Genomic characterization of a cell-culture-adapted Korean human G9P[8] rotavirus, CAU05-202

The human rotavirus G9 strain is the fifth most common rotavirus worldwide. A human rotavirus G9P[8] strain CAU05-202 was isolated from a young child with diarrhea using a cell culture system, and its major gene sequences were determined. Phylogenetic analysis of the VP7 gene revealed that CAU05-202 clustered into genetic lineage III-d and was most closely related to G9 rotaviruses from Turkey (strain GUH13) and Sri Lanka (strain 05SLC056 and 05SLC057). VP4 and NSP4 gene analysis showed that CAU05-202 belongs to the P[8]-3 lineage and genotype B, respectively. In addition, CAU05-202 has a long RNA electropherotype, supported by VP6 gene analysis, which is clearly associated with subgroup II specificity. Analysis of the G9 rotavirus strain CAU05-202 provides information concerning the genetic relationships among global rotavirus G9 strains, suggesting that closely related G9 strains are persistent and widespread in Asian countries.     

Phylogenetic analysis of rotaviruses with predominant G3 and emerging G9 genotypes from adults and children in Wuhan,China

Prevalence and phylogenetic relatedness of rotaviruses causing diarrheal diseases in children and adults were analyzed in Wuhan, China. During a period between June 2006 and February 2008, group A rotavirus was identified in 24.9% (280/1126) and 7.6% (83/1088) of specimens taken from children and adults, respectively. G3P[8] was the most frequent genotype in both children (66.3%) and adults (62.7%), followed by G1P[8] (20.3% and 26.2%, respectively). G9 was detected in specimens from six children (2.0%) and seven adults (5.6%). The VP7 genes of G3P[8] rotaviruses from children and adults showed extremely high sequence identities to each other (98.9–100%) and also to those of G3 viruses isolated in Wuhan in 2003–2004. In the phylogenetic analysis of the VP7 gene, the G3P[8] rotaviruses in Wuhan were clustered into a single lineage with some G3 viruses, which had been referred to as “the new variant G3” rotaviruses, reported recently in East Asia and Southeast Asia. Similar to G3P[8] rotaviruses, extremely high sequence identities between children and adults were observed for VP7 genes of G1 and G9 rotaviruses. The G9 viruses were clustered in the lineage of globally spreading strains, while G1 viruses were genetically close to those reported previously in China and Japan. These findings indicated the persistence of the variant G3 rotaviruses and spread of G9 rotaviruses derived from the global G9 lineage in Wuhan, and suggested that the rotaviruses were circulating among children and adults, irrelevant to the G types. J. Med. Virol. 81:382–389, 2009. © 2008 Wiley‐Liss, Inc.     

Prevalence of human rotavirus genotypes in Wuhan, China, during 2008-2011: changing trend of predominant genotypes and emergence of strains with the P[8]b subtype of the VP4 gene

Hospital-based surveillance of rotavirus genotypes was conducted in Wuhan, China, between March 2008 and May 2011. The detection rates of group A rotavirus were 24.6% (458/1859) and 12.1% (96/795) in children and adults, respectively, with diarrhea. Among the 554 positive specimens, the most frequent genotype was G3P[8] (57.9%), followed by G1P[8] (29.4%). Compared with previous studies in Wuhan (2000-2008), the relative frequency of G3P[8] has been decreasing year by year, while the predominant genotype G3 shifted to G1 in 2011. In the present study, a rare P[8]b subtype of the VP4 gene (OP354-like P[8]) was identified in nine strains. Full-length sequences of VP7, VP4, VP6 and NSP4 genes of two G9P[8]b strains (RVA/Human-wt/CHN/E1545/2009/G9P[8]b and RVA/Human-wt/CHN/Z1108/2008/G9P[8]b) were determined for phylogenetic analysis. The four genes of these strains were closely related to one another, and the G9-VP7 genes of these strains belonged to lineage III, which contains globally spreading G9 rotaviruses. The full-length sequence of VP4 gene segments of the P[8]b strains in Wuhan clustered with those of P[8]b strains in Vietnam, Russia and Belgium, while they were distinct from those of the OP354 strain from Malawi and Bangladeshi strains. The VP6 and NSP4 genes of two P[8]b strains belonged to the I1 and E1 genotype, respectively, and clustered with those of strains belonging to Wa-like human rotaviruses from various Asian countries. These findings indicate the changing epidemiologic trend of rotavirus genotypes in Wuhan, i.e., the shift of the predominant type from G3 to G1 and the emergence of P[8]b strains genetically related to those distributed in other Asian countries.     

Molecular epidemiology of rotavirus diarrhea among children in Saudi Arabia: first detection of G9 and G12 strains

In anticipation of rotavirus vaccine introduction in Saudi Arabia, this study was undertaken to determine the distribution of the G and P genotypes of rotaviruses in order to examine whether there was any emerging serotype or unusual strain circulating in children in Saudi Arabia. Of 984 stool specimens collected between 17 April 2004 and 16 April 2005, rotavirus was detected by an enzyme-linked immunosorbent assay in 187 (19%) diarrheal children less than 5 years of age. Of these, 160 (86%) were classified into G and P genotypes as follows: G1P[8] (44%), G2P[4] (20%), G9P[8] (11%), G12P[8] (4%), and G3P[8] (4%). RNA polyacrylamide gel electrophoresis identified 94 (50%) specimens as long RNA patterns, 30 (16%) specimens as short RNA patterns, and 1 mixed infection. Only a single long RNA electropherotype was identified for seven specimens containing G12P[8] rotavirus. RNA-RNA hybridization demonstrated that the G12P[8] strains were similar in their genomic constellation to locally cocirculating strains and to a Nepalese G12P[8] strain. The Saudi Arabian G12 VP7 gene had a 99% nucleotide sequence identity with Nepalese and Indian G12 VP7 genes and belonged to the third lineage. This study is the first to describe the distribution of rotavirus G and P types and also the first to identify G9P[8] and G12P[8] strains in the country.     

Molecular epidemiology of G9 rotaviruses in Taiwan between 2000 and 2002

Since the mid-1990s, novel G9 rotaviruses have been detected in many countries, suggesting that G9 is a globally important serotype. The molecular epidemiology of G9 rotaviruses in Taiwan from 2000 to 2002 was investigated in this study. G9 rotavirus first appeared in 2000 with 4 cases and constituted 33.8% and 54.8% of the rotavirus-positive samples in 2001 and 2002, respectively. These G9 strains belonged to P[8]G9, subgroup II, and long electropherotype, except one belonged to P[4]G9, subgroup II, and short electropherotype. Nucleotide sequencing and phylogenetic analysis of 52 Taiwanese G9 rotaviruses showed that the VP7 genes shared a high degree of identity to overseas G9 rotaviruses detected after 1993 and that the VP8* portions of the VP4 genes were more closely related to those of local rotaviruses of other G types. The two P[8]G9 strains with high nucleotide identities in the VP7 and the partial VP4 genes, 01TW591 of Taiwan from 2001 and 95H115 of Japan from 1995, varied in four genes, genes 2, 3, 7, and 8, which was revealed by RNA-RNA hybridization. Representative strains for different RNA patterns were also analyzed in the partial VP2 and VP3 genes; the nucleotide identities were high between Taiwanese G9 strains and local G3 or G2 strains. These results suggested that Taiwanese G9 rotaviruses possibly had evolved through reassortment between overseas G9 strains and circulating rotaviruses of other G types.     

Genetic variation of prevalent G1P[8] human rotaviruses in South Korea

The human rotavirus G1P[8] strain is one of the most common rotaviruses worldwide, including Korea. Six Korean G1P[8] human rotaviruses, isolated using cell culture techniques, were characterized on the basis of sequence differences in VP7, VP4, VP6, and NSP4 genes to elucidate the evolutionary relationships in the community. All strains had a long RNA electropherotype, supported by VP6 gene analysis, clearly associated with subgroup II specificity. The phylogenetic analysis of VP7 gene sequences showed that they all clustered into lineage I, as reported for G1 strains in Japan, China, Vietnam, and Thailand. In addition, phylogenetic analysis of the VP4 gene showed that they belong to two distinct lineages, P[8]‐II and P[8]‐III. With respect to the NSP4 gene, all strains belonged to genotype B. An understanding of the ecology and molecular evolution of rotaviruses circulating in the country is very important for the development of vaccines and vaccination strategies. This study provides new information concerning the genetic variability of the rotavirus strain G1P[8] occurring most commonly as a vaccine candidate. J. Med. Virol. 82: 886–896, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of full-length VP4 genes of OP354-like P[8] human rotavirus strains detected in Bangladesh representing a novel P[8] subtype

The G1 and G9 rotavirus strains MMC71 and MMC38 (subgroup II, NSP4 genogroup B), respectively, isolated from children in Bangladesh, were analyzed genetically. Full-length VP4 genes of these strains had 98.9% identity to each other and showed 83.9–89.4% identity to those of the P[4] and P[8] rotaviruses. Phylogenetic analysis of VP4 nucleotide sequences revealed that strains MMC38 and MMC71 were located in a lineage of P[8] strains. However, the cluster was highly divergent from the previously established P[8] strains. The VP8* portions of strains MMC38 and MMC71 showed more than 93.9% nucleotide sequence identity to OP354-like P[8] strains, and these strains were clustered into the same lineage. These findings indicate that the VP4 of these strains should be classified into a subtype of the P[8] genotype (P[8]b) that is distinct from that of common P[8] rotaviruses (P[8]a).     

Molecular characterization of a rare G9P[23] porcine rotavirus isolate from China

The fifth most important G genotype, G9 rotavirus, is recognized as an emerging genotype that is spreading around the world. Sequence analysis was completed of a rare group A rotavirus, strain G9P[23], that was designated rotavirus A pig/China/NMTL/2008/G9P[23] and abbreviated as NMTL. It was isolated from a piglet with diarrhea in China. Nucleotide sequence analysis revealed that the VP7 gene clustered within the G9 lineage VId. The VP4 gene clustered within the rare P[23] genotype. NMTL is the first porcine G9 stain reported in China. Thus, to further characterize the evolutionary diversity of the NMTL strain, all gene segments were used to draw a phylogenetic tree. Based on the new classification system of rotaviruses, the NMTL sequence revealed a G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1 genotype with close similarity to human Wa-like and porcine strains. The results showed that (i) NSP2 and NSP4 genes of NMTL exhibited higher genetic relatedness to human group A rotaviruses than to porcine strains, (ii) the VP2 and VP4 genes clustered with porcine and porcine-like human strains, and (iii) VP1 genes clustered apart from the Wa-like human and porcine clusters. In view of rotavirus evolution, this report provides additional evidence to support the notion that the human and porcine rotavirus genomes might be related.     

Clinical and molecular observations of two fatal cases of rotavirus-associated enteritis in children in Italy

Two fatal cases of infantile rotavirus enteritis occurred in northern Italy in 2005. Both children were severely dehydrated, and death was related to severe cerebral edema. Histological examination demonstrated extensive damage of the intestinal epithelium, villous atrophy or blunting, and macrophage infiltration. The two rotavirus strains were of the G1P[8] type and the long electropherotype. The 2005 G1P[8] rotaviruses differed in the NSP4, VP3, VP4, and VP7 genes from G1P[8] rotaviruses circulating in 2004, suggesting the onset of a new G1P[8] strain in the local population.     

Detection and full genomic analysis of G6P[9] human rotavirus in Japan

A rare genotype G6P[9] was identified in two human group A rotavirus strains designated as KF14 and KF17, that were detected in stool specimens from children with diarrhea in Japan. VP7 gene sequences of these two strains were identical and genetically closely related to G6 human rotavirus strains reported in European countries and the United States. To our knowledge, this is the first report of detection of a G6 human rotavirus in Japan. For further genetic analysis to elucidate the origin of the G6 rotavirus, nearly full-length sequences of all 11 RNA segments were determined for the KF17 strain. The complete genomic constellation of KF17 was determined as G6-P[9]-I2-R2-C2-M2-A3-N2-T3-E3-H3, a novel genotype constellation for human rotavirus. Phylogenetic analysis indicated that VP6, VP1-3, and NSP2 genes of KF17 clustered with bovine-like G6 human strains and some animal strains into sub-lineages distinct from those of common DS-1-like G2 human rotaviruses. On the other hand, KF17 genes encoding VP4, NSP1, and NSP3-5 showed high sequence identities to the human G3P[9] strain AU-1, and clustered with AU-1 and some feline strains within the same lineage. These findings suggested that the G6P[9] human rotavirus detected in Japan may have occurred through reassortment among uncommon bovine-like human rotaviruses and human/feline AU-1-like rotaviruses.     

Rotavirus genotypes in Slovenia: unexpected detection of G8P[8] and G12P[8] genotypes

A rotavirus surveillance study was undertaken in Slovenia from December 2005 to March 2006. Stool samples from 114 children hospitalized with acute viral gastroenteritis were collected from two main Slovenian hospitals. These confirmed rotavirus-positive samples were selected for a rotavirus G and P genotype prevalence study. Six untypable strains of genotype G were further analyzed with sequencing of the VP7, VP8*, and NSP4 genes. The findings of the study were that the G1 genotype was the most prevalent, found in 72 samples (63.2%), followed by G9 in 26 samples (22.8%), G4 in 10 samples (8.8%), and G3 in 2 samples (1.7%). All G genotypes were combined with the P[8] genotype specificity. After sequence analysis, one G8 and two G12 genotypes were also characterized. In a VP7-based phylogenetic analysis, the G8P[8] strain (SI-885/06) was more closely related to the Cody I801 bovine strain than to other human strains. Both G12 strains (SI-264/06 and SI-403/06) were shown to belong to the Se585 G12 cluster. In the VP8* phylogenetic tree, all analyzed strains except one, belonged to the P[8] lineage II and shared high identity in amino acid sequence. All characterized strains were clustered into the NSP4 genotype B. The molecular characterization of this G8 strain supports the theory of interspecies transmission of rotaviruses and animal-human genome reassortment. This is the first report on rotavirus G12 detection in Slovenia.     

Genetic variation in the VP7 gene of rotavirus G1P[8] strains isolated in Vietnam, 1998-2009

Group A rotavirus genotype G1P[8] is the most common strain affecting humans around the world over the past few decades. In this study, we examined genetic variation in the VP7 gene of rotavirus G1P[8] strains, detected in children of four major cities of Vietnam during three different rotavirus seasons: 1998-1999, 2007-2008 and 2008-2009 in order to assess the evolution of the virus over 11 years. Fecal samples (n=73) from children hospitalized for gastroenteritis caused by G1P[8] rotavirus were analyzed by DNA sequencing of gene 9 encoding the VP7 capsid protein. Phylogenetic analyses indicated that VP7 gene of the G1 strains from 1999 contained a lineage I, while rotaviruses from 2009 clustered in lineage II. Both of these lineages were found co-circulating in 2007-2008 season. While different sublineages of lineage I and II co-circulated in the 1998-1999 and 2007-2008 seasons, almost all strains in 2009 belonged to sub-lineage II-C. In the analysis using selected 10 strains, the VP4 genes of these 2 VP7-G1 lineages were all grouped in F45-like cluster. Deduced amino acid analyses indicated that there were thirteen amino acid substitutions between strains of two lineages. Of those, two were found in antigenic regions A and C, implying possible antigenic differences between these two lineages. The G1P[8] strains in Vietnam are very genetically diverse and dynamic, implying the frequent monitoring on evolution of rotavirus will be important to assess efficacy of rotavirus vaccine in Vietnam.     

Sequence analysis of Vietnamese P[6] rotavirus strains suggests evidence of interspecies transmission

Nucleotide and amino acid sequences of the VP8* gene of five Vietnamese P[6] rotavirus strains detected from hospitalized patients with acute gastroenteritis were analyzed and compared with other human and porcine P[6] rotaviruses. It is of interest that these strains had greatest identity with two Italian porcine rotavirus strains, 134/04-10 and 134/04-11. To our knowledge, these five Vietnamese rotaviruses are the rare P[6] rotavirus strains belonging to lineage I that cluster into sublineage Ic with porcine rotaviruses, and not into sublineage Ia, as other human P[6] rotaviruses have done so far. Sequence analysis of the VP7 gene of these P[6] rotavirus strains was also performed. The results showed that the Vietnamese G9P[6] strain had high similarity with other human G9 rotaviruses, confirming a human-animal reassortant virus, whereas other three G4P[6] strains had best identity with porcine G4 rotavirus strains, suggesting interspecies transmission of rotavirus between porcine and humans. This result provides the important data on molecular characteristics of Vietnamese rotaviruses, and highlights interspecies transmission events of rotaviruses in Vietnam as well as in Asia.     

Use of sequence analysis of the VP4 gene to classify recent Vietnamese rotavirus isolates

Twenty-eight strains of P(8), four of P(4) and one of P(19) rotavirus, isolated in Ho Chi Minh City, Vietnam, during 2002-2003, were investigated by sequence analysis of the VP4 gene. Seven of the 28 P(8) rotavirus VP4 sequences clustered in the P(8)-3 lineage, or the rare, so-called OP354-like lineage. Amino-acid sequence comparison revealed that Vietnamese P(8)-3 rotaviruses were generally very similar to Malawian strains, including the prototype OP354 strain. The numerical severity scores of diarrhoeal disease caused by the Vietnamese P(8)-3 rotaviruses were statistically higher than those of diarrhoeal disease caused by rotaviruses in the more common P(8)-2 lineage. Sequence and phylogenetic analysis of the VP4 gene of a Vietnamese G9P(19) rotavirus isolate showed a high degree of homology with the cognate genes of other human and porcine rotaviruses, including the prototype 4F strain.     

Molecular characterization of rare G3P[9] rotavirus strains isolated from children hospitalized with acute gastroenteritis

In 2004, an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand detected two uncommon human rotavirus strains (CMH120/04 and CMH134/04) bearing AU-1-like G3P[9] genotypes in 1 year old children hospitalized with acute gastroenteritis. The CMH120/04 and CMH134/04 rotavirus strains were characterized by molecular analyses of their VP6, VP7, VP8*, and NSP4 gene segments as well as the determination of RNA patterns by polyacrylamide gel electrophoresis (PAGE). Analysis of the VP8* gene revealed a high level of amino acid sequence identities with those of P[9] rotavirus reference strains, ranging from 94.9% to 98.3%. The highest identities were shared with the human rotavirus AU-1 strain at 97.8% and 98.3% for CMH120/04 and CMH134/04 strains, respectively. Analysis of the VP7 gene sequence revealed the highest identities with G3 human rotavirus strain KC814 at 96.6% and 96.2% for CMH120/04 and CMH134/04 strains, respectively. Based on the analyses of VP7 and VP8* genes, CMH120/04 and CMH134/04 belonged to G3P[9] genotypes. In addition, analyses of VP6 and NSP4 sequences revealed a VP6 subgroup (SG) I, with NSP4 genetic group C specificities. Moreover, both strains displayed a long RNA electrophoretic pattern. The finding of uncommon G3P[9] rotaviruses in pediatric patients provided additional evidence of the genetic/antigenic diversities of human group A rotaviruses in the Chiang Mai area of Thailand.     

Molecular characterization of VP7 gene of human rotaviruses from Bangladesh

This study was carried out during July 2005–June 2006, to characterize rotaviruses circulating in Bangladeshi children less than 5 years attended a peri-urban hospital. The proportion of rotavirus diarrhea was 39.5%. Genotype G2 was dominant (45.5%) followed by G1 (24.8%), G12 (9.6%), G9 (8.5%), and G4 (2.1%). G2 were mainly in combination with P[4], G1 and G9 with P[8], and G12 with P[6]. Phylogenetically Bangladeshi G1, G2, and G12 were closely related with the respective types from India, whereas Bangladeshi G9s of lineage III were with strains from Belgium and Australia. A G9 strain of lineage IV was clustered with strains from Sri Lanka and Turkey. Compared with prototype rotaviruses, Bangladeshi strains showed several amino acid substitutions at the antigenic sites of VP7. This study showed that the generation of diverse strains continued as evidenced by long G2, short G1 and G9 strains, and various combinations of G and P types.     

Molecular epidemiology of group A rotavirus in outpatient diarrhea infants and children in Chongqing,China, 2011-2015

Human group A rotavirus (RVA) is the leading cause of acute viral gastroenteritis in children under 5 years old worldwide. The aim of this study was to investigate the genotype distribution of RVA in the Midwest of China. Sentinel-based surveillance of acute diarrhea was conducted at Children's Hospital of Chongqing Medical University from 2011 to 2015. RVA was tested by using enzyme-linked immunosorbent assays. The partial VP4 genes and VP7 genes of rotavirus were amplified and sequenced, and genotyping and phylogenetic analyses were performed. Among the 2236 stool specimens collected from children with acute gastroenteritis, 681 (30.46%) were positive for RVA. The majority of children (89.28%) who tested positive for RVA were children aged ≤2 years. The seasonal peak of RVA was in the winter. As for genotype, four strain combinations, G9P[8], G3P[8], G1P[8], and G2P[4] contributed to 75.62% (515/681) of the RVA-associated diarrhea cases. After a marked increase in G9P[8] (30.77%) in 2013, G9P[8] became the predominant genotype in 2014 and 2015, whilst the prevalence of G1P[8] was decreased to 2.72% in 2015. Unusual G-P combinations (eg, G1P[4], G9P[4], G4P[6], G3P[4], G2P[8]) were also detected sporadically over the study period. Phylogenetic tree analysis results showed that the VP7 sequences of G9 strains were clustered into two main lineages, and 77.34% of them were clustered into lineage VI, with the highest nucleotide similarity to the strain JS12-17(China). VP4 gene sequences of P[8] strains were almost P[8]-lineage 3. Substantial temporal variation in the circulation of various genotypes of rotavirus in Chongqing was observed during 2011-2015, and highlights the need for continuous surveillance of RVA infection for better understanding and control of RVA infection.     

Predominance of G9P[8] rotavirus strains throughout France, 2014–2017

ObjectivesGroup A rotavirus is a major cause of acute gastroenteritis in young children worldwide. A prospective surveillance network has been set up in France to investigate rotavirus infections and to detect the emergence of potentially epidemic strains.MethodsFrom 2014 to 2017, rotavirus-positive stool samples were collected from 2394 children under 5 years old attending the paediatric emergency units of 13 large hospitals. Rotaviruses were genotyped by RT-PCR with regard to their outer capsid proteins VP4 and VP7.ResultsGenotyping of 2421 rotaviruses showed that after a marked increase in G9P[8] (32.1%) during the 2014–2015 season, G9P[8] became the predominant genotype during the 2015–2016 and 2016–2017 seasons with detection rates of 64.1% and 77.3%, respectively, whereas G1P[8] were detected at low rates of 16.8% and 6.6%, respectively. Phylogenetic analysis of the partial rotavirus VP7 and VP4 coding genes revealed that all of these G9P [8] strains belonged to the lineage III and the P [8]-3 lineage, respectively, and shared the same genetic background (G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1) as did most of previously detected G9P[8] strains and particularly the emerging G9P[8] strains from the 2004–2005 season in France.ConclusionsG9P[8] rotaviruses have become the predominant circulating genotype for the first time since their emergence a decade ago. In the absence of rotavirus immunization programmes in France, our data give an insight into the natural fluctuation of rotavirus genotypes in a non-vaccinated population and provide a base line for a better interpretation of data in European countries with routine rotavirus vaccination.     

Unexpected substitution of dominant rotavirus G genotypes in French hospitalized children over five consecutive seasons

The study was designed to evaluate the circulation of group A rotaviruses in French hospitalized children, and to detect unusual strains. This prospective study was conducted from 2001 to 2006 in children consulting for acute diarrhea at the pediatric emergency department in three French University Hospitals. The rotaviruses were detected by rapid test and genotyped by RT-PCR on the basis of their outer capsid proteins VP4 (P-type) and VP7 (G-type). The stools from 757 children were analyzed. G1P[8] strains were predominant (44.0%), followed by G9P[8] (17.7%), G3P[8] 13.1%, G4P[8] (9.5%), and G2P[4] (1.8%); mixed rotavirus infections occurred in 2.3%. G9 rotaviruses emerged during the 2004–2005 season (73.4%) and remained the second most prevalent strains. Few unusual strains, G6, G8, G12 and P[6]-types, were detected. The monitoring of rotavirus infections should be maintained to document strain distribution and to assess the emergence of new reassortants that may not respond to current rotavirus vaccines.