 or G8 RVA strains with DS-1-like background, acquiring the G6 VP7 gene from a human G6P[9] RVA and not from an artiodactyl G6 RVA strain.

Whole genotype constellation of prototype feline rotavirus strains FRV-1 and FRV64 and their phylogenetic relationships with feline-like human rotavirus strains.

Feline rotaviruses, members of the species Rotavirus A, are an infrequent source of zoonotic infections, and were previously shown by RNA-RNA hybridization assays to possess two distinct genomic RNA constellations, represented by strains FRV-1 and FRV64. Due to the lack of whole genome sequence information for FRV-1, human rotavirus strain AU-1 has been used as a surrogate for the genotype constellation of feline rotaviruses. The aim of this study was to determine the whole genome sequence of FRV-1 and FRV64 to help understand the genetic relationships among existing feline rotaviruses from the evolutionary perspective. The genotype constellations of FRV-1 and FRV64 were G3-P[9]-I3-R3-C3-M3-A3-N3-T3-E3-H3 and G3-P[3]-I3-R3-C2-M3-A9-N2-T3-E3-H6, respectively. FRV-1 has a genotype constellation identical to that of the AU-1 strain. Although for individual genes they shared lineages, with the exception of genes encoding VP2, VP6 and VP7, the sequence identity between FRV-1 and AU-1 was considered to be sufficiently high for the AU-1 to be regarded as an example of the direct transmission of a feline rotavirus to a child. On the other hand, the FRV64 strain was not only similar in all the 11 genome segments to another feline rotavirus strain, Cat97, but also to canine rotavirus strains (K9 and CU-1) and feline/canine-like human rotavirus strains (Ro1845 and HCR3A). In conclusion, this study revealed intermingled sharing of genotypes and lineages among feline rotaviruses, suggesting the occurrence of frequent reassortment events over the course of evolution to emerge in four genotype constellations represented by FRV-1, FRV64/Cat97, Cat2 and BA222 strains.

Culture-independent evaluation of nonenveloped-virus infectivity reduced by free-chlorine disinfection.

The inability of molecular detection methods to distinguish disinfected virions from infectious ones has hampered the assessment of infectivity for enteric viruses caused by disinfection practices. In the present study, the reduction of infectivity of murine norovirus S7-PP3 and mengovirus vMC0, surrogates of human noroviruses and enteroviruses, respectively, caused by free-chlorine treatment was characterized culture independently by detecting carbonyl groups on viral capsid protein. The amount of carbonyls on viral capsid protein was evaluated by the proportion of biotinylated virions trapped by avidin-immobilized gel (percent adsorbed). This culture-independent approach demonstrated that the percent adsorbed was significantly correlated with the logarithm of the infectious titer of tested viruses. Taken together with the results of previous reports, the result obtained in this study indicates that the amount of carbonyls on viral capsid protein of four important families of waterborne pathogenic viruses, Astroviridae, Reoviridae, Caliciviridae, and Picornaviridae, is increased in proportion to the received oxidative stress of free chlorine. There was also a significant correlation between the percent adsorbed and the logarithm of the ratio of genome copy number to PFU, which enables estimation of the infectious titer of a subject virus by measuring values of the total genome copy number and the percent adsorbed. The proposed method is applicable when the validation of a 4-log reduction of viruses, a requirement in U.S. EPA guidelines for virus removal from water, is needed along with clear evidence of the oxidation of virus particles with chlorine-based disinfectants.

Molecular identification of a G2 rotavirus that provided a G1P[4] mono-reassortant with a DS-1-like genotype constellation.

The segmented nature of the rotavirus genome provides an opportunity for the virus to reassort upon co-infection of more than one rotavirus strain. Previously, two G1P[4] strains isolated from children with diarrhoea, AU64, and AU67, were shown by RNA-RNA hybridization to be a VP7 mono-reassortant possessing a DS-1 genogroup background. However, the origin of the parental G2 strain was not sought for at that time. The aim of this study, therefore, was to identify the G2 strain that provided AU64/AU67 with the DS-1-like genotype constellation. By carefully comparing the genomic RNA migration patterns on polyacrylamide gels of strains circulating at the same and nearby geographic locations around the time of the mono-reassortant detection (1989), a G2 strain designated 88H449 was shown to possess 10 genome segments co-migrating with those of AU64. Sequencing of all genome segments of AU64 and 88H449 showed that those two strains were 99.6-100% identical at the nucleotide level in all except the VP7 gene, indicating that the parental G2 strain that provided AU64 with the 10 genome segments was a sibling of 88H449. Sequencing of the VP7 genes of 36 G1P[8] strains circulating locally between 1981 and 1990 revealed the presence of strains bearing diverse VP7 sequences among which the closest nucleotide identity to AU64 was 98.6% and the closest amino acid identity in the major antigenic regions was 100%. This unusual mono-reassortant was concluded to be the result of a contemporary reassosrtment event occurring between locally co-circulating strains.

Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan.

Norovirus (NoV) and sapovirus (SaV) are important causes of human diarrhea. In this study, between 2007 and 2014 fecal samples were collected from 97 dogs and 83 cats with diarrhea and examined to determine the prevalence of NoV and SaV infections in Japan. To detect caliciviruses, approximately 300 bases targeting the polymerase gene were amplified using RT-PCR and subjected to phylogenetic and homology analyses. Specific PCR products were obtained from four canine and nine feline samples: two canine and one feline isolate were classified as NoV, two canine isolates as SaV and the remaining eight feline isolates as vesivirus (VeV). The three NoV isolates were classified into the same clade as that of known canine and feline NoVs; their homologies (75.9-92.3%) were higher than those with human genogroup IV (GIV) NoVs (59.1-65.9%). The homology of the feline NoV isolate with previously reported feline NoV isolates was particularly high (91.7-92.3%). Regarding SaV, the two canine isolates were classified into the same clade as known canine SaVs and their homologies (72.5-86.5%) were higher than those with other mammal SaVs (20.7-58.0%). The eight feline VeV isolates were assumed to be feline calicivirus. The present study is the first report of the presence of NoV- and SaV-infected dogs and cats in Japan. The findings suggest there are species-specific circulations of NoV and SaV among dogs and cats, in Japan.

Whole genome analysis of Vietnamese G2P[4] rotavirus strains possessing the NSP2 gene sharing an ancestral sequence with Chinese sheep and goat rotavirus strains.

Because imminent introduction into Vietnam of a vaccine against Rotavirus A is anticipated, baseline information on the whole genome of representative strains is needed to understand changes in circulating strains that may occur after vaccine introduction. In this study, the whole genomes of two G2P[4] strains detected in Nha Trang, Vietnam in 2008 were sequenced, this being the last period during which virtually no rotavirus vaccine was used in this country. The two strains were found to be >99.9% identical in sequence and had a typical DS-1 like G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2 genotype constellation. Analysis of the Vietnamese strains with >184 G2P[4] strains retrieved from GenBank/EMBL/DDBJ DNA databases placed the Vietnamese strains in one of the lineages commonly found among contemporary strains, with the exception of the NSP2 and NSP4 genes. The NSP2 genes were found to belong to a previously undescribed lineage that diverged from Chinese sheep and goat rotavirus strains, including a Chinese rotavirus vaccine strain LLR with 95% nucleotide identity; the time of their most recent common ancestor was 1975. The NSP4 genes were found to belong, together with Thai and USA strains, to an emergent lineage (VIII), adding further diversity to ever diversifying NSP4 lineages. Thus, there is a need to enhance surveillance of locally-circulating strains from both children and animals at the whole genome level to address the effect of rotavirus vaccines on changing strain distribution.

Reduction in Rotavirus Disease and Sustained Predominance of G2P[4] Rotavirus Strain following Introduction of Rotavirus Vaccine in Recife, Brazil.

Rotavirus vaccination was introduced in Brazil in March 2006. We describe the distribution of rotavirus genotypes in children with acute gastroenteritis in a hospital in Recife, Brazil, during pre- and post-vaccination periods. There was a 43.8% reduction in the proportion of diarrhea episodes due to rotavirus. Nevertheless, we observed a sustained predominance of G2P[4] as the main genotype identified in the post-vaccination period.

Histo-blood group antigen-like substances of human enteric bacteria as specific adsorbents for human noroviruses.

Histo-blood group antigens (HBGAs) have been suggested to be receptors or coreceptors for human noroviruses (HuNoVs) expressed on the intestinal epithelium. We isolated an enteric bacterium strain (SENG-6), closely related to Enterobacter cloacae, bearing HBGA-like substances from a fecal sample of a healthy individual by using a biopanning technique with anti-HBGA antibodies. The binding capacities of four genotypes of norovirus-like particles (NoVLPs) to Enterobacter sp. SENG-6 cells were confirmed by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy demonstrated that NoVLPs bound mainly to extracellular polymeric substances (EPS) of Enterobacter sp. SENG-6, where the HBGA-like substances were localized. EPS that contained HBGA-like substances extracted from Enterobacter sp. SENG-6 was shown by enzyme-linked immunosorbent assay (ELISA) to be capable of binding to NoVLPs of a GI.1 wild-type strain (8fIIa) and a GII.6 strain that can recognize A antigen but not to an NoVLP GI.1 mutant strain (W375A) that loses the ability to bind to A antigen. Enzymatic cleavage of terminal N-acetyl-galactosamine residues in the bacterial EPS weakened bacterial EPS binding to the GI.1 wild-type strain (8fIIa). These results indicate that A-like substances in the bacterial EPS play a key role in binding to NoVLPs. Since the specific binding of HuNoVs to HBGA-positive enteric bacteria is likely to affect the transmission and infection processes of HuNoVs in their hosts and in the environment, further studies of human enteric bacteria and their binding capacity to HuNoVs will provide a new scientific platform for understanding interactions between two types of microbes that were previously regarded as biologically unrelated.