Human Rotavirus Replicates in Salivary Glands and Primes Immune Responses in Facial and Intestinal Lymphoid Tissues of Gnotobiotic Pigs.

Human rotavirus (HRV) is a leading cause of viral gastroenteritis in children across the globe. The virus has long been established as a pathogen of the gastrointestinal tract, targeting small intestine epithelial cells and leading to diarrhea, nausea, and vomiting. Recently, this classical infection pathway was challenged by the findings that murine strains of rotavirus can infect the salivary glands of pups and dams and transmit via saliva from pups to dams during suckling. Here, we aimed to determine if HRV was also capable of infecting salivary glands and spreading in saliva using a gnotobiotic (Gn) pig model of HRV infection and disease. Gn pigs were orally inoculated with various strains of HRV, and virus shedding was monitored for several days post-inoculation. HRV was shed nasally and in feces in all inoculated pigs. Infectious HRV was detected in the saliva of four piglets. Structural and non-structural HRV proteins, as well as the HRV genome, were detected in the intestinal and facial tissues of inoculated pigs. The pigs developed high IgM antibody responses in serum and small intestinal contents at 10 days post-inoculation. Additionally, inoculated pigs had HRV-specific IgM antibody-secreting cells present in the ileum, tonsils, and facial lymphoid tissues. Taken together, these findings indicate that HRV can replicate in salivary tissues and prime immune responses in both intestinal and facial lymphoid tissues of Gn pigs.

Next-generation sequencing of a human-animal reassortant G6P[14] rotavirus A strain from a child hospitalized with diarrhoea.

We previously reported the VP4 and the VP7 genotypes of the first G6P[14] rotavirus strain (RVA/Human-wt/GHA/M0084/2010/G6P[14]) from the stool of an infant with diarrhoea in Ghana. In the current study, we obtained the complete genome sequences using Illumina MiSeq next-generation sequencing to enable us to determine the host species origin of the genes by phylogenetic analysis. The genotype constellation was G6-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3. Phylogenetic analysis showed that M0084 was a reassortant strain from RVAs of both artiodactyl and human host species origin. The level of sequence identity of the individual genes of M0084 to other sequences in the GenBank ranged from 95.2 to 99.5%; however, there was no single strain from the GenBank database with a complete genome sequence that was highly similar to that of M0084. To help trace the source of such unique gene pools being introduced into human RVAs, it will be useful to examine RVA sequences from potential reservoirs such as sheep and goats, which are common domestic animals in this locality.

Whole genome characterization and evolutionary analysis of OP354-like P[8] Rotavirus A strains isolated from Ghanaian children with diarrhoea.

In 2010, the rare OP354-like P[8]b rotavirus subtype was detected in children less than 2 years old in Ghana. In this follow-up study, to provide insight into the evolutionary history of the genome of Ghanaian P[8]b strains RVA/Human-wt/GHA/GHDC949/2010/G9P[8] and RVA/Human-wt/GHA/GHM0094/2010/G9P[8] detected in an infant and a 7-month old child hospitalised for acute gastroenteritis, we sequenced the complete genome using both Sanger sequencing and Illumina MiSeq technology followed by phylogenetic analysis of the near-full length sequences. Both strains possessed the Wa-like/genotype 1 constellation G9P[8]b-I1-R1-C1-M1-A1-N1-T1-E1-H1. Sequence comparison and phylogenetic inference showed that both strains were identical at the lineage level throughout the 11 genome segments. Their VP7 sequences belonged to the major sub-lineage of the G9-lineage III whereas their VP4 sequences belonged to P[8]b cluster I. The VP7 and VP4 genes of the study strains were closely related to a Senegalese G9P[8]b strain detected in 2009. In the remaining nine genome segments, both strains consistently clustered together with Wa-like RVA strains possessing either P[8]a or P[8]b mostly of African RVA origin. The introduction of a P[8]b subtype VP4 gene into the stable Wa-like strain backbone may result in strains that might propagate easily in the human population, with a potential to become an important public health concern, especially because it is not certain if the monovalent rotavirus vaccine (Rotarix) used in Ghana will be efficacious against such strains. Our analysis of the full genomes of GHM0094 and GHDC949 adds to knowledge of the genetic make-up and evolutionary dynamics of P[8]b rotavirus strains.

Genetic analysis of Ghanaian G1P[8] and G9P[8] rotavirus A strains reveals the impact of P[8] VP4 gene polymorphism on P-genotyping.

The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006-2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.

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 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.

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.

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.

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.

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.

Phylogenetic inference of the porcine Rotavirus A origin of the human G1 VP7 gene.

Rotavirus A (RVA) is an important cause of acute gastroenteritis in children worldwide. The most common VP7 genotype of human RVA is G1, but G1 is rarely detected in porcine strains. To understand the evolutionary relationships between human and porcine G1 VP7 genes, we sequenced the VP7 genes of three Japanese G1 porcine strains; the first two (PRV2, S80B) were isolated in 1980 and the third (Kyusyu-14) was isolated in 2001. Then, we performed phylogenetic and in-silico structural analyses. All three VP7 sequences clustered into lineage VI, and the mean nucleotide sequence identity between any pair of porcine G1 VP7 sequences belonging to lineage VI was 91.9%. In contrast, the mean nucleotide sequence identity between any pair of human G1 VP7 sequences belonging to lineages I-V was 95.5%. While the mean nucleotide sequence identity between any pair of porcine lineage VI strain and human lineage I-V strain was 85.4%, the VP7 genes of PRV2 and a rare porcine-like human G1P[6] strain (AU19) were 98% identical, strengthening the porcine RVA origin of AU19. The phylogenetic tree suggests that human G1 VP7 genes originated from porcine G1 VP7 genes. The time of their most recent common ancestor was estimated to be 1948, and human and porcine RVA strains evolved along independent pathways. In-silico structural analyses identified 7 amino acid residues within the known neutralisation epitopes that show differences in electric charges and shape between different porcine and human G1 strains. When compared with much divergent porcine G1 VP7 lineages, monophyletic, less divergent human G1 VP7 lineages support the hypothesis that all human G1 VP7 genes included in this study originated from a rare event of a porcine RVA transmitting to humans that was followed by successful adaptation to the human host. By contrast, AU19 represents interspecies transmission that terminated in dead-end infection.

Whole genomic constellation of the first human G8 rotavirus strain detected in Japan.

Human G8 Rotavirus A (RVA) strains are commonly detected in Africa but are rarely detected in Japan and elsewhere in the world. In this study, the whole genome sequence of the first human G8 RVA strain designated AU109 isolated in a child with acute gastroenteritis in 1994 was determined in order to understand how the strain was generated including the host species origin of its genes. The genotype constellation of AU109 was G8-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analyses of the 11 genome segments revealed that its VP7 and VP1 genes were closely related to those of a Hungarian human G8P[14] RVA strain and these genes shared the most recent common ancestors in 1988 and 1982, respectively. AU109 possessed an NSP2 gene closely related to those of Chinese sheep and goat RVA strains. The remaining eight genome segments were closely related to Japanese human G2P[4] strains which circulated around 1985-1990. Bayesian evolutionary analyses revealed that the NSP2 gene of AU109 and those of the Chinese sheep and goat RVA strains diverged from a common ancestor around 1937. In conclusion, AU109 was generated through genetic reassortment event where Japanese DS-1-like G2P[4] strains circulating around 1985-1990 obtained the VP7, VP1 and NSP2 genes from unknown ruminant G8 RVA strains. These observations highlight the need for comprehensive examination of the whole genomes of RVA strains of less explored host species.

Changes in the distribution of lineage constellations of G2P[4] Rotavirus A strains detected in Japan over 32 years (1980-2011).

Rotavirus A (RVA) is a leading cause of acute gastroenteritis in young children worldwide. Most human RVA strains are classified into three major genotype constellations: Wa-like, DS-1-like and AU-1-like. The evolution of G2P[4] strains possessing the DS-1-like genetic background was described in a few recent studies. However, the strains analyzed in these studies were almost exclusively the ones detected after 2000. In recognition of the scarcity of G2P[4] strains detected before 2000 for which whole genome information was available, this study was undertaken to characterize 19 Japanese G2P[4] strains detected between 1983 and 1990 (14 strains) and between 2001 and 2011 (5 strains), and to compare them with 131 G2P[4] strains from across the world. The Japanese strains along with the strains elsewhere in the world underwent stepwise changes from lineage I to IVa in 5 genes (the VP7, VP4, VP2, NSP1 and NSP5 genes) and from lineage I to V in 6 genes (the VP6, VP1, VP3, NSP2, NSP3 and NSP4 genes). Furthermore, G2P[4] strains detected after 2004 appeared to have undergone further intragenotype reassortment, resulting in the emergence of lineage V in the VP7 gene, and VI and VII in the VP3 and NSP4 genes. The time of the most recent common ancestor (tMRCA) for the emergent lineages VI and VII was estimated to be around the early 2000s. However, the year when the ancestor of the emergent lineages diverged from that of the rest of the lineages in the respective genes preceded the tMRCA 80-90 years. The origin of the emergent lineages is likely to be human RVA strains possessing genotypes other than G2P[4], and not RVA strains of an animal origin. In conclusion, stepwise changes in lineages imparted new genomic constellations to G2P[4] strains, which appears to have contributed to their successful spread across the globe, most notably since 2004.