Sequence analysis of the whole genomes of five African human G9 rotavirus strains

The G9 rotaviruses are amongst the most common global rotavirus strains causing severe childhood diarrhoea. However, the whole genomes of only a few G9 rotaviruses have been fully sequenced and characterised of which only one G9P[6] and one G9P[8] are from Africa. We determined the consensus sequence of the whole genomes of five African human group A G9 rotavirus strains, four G9P[8] strains and one G9P[6] strain collected in Cameroon (central Africa), Kenya (eastern Africa), South Africa and Zimbabwe (southern Africa) in 1999, 2009 and 2010. Strain RVA/Human-wt/ZWE/MRC-DPRU1723/2009/G9P[8] from Zimbabwe, RVA/Human-wt/ZAF/MRC-DPRU4677/2010/G9P[8] from South Africa, RVA/Human-wt/CMR/1424/2009/G9P[8] from Cameroon and RVA/Human-wt/KEN/MRC-DPRU2427/2010/G9P[8] from Kenya were on a Wa-like genetic backbone and were genotyped as G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Strain RVA/Human-wt/ZAF/MRC-DPRU9317/1999/G9P[6] from South Africa was genotyped as G9-P[6]-I2-R2-C2-M2-A2-N1-T2-E2-H2. Rotavirus A strain MRC-DPRU9317 is the second G9 strain to be reported on a DS-1-like genetic backbone, the other being RVA/Human-wt/ZAF/GR10924/1999/G9P[6]. MRC-DPRU9317 was found to be a reassortant between DS-1-like (I2, R2, C2, M2, A2, T2, E2 and H2) and Wa-like (N1) genome segments. All the genome segments of the five strains grouped strictly according to their genotype Wa- or DS-1-like clusters. Within their respective genotypes, the genome segments of the three G9 study strains from southern Africa clustered most closely with rotaviruses from the same geographical origin and with those with the same G and P types. The highest nucleotide identity of genome segments of the study strains from eastern and central Africa regions on a Wa-like backbone was not limited to rotaviruses with G9P[8] genotypes only, they were also closely related to G12P[6], G8P[8], G1P[8] and G11P[25] rotaviruses, indicating a close inter-genotype relationship between the G9 and other rotavirus genotypes. Rotavirus strain MRC-DPRU9317 is the first G9P[6] to be characterised on a DS-1-like genetic backbone with a reassortant segment 8 (NSP2) and fourth G9P[6] to be fully sequenced globally.     

Complete genomic analysis of a Bangladeshi G1P[8] rotavirus strain detected in 2003 reveals a close evolutionary relationship with contemporary human Wa-like strains

More than 120 variants of rotavirus strains with different VP7 (G type) and VP4 (P type) combinations are reported thus far. Among them Wa-like G1P[8] rotaviruses are the most common human strains worldwide. However, characterization of their entire genome complement is limited to a few old prototype strains, and no complete genome data for any G1P[8] strain isolated in the last decade are available. Both the currently licensed rotavirus vaccines Rotarix? and RotaTeq? possess the G1 and P[8] specificities. Therefore, comprehensive genetic information of the currently circulating G1P[8] strain is important to assess the impact of rotavirus vaccines on the circulating rotavirus strains. Here we report the complete genome sequence of a G1P[8] rotavirus strain Dhaka16-03 isolated in 2003 from a Bangladeshi child hospitalized with severe diarrhea. Based on a full-genome classification system, Dhaka16-03 was shown to posses the typical Wa-like genotype constellation: G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-E1-H1. The strain was phylogenetically more closely related to contemporary human rotavirus strains (isolated in the 2000s) with a range of G and P-genotypes than to those of the prototype G1P[8] strains. Since the vaccine strains are developed based on strains isolated several decades ago, it is important to know how much the vaccine strains differ from the currently circulating G1P[8] and other Wa-like strains. Our complete genome characterization of a recent G1P[8] strain will be helpful to assess the ongoing rotavirus vaccine trials and their implementation programs in the forthcoming years.     

Molecular characterization of rotavirus strains detected during a clinical trial of a human rotavirus vaccine in Blantyre, Malawi

The human, G1P[8] rotavirus vaccine (Rotarix?) significantly reduced severe rotavirus gastroenteritis episodes in a clinical trial in South Africa and Malawi, but vaccine efficacy was lower in Malawi (49.5%) than reported in South Africa (76.9%) and elsewhere. The aim of this study was to examine the molecular relationships of circulating wild-type rotaviruses detected during the clinical trial in Malawi to RIX4414 (the strain contained in Rotarix?) and to common human rotavirus strains. Of 88 rotavirus-positive, diarrhoeal stool specimens, 43 rotaviruses exhibited identifiable RNA migration patterns when examined by polyacrylamide gel electrophoresis. The genes encoding VP7, VP4, VP6 and NSP4 of 5 representative strains possessing genotypes G12P[6], G1P[8], G9P[8], and G8P[4] were sequenced. While their VP7 (G) and VP4 (P) genotype designations were confirmed, the VP6 (I) and NSP4 (E) genotypes were either I1E1 or I2E2, indicating that they were of human rotavirus origin. RNA-RNA hybridization using 21 culture-adapted strains showed that Malawian rotaviruses had a genomic RNA constellation common to either the Wa-like or the DS-1 like human rotaviruses. Overall, the Malawi strains appear similar in their genetic make-up to rotaviruses described in countries where vaccine efficacy is greater, suggesting that the lower efficacy in Malawi is unlikely to be explained by the diversity of circulating strains.     

Molecular characterization of group A rotaviruses detected in children with gastroenteritis in Ireland in 2006-2009

Community and hospital-acquired cases of human rotavirus are responsible for millions of gastroenteritis cases in children worldwide, chiefly in developing countries, and vaccines are now available. During surveillance activity for human rotavirus infections in Ireland, between 2006 and 2009, a total of 420 rotavirus strains were collected and analysed. Upon either PCR genotyping and sequence analysis, a variety of VP7 (G1-G4 and G9) and VP4 (P[4], P[6], P[8] and P[9]) genotypes were detected. Strains G1P[8] were found to be predominant throughout the period 2006-2008, with slight fluctuations seen in the very limited samples available in 2008-2009. Upon either PCR genotyping and sequence analysis of selected strains, the G1, G3 and G9 viruses were found to contain E1 (Wa-like) NSP4 and I1 VP6 genotypes, while the analysed G2 strains possessed E2 NSP4 and I2 VP6 genotypes, a genetic make-up which is highly conserved in the major human rotavirus genogroups Wa- and Kun-like, respectively. Upon sequence analysis of the most common VP4 genotype, P[8], at least two distinct lineages were identified, both unrelated to P[8] Irish rotaviruses circulating in previous years, and more closely related to recent European humans rotaviruses. Moreover, sequence analysis of the VP7 of G1 rotaviruses revealed the onset of a G1 variant, previously unseen in the Irish population.     

Genetic diversity of species A rotaviruses detected in clinical and environmental samples,including porcine-like rotaviruses from hospitalized children in the Philippines

Rotaviruses are the major cause of severe acute diarrhea in infants and young children. Rotaviruses exhibit zoonosis and thereby infect both humans and animals. Viruses detected in urban rivers possibly reflect the presence of circulating viruses in the catchment. The present study investigates the genetic diversity of species A rotaviruses detected from river water and stool of hospitalized children with acute diarrhea in Tacloban City, the Philippines. Species A rotaviruses were detected by real-time RT-PCR and their genotypes were identified by multiplex PCR and sequencing of partial regions of VP7 and VP4. Rotaviruses were detected in 85.7% (30/35) of the river water samples and 62.7% (151/241) of the clinical samples. Genotypes of VP7 in the river water samples were G1, G2, G3, G4, G5, and G9, and those of VP4 were P[3], P[4], P[6], P[8], and P[13]. Genotypes of viruses from the clinical samples were G2P[4], G1P[8], G3P[8], G4P[6], G5P[6], and G9P[8]. Among those, G2P[4] in clinical samples (77.9%, 81/104) and P[4] of VP4 in river water samples (67.5%, 56/83)) were the most frequently detected rotavirus genotypes. However, G5 was the more frequently detected than G2 in the river water samples (42% vs. 13%) which may be originated from porcine rotavirus. Sequence analyses of eleven gene segments revealed one G5P[6] and two G4P[6] rotaviruses in the clinical samples, wherein, several gene segments were closely related to porcine rotaviruses. The constellation of these rotavirus genes suggests the emergence of reassortment between human and porcine rotavirus due to interspecies transmission.Although two commercial rotavirus vaccines are available now, these vaccines are designed to confer immunity against the major human rotaviruses. Constant monitoring of viral variety in populated areas where humans and domestic animals live in close proximity provides vital information related to the diversity of rotaviruses in a human population.     

Surveillance and molecular characterization of rotavirus strains circulating in Manipur,North-Eastern India: Increasing prevalence of emerging G12 strains

To determine the frequency and genotypes of rotavirus strains, samples were collected from children hospitalized with acute diarrhea at the Regional Institute of Medical Sciences, Manipur. The globally common genotypes G1P[8] and G2P[4] constituted 58% of the total positive strains, while 3% and 8% strains were emerging genotypes, G9P[6] and G12P[6]. This is the first report of genotype G12 in Manipur. The G12 strains clustered with lineage III strains and had >98% identity with corresponding rotaviruses from Bangladesh, Thailand and the USA. Other uncommon G–P combinations including G4P[4], G4P[6], G10P[6] and G9P[19], along with a few strains that could not be typed were also found. The VP7 genes of G4 and G10 strains clustered with porcine and bovine strains, indicating possible zoonotic transmission. High frequency (36–62%) of rotavirus infection and predominance of G1P[8] and G2P[4] among children with acute diarrhea emphasized the need for implementation of currently available vaccines to reduce the burden of rotavirus induced diarrhea in India.     

Reassortment of Human Rotavirus Gene Segments into G11 Rotavirus Strains

G11 rotaviruses are believed to be of porcine origin. However, a limited number of G11 rotaviruses have been recently isolated from humans in combination with P[25], P[8], P[6], and P[4]. To investigate the evolutionary relationships of these strains, we analyzed the complete genomes of 2 human G11P[25] strains, 2 human G11P[8] strains, and 3 porcine reference strains. Most of the 11 gene segments of these 7 strains belonged to genotype 1 (Wa-like). However, phylogenetic clustering patterns suggested that an unknown G11P[25] strain with a new I12 VP6 genotype was transmitted to the human population, in which it acquired human genotype 1 gene segments through reassortment, resulting in a human G11P[8] rotavirus strain with an entire human Wa-genogroup backbone. This Wa-like backbone is believed to have caused the worldwide spread of human G9 and G12 rotaviruses. G11 human rotavirus strains should be monitored because they may also become major human pathogens.     

Novel NSP1 genotype characterised in an African camel G8P[11] rotavirus strain

Animal–human interspecies transmission is thought to play a significant role in influencing rotavirus strain diversity in humans. Proving this concept requires a better understanding of the complete genetic constellation of rotaviruses circulating in various animal species. However, very few whole genomes of animal rotaviruses, especially in developing countries, are available. In this study, complete genetic configuration of the first African camel rotavirus strain (RVA/Camel-wt/SDN/MRC-DPRU447/2002/G8P[11]) was assigned a unique G8-P[11]-I2-R2-C2-M2-A18-N2-T6-E2-H3 genotype constellation that has not been reported in other ruminants. It contained a novel NSP1 genotype (genotype A18). The evolutionary dynamics of the genome segments of strain MRC-DPRU447 were rather complex compared to those found in other camelids. Its genome segments 1, 3, 7–10 were closely related (>93% nucleotide identity) to those of human–animal reassortant strains like RVA/Human-tc/ITA/PA169/1988/G6P[14] and RVA/Human-wt/HUN/Hun5/1997/G6P[14], segments 4, 6 and 11 shared common ancestry (>95% nucleotide identity) with bovine rotaviruses like strains RVA/Cow-wt/CHN/DQ-75/2008/G10P[11] and RVA/Cow-wt/KOR/KJ19-2/XXXX/G6P[7], whereas segment 2 was closely related (94% nucleotide identity) to guanaco rotavirus strain RVA/Guanaco-wt/ARG/Rio_Negro/1998/G8P[1]. Its genetic backbone consisted of DS-1-like, AU-1-like, artiodactyl-like and a novel A18 genotype. This suggests that strain MRC-DPRU447 potentially emerged through multiple reassortment events between several mammalian rotaviruses of at least two genogroups or simply strain MRC-DPRU447 display a unique progenitor genotypes. Close relationship between some of the genome segments of strain MRC-DPRU447 to human rotaviruses suggests previous occurrence of reassortment processes combined with interspecies transmission between humans and camels. The whole genome data for strain MRC-DPRU447 adds to the much needed animal rotavirus data from Africa which is limited at the moment.     

Genotype distribution of Group A rotavirus from southern India, 2005–2016

Diarrheal disease due to Group A rotaviruses remain a leading cause of mortality and morbidity in the less developed parts of the world. India has started a phased roll out of rotavirus vaccine in the national immunization program. This analysis summarizes the rotavirus genotype strain distribution pre-vaccine introduction in Vellore, India from December 2005 to June 2016. Rotavirus was responsible for 32% of all diarrheal admission to the hospital. G2P[4] was the predominant strain in the initial years and was gradually replaced by G1P[8]. The emergence of G9P[4] replacing G9P[8], and the detection of G12 strains over several years were documented. There was no clear seasonality of disease. These data form the baseline to monitor genotype distribution post-vaccine introduction in Tamil Nadu.     

Genotypic linkages of VP4, VP6, VP7, NSP4, NSP5 genes of rotaviruses circulating among children with acute gastroenteritis in Thailand

Rotavirus is the main cause of acute viral gastroenteritis in infants and young children worldwide. Surveillance of group A rotavirus has been conducted in Chiang Mai, Thailand since 1987 up to 2004 and those studies revealed that group A rotavirus was responsible for about 20-61% of diarrheal diseases in hospitalized cases. In this study, we reported the continuing surveillance of group A rotavirus in 2005 and found that group A rotavirus was detected in 43 out of 147 (29.3%) stool samples. Five different G and P genotype combinations were detected, G1P[8] (27 strains), G2P[4] (12 strains), G9P[8] (2 strains), G3P[8] (1 strain), and G3P[10] (1 strain). In addition, analysis of their genotypic linkages of G (VP7), P (VP4), I (VP6), E (NSP4), and H (NSP5) genotypes demonstrated that the rotaviruses circulating in Chiang Mai, Thailand carried 3 unique linkage patterns. The G1P[8], G3P[8], and G9P[8] strains carried their VP6, NSP4, NSP5 genotypes of I1, E1, H1, respectively. The G2P[4] strains were linked with I2, E2, H2 genotypes, while an uncommon G3P[10] genotype carried unique genotypes of I8, E3 and H6. These findings provide the overall picture of genotypic linkage data of rotavirus strains circulating in Chiang Mai, Thailand.     

Human group A rotavirus infections in children in Denmark; detection of reassortant G9 strains and zoonotic P[14] strains

One of the leading causes of severe childhood gastroenteritis are group A rotaviruses, and they have been found to be associated with ∼40% of the annual gastroenteritis-associated hospitalizations in young Danish children <5 years of age (Fischer et al., 2011). In this study, we investigated the diversity of rotavirus strains circulating among young children <5 years of age, presenting with gastroenteritis disease either at the general practitioner or in the hospital, during the period 2009–2013. A total of 831 rotavirus positive stool samples were genotyped in the study period, and the majority of samples (74%) were from hospitalized children. G and P genotypes were successfully determined for 826 of samples, with G1P[8] being the most commonly detected genotype. Detection of G1 showed a decreasing trend over time, and an inverse trend was seen for the emerging G9P. The common human genotypes (G1/G3/G4/G9P[8] and G2P[4]) were detected in the majority of samples (n = 733, 88.2%). Rare genotype combinations such as G6P[14] were detected in <1% of samples. Rare genotype strains and strains which failed to amplify in genotyping RT-PCR were subjected to genetic characterization by sequencing one or all of the following genes; VP7, VP4, VP6 and NSP4. Sequences of sufficient length and quality were available for all 4 genes for 28 strains. Phylogenetic analysis revealed that reassortant G9P[4] strains circulated with 3 different genotype combinations. As rotavirus vaccines are not widely used in Denmark or its neighboring countries, the diversity of rotavirus strains identified in this study most likely reflects naturally occurring selection pressures and viral evolution.     

A rare G1P[6] super-short human rotavirus strain carrying an H2 genotype on the genetic background of a porcine rotavirus

Rotavirus strains with a rearranged 11th genome segment may show super-short RNA electropherotypes. Examples from human strains were limited to seven strains, 69M, 57M, B37, Mc345, AU19, B4106 and BE2001, which have a variety of G and P genotypes. AU19 is a rare G1P[6] human rotavirus strain detected in a Japanese infant with severe acute gastroenteritis. This study was undertaken to better understand the origin of AU19 by determining the genotype constellation of AU19. Upon nearly-full genome sequencing, AU19 had a G1-P[6]-I5-R1-C1-M1-A8-N1-T1-E1-H2 genotype constellation. Possession of I5 and A8 genotypes is indicative of its porcine rotavirus origin, whereas possession of H2 genotype is indicative of its DS-1 like human rotavirus origin. At the phylogenetic lineage level for the genome segments that share the genotype between porcine and human rotaviruses, the VP1-4, VP7, NSP3-4 genes were most closely related to those of porcine rotaviruses, but the origin of the NSP2 gene was inconclusive. As to the NSP5 gene, the lineage containing AU19 and the other three super-short human strains, 69M, 57M and B37, carrying the H2 genotype (H2b) clustered with the lineage to which DS-1- like short strains belonged (H2a) albeit with an insignificant bootstrap support. Taken all these observations together, AU19 was likely to emerge as a consequence of interspecies transmission of a porcine rotavirus to a child coupled with the acquisition of a rare H2b genotype by genetic reassortment probably from a co-circulating human strain. The addition of the AU19 NSP5 sequence to much homogeneous H2b genotypes shared by previous super-short rotavirus strains made the genetic diversity of H2b genotypes as diverse as that of the H2a genotype, lending support to the hypothesis that super-short strains carrying H2b genotype have long been circulating unnoticed in the human population.     

The laboratory test procedure to confirm rotavirus vaccine infection in severe complex immunodeficiency patients

The rotavirus vaccine is a live vaccine, and there is a possibility of infection by the virus strain used in the vaccine. We investigated the process of determining whether an infection was caused by the vaccine strain in a severe complex immunodeficiency (SCID) patient with rotavirus infection. The patient was vaccinated with RotaTeq prior to being diagnosed with SCID. The testing process was conducted in the following order: confirming rotavirus infection, determining its genotype, and confirming the vaccine strain. Rotavirus infection was confirmed through enzyme immunoassay and VP6 gene detection. G1 and P[8] were identified by multiplex polymerase chain reaction for the genotype, and G3 was further identified using a single primer. By detecting the fingerprint gene (WC3) of RotaTeq, it was confirmed that the detected virus was the vaccine strain. Genotypes G1 and P[8] were identified, and the infection was suspected of having been caused by rotavirus G1P[8]. G1P[8] is the most commonly detected genotype worldwide and is not included in the recombinant strains used in vaccines. Therefore, the infection was confirmed to have been caused by the vaccine strain by analyzing the genetic relationship between VP4 and VP7. Rotavirus infection by the vaccine strain can be identified through genotyping and fingerprint gene detection. However, genetic linkage analysis will also help to identify vaccine strains.     

Full genomic characterization of a novel genotype combination,G4P[14], of a human rotavirus strain from Barbados

Since 2004, the Pan American Health Organization (PAHO) has carried out rotavirus surveillance in Latin America and the Caribbean. Here we report the characterization of human rotavirus with the novel G–P combination of G4P[14], detected through PAHO surveillance in Barbados. Full genome sequencing of strain RVA/Human-wt/BRB/CDC1133/2012/G4P[14] revealed that its genotype is G4-P[14]-I1-R1-C1-M1-A8-N1-T1-E1-H1. The possession of a Genogroup 1 (Wa-like) backbone distinguishes this strain from other P[14] rotavirus strains. Phylogenetic analyses suggested that this strain was likely generated by genetic reassortment between human, porcine and possibly other animal rotavirus strains and identified 7 lineages within the P[14] genotype. The results of this study reinforce the potential role of interspecies transmission in generating human rotavirus diversity through reassortment. Continued surveillance is important to determine if rotavirus vaccines will protect against strains that express the P[14] rotavirus genotype.     

Burden of rotavirus gastroenteritis and distribution of rotavirus strains in Asia: a systematic review

Background

Rotavirus is the leading cause of severe diarrhea in children worldwide. We systematically reviewed the burden of rotavirus gastroenteritis (RVGE) and distribution of rotavirus strains in Asia.

Methods

We searched MEDLINE, EMBASE and the World Health Organization (WHO) website for the term “rotavirus” and the name of each country. We included studies that were conducted in children between 2000 and 2011 and that examined the epidemiology, health and/or economic burden of RVGE, and G and P-type distribution in Eastern, South East, Southern and Central Asia. Random effects models were used to pool the proportions of RVGE. We also estimated child mortality due to RVGE using the updated WHO and United Nations Children's Fund's mortality estimates in 2008.

Results

The search identified 113 eligible articles. The incidence rates of rotavirus-related hospitalizations in children under 5 years of age ranged from 2.1 to 20.0 cases per 1000 children per year with the highest rates reported in Bangladesh, South Korea, Taiwan, Thailand, and Vietnam. Rotavirus accounted for 37.5% of year-round hospitalized gastroenteritis cases, with higher proportions reported in South East Asia. Rotavirus was associated with approximately 145,000 deaths every year in Asia, with the greatest numbers occurring in India, Pakistan, and Indonesia. The highest annual societal costs of treating RVGE were reported in China (US$365 million), followed by Japan (US$254 million) and India (US$41–72 million). A diversity of rotavirus G and P-types was observed across Asia and the distribution of strains differed by country and year. The most common strains were G1P[8] (23.6%), G2P[4] (11.8%), G3P[8] (18.9%), and G9P[8] (7.4%).

Conclusions

Rotavirus is associated with substantial hospitalizations and deaths among children and causes large healthcare expenditures throughout Asia. Safe and effective rotavirus vaccines could substantially reduce the burden of disease.     

Sequence and phylogenetic analyses of human rotavirus strains: Comparison of VP7 and VP81 antigenic epitopes between Tunisian and vaccine strains before national rotavirus vaccine introduction

Group A rotaviruses (RVA) are the leading cause of severe gastroenteritis in infants and young children worldwide. Due to their epidemiological complexity, it is important to compare the genetic characteristics of vaccine strains with the RVA strains circulating before the introduction of the vaccine in the Tunisian immunization program. In the present study, the nucleotide sequences of VP7 and VP8¹ (n = 31), the main targets for neutralizing antibodies, were determined. Comparison of antigenic epitopes of 11 G1P[8], 12 G2P[4], 4 G3P[8], 2 G4P[8], 1 G6P[9] and 1 G12P[8] RVA strains circulating in Tunisia from 2006 to 2011 with the RVA strains present in licensed vaccines showed that multiple amino acid differences existed in or near putative neutralizing domains of VP7 and VP8¹. The Tunisian G3 RVA strains were found to possess a potential extra N-linked glycosylation site. The Tunisian G4 RVA were closely related to the G4 vaccine strain in RotaTeq, belonging to the same lineage, but the alignment of their VP7 amino acids revealed an insertion of an asparagine residue at position 76 which is close to a glycosylation site (aa 69–71). Despite several differences detected between Tunisian and vaccine strains, which may affect binding of neutralizing antibodies, both vaccines are known to protect against the vast majority of the circulating genotypes, providing an indication of the high vaccine efficiency that can be expected in a future rotavirus immunization program.     

Spread and predominance in Japan of novel G1P[8] double-reassortant rotavirus strains possessing a DS-1-like genotype constellation typical of G2P[4] strains

Rotavirus is a major cause of severe gastroenteritis in children <5 years of age worldwide, and two, live attenuated rotavirus vaccines are globally available. As rotavirus vaccines are introduced into national immunization programs, there is an increasing need to monitor circulating wild-type strains. However, few studies have systematically examined their full genotype constellation. This study was therefore undertaken to characterize the whole genotype constellation of circulating rotavirus strains in three widely-separated locations in Japan during the 2012 rotavirus season when rotavirus vaccines became available in the country for the first time. Of 107 rotavirus-positive specimens, 50 (46.7%) strains collected from all three locations possessed an unusual G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 constellation in which a typical G2P[4] strain appeared to have acquired its two surface protein genes from the most common G1P[8] strain. These G1P[8] double-reassortant strains were shown to possess the 11 genome segments virtually indistinguishable from each other in their nucleotide sequences and phylogenetic lineages except for two strains that underwent further intra-genotype reassortment. Successful spread to and predominance in broad locations across Japan of novel rotavirus strains possessing a genotype constellation that was previously thought not to be preferred suggests unexpected genomic flexibility of the genotype constellation.