Dominance of Emerging G9 and G12 Genotypes and Polymorphism of VP7 and VP4 of Rotaviruses from Bhutanese Children with Severe Diarrhea Prior to the Introduction of Vaccine

A prospective study was performed to determine the molecular characteristics of rotaviruses circulating among children aged <5 years in Bhutan. Stool samples were collected from February 2010 through January 2011 from children who attended two tertiary care hospitals in the capital Thimphu and the eastern regional headquarters, Mongar. The samples positive for rotavirus was mainly comprised genotype G1, followed by G12 and G9. The VP7 and VP4 genes of all genotypes clustered mainly with those of neighboring countries, thereby indicating that they shared common ancestral strains. The VP7 gene of Bhutanese G1 strains belonged to lineage 1c, which differed from the lineages of vaccine strains. Mutations were also identified in the VP7 gene of G1 strains, which may be responsible for neutralization escape strains. Furthermore, we found that lineage 4 of P[8] genotype differed antigenically from the vaccine strains, and mutations were identified in Bhutanese strains of lineage 3. The distribution of rotavirus genotypes varies among years, therefore further research is required to determine the distribution of rotavirus strain genotypes in Bhutan.     

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.     

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.     

新成人腹泻轮状病毒J19株VP2、VP3的编码基因序列分析

利用一种改良的非依赖核酸序列的单引物扩增方法,从新成人腹泻轮状病毒J19株的核酸中扩增基因,克隆至pMD18-T中并进行测序和基因序列分析。J19株的VP2、VP3的编码基因为基因2、4,分别长2 969bp、2 204bp,它们分别编码973个氨基酸和719个氨基酸。J19株的VP2蛋白序列对B组人轮状病毒IDIR株的一致性为47.2%;J19株的VP3蛋白序列对C组人轮状病毒Cowden株一致性为25.1%。对J19株VP2的遗传进化分析表明,J19株在进化树上的位置靠近外群蛋白以及A、B和C组轮状病毒分枝的根部,并且它比较偏向于B组轮状病毒的分枝。这与VP6的遗传进化分析结果相一致。根据上述结果推测J19株可能是一个与B组轮状病毒的起源和进化密切相关的毒株之一;同时,这表明VP2在研究轮状病毒的遗传进化上具有重要价值。关于新成人腹泻轮状病毒J19株VP2、VP3的编码基因的序列分析,这是首次报道。     

Interspecies Transmission of Animal Rotaviruses to Humans as Evidenced by Phylogenetic Analysis of the Hypervariable Region of the VP4 Protein

A phylogenetic tree constructed for the hypervariable region (aa 71–203) of the VP4 protein of 28 human and animal rotaviruses that were previously reported to belong to 13 distinct VP4 genotypes revealed unique positions of human rotavirus strains HCR3 and Ro1845, together with feline strain FRV64 and canine strains K9 and CU-1, in the animal rotavirus lineages, lending strong support to the view that both HCR3 and Ro1845 were of animal rotavirus origin.     

新成人腹泻轮状病毒J19株的全基因组克隆和VP4、VP6和VP7基因序列分析

为了进一步了解新成人腹泻轮状病毒J19株的基因和蛋白特征,利用一种改良的非依赖核酸序列的单引物扩增方法扩增J19株的11个基因,克隆到pMD18-T载体中并进行测序。在此基础上,将主要抗原蛋白VP4、VP6和VP7的蛋白序列与其它轮状病毒的相关蛋白序列进行比较分析并对VP6蛋白序列做遗传进化分析。结果获得J19株11个基因的全长基因序列。基因序列分析表明J19株的第3、6和第9基因分别长2 512bp、1 287bp和820bp,它们分别预测编码抗原蛋白VP4(823aa)、VP6(396aa)和VP7(258aa)。组成J19株的VP4、VP6和VP7蛋白序列对B组轮状病毒的CAL株、IDIR株以及ADRV株的相关蛋白序列的一致性分别是27.6%、38.5%和22.3%。对分组抗原蛋白VP6的遗传进化分析表明,J19株在进化树上的位置靠近外群蛋白分支以及A、B和C组轮状病毒分支的根部,而且它比较偏向于B组轮状病毒的分支。J19株的VP4、VP6和VP7蛋白序列与其它轮状病毒的相应蛋白序列存在显著差异。VP6蛋白序列的遗传进化分析表明J19株可能是一个新组轮状病毒的代表性毒株;同时,它也可能是一个与B组轮状病毒的起源和进化密切相关的毒株之一。关于新成人腹泻轮状病毒J19株11个基因的克隆及VP4、VP6和VP7基因的序列分析,这是第一次报道。     

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

The predominant rotavirus electropherotypes (e-types) during 17 epidemic seasons (1980 through 1997) in Finland were established, and representative virus isolates were studied by nucleotide sequencing and phylogenetic analysis. The virus isolates were either P[8]G1 or P[8]G4 types. The G1 and G4 strains formed one G1 lineage (VP7-G1-1) and one G4 lineage, respectively. Otherwise, they belonged to two P[8] lineages (VP4-P[8]-1 and -2) unrelated to their G types. Phylogenetic analysis of partial sequences of all 11 RNA segments obtained from the strains also revealed genetic diversity among gene segments other than those defining P and G types. With the exception of segments 1, 3, and 10, the sequences of the other segments could be assigned to 2 to 4 different genetic clusters. The results of this study suggest that, in addition to the RNA segments encoding VP4 and VP7, the other RNA segments may segregate independently as well. In total, the 9 predominant e-types represented 7 different RNA segment combinations when the phylogenetic clusters of their 11 genes were determined. The extensive genetic diversity and number of e-types among rotaviruses are best explained by frequent genetic reassortment.     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Rotavirus serotype G9 strains belonging to VP7 gene phylogenetic sequence lineage 1 may be more suitable for serotype G9 vaccine candidates than those belonging to lineage 2 or 3

A safe and effective group A rotavirus vaccine that could prevent severe diarrhea or ameliorate its symptoms in infants and young children is urgently needed in both developing and developed countries. Rotavirus VP7 serotypes G1, G2, G3, and G4 have been well established to be of epidemiologic importance worldwide. Recently, serotype G9 has emerged as the fifth globally common type of rotavirus of clinical importance. Sequence analysis of the VP7 gene of various G9 isolates has demonstrated the existence of at least three phylogenetic lineages. The goal of our study was to determine the relationship of the phylogenetic lineages to the neutralization specificity of various G9 strains. We generated eight single VP7 gene substitution reassortants, each of which bore a single VP7 gene encoding G9 specificity of one of the eight G9 strains (two lineage 1, one lineage 2 and five lineage 3 strains) and the remaining 10 genes of bovine rotavirus strain UK, and two hyperimmune guinea pig antisera to each reassortant, and we then analyzed VP7 neutralization characteristics of the eight G9 strains as well as an additional G9 strain belonging to lineage 1; the nine strains were isolated in five countries. Antisera to lineage 1 viruses neutralized lineage 2 and 3 strains to at least within eightfold of the homotypic lineage viruses. Antisera to lineage 2 virus neutralized lineage 3 viruses to at least twofold of the homotypic lineage 2 virus; however, neutralization of lineage 1 viruses was fourfold (F45 and AU32) to 16- to 64-fold (WI61) less efficient. Antisera to lineage 3 viruses neutralized the lineage 2 strain 16- to 64-fold less efficiently, the lineage 1 strains F45 and AU32 8- to 128-fold less efficiently, and WI61 (prototype G9 strain) 128- to 1024-fold less efficiently than the homotypic lineage 3 viruses. These findings may have important implications for the development of G9 rotavirus vaccine candidates, as the strain with the broadest reactivity (i.e., a prime strain) would certainly be the ideal strain for inclusion in a vaccine.     

Similarity of the outer capsid protein VP4 of the Gottfried strain of porcine rotavirus to that of asymptomatic human rotavirus strains.

Genomic segment 4 of the porcine Gottfried strain (serotype 4) of porcine rotavirus, which encodes the outer capsid protein VP4, was sequences, and its deduced amino acid sequence was analyzed. Amino acid homology of the porcine rotavirus VP4 to the corresponding protein of asymptomatic or symptomatic human rotaviruses representing serotypes 1 to 4 ranged from 87.1 to 88.1% for asymptomatic strains and from 77.5 to 77.8% for symptomatic strains. Amino acid homology of the Gottfried strain to simian rhesus rotavirus, simian SA11 virus, bovine Nebraska calf diarrhea virus, and porcine OSU strains ranged from 71.5 to 74.3%. Antigenic similarities of VP4 epitopes between the Gottfried strain and human rotaviruses were detected by a plaque reduction neutralization test with hyperimmune antisera produced against the Gottfried strain or a Gottfried (10 genes) x human DS-1 rotavirus (VP7 gene) reassortant which exhibited serotype 2 neutralization specificity. In addition, a panel of six anti-VP4 monoclonal antibodies capable of neutralizing human rotaviruses belonging to serotype 1, 3, or 4 was able to neutralize the Gottfried strain. These observations suggest that the VP4 outer capsid protein of the Gottfried rotavirus is more closely related to human rotaviruses than to animal rotaviruses.     

Isolation of human monoclonal antibodies that neutralize human rotavirus

A human antibody library constructed by utilizing a phage display system was used for the isolation of human antibodies with neutralizing activity specific for human rotavirus. In the library, the Fab form of an antibody fused to truncated cp3 is expressed on the phage surface. Purified virions of strain KU (G1 serotype and P[8] genotype) were used as antigen. Twelve different clones were isolated. Based on their amino acid sequences, they were classified into three groups. Three representative clones-1-2H, 2-3E, and 2-11G-were characterized. Enzyme-linked immunosorbent assay with virus-like particles (VLP-VP2/6 and VLP-VP2/6/7) and recombinant VP4 protein produced from baculovirus recombinants indicated that 1-2H and 2-3E bind to VP4 and that 2-11G binds to VP7. The neutralization epitope recognized by each of the three human antibodies might be human specific, since all of the antigenic mutants resistant to mouse monoclonal neutralizing antibodies previously prepared were neutralized by the human antibodies obtained here. After conversion from the Fab form of an antibody into immunoglobulin G1, the neutralizing activities of these three clones toward various human rotavirus strains were examined. The 1-2H antibody exhibited neutralizing activity toward human rotaviruses with either the P[4] or P[8] genotype. Similarly, the 2-3E antibody showed cross-reactivity against HRVs with the P[6], as well as the P[8] genotype. In contrast, the 2-11G antibody neutralized only human rotaviruses with the G1 serotype. The concentration of antibodies required for 50% neutralization ranged from 0.8 to 20 micro g/ml.     

Molecular characterization of two strains of porcine group C rotavirus

Group C rotaviruses are an important cause of acute gastroenteritis in humans and animals. Fecal samples were collected from a porcine herd in July, 2009. Group C rotavirus RNA was detected using RT-PCR for the VP6 gene. The identified strain was further characterized by sequencing and phylogenetic analysis of the partial VP4, and complete VP6 and VP7 gene sequences. The partial VP4 and complete VP6 gene sequences of the CUK-5 strain were most closely related to those of the CUK-6 strain of group C rotaviruses. Phylogenetic analysis of the VP7 gene of the 2 strains (CUK-5 and CUK-6) and reference strains of group G rotavirus by the neighbor-joining method also confirmed that CUK-5 and CUK-6 belonged to type G5 and G1 strains, respectively. This study provides useful data for the prediction of newly appearing variants of porcine group C rotaviruses in neighboring countries through comparisons with GCRVs and fundamental research for vaccine development.     

Full Genome Characterization of Novel DS-1-Like G8P[8] Rotavirus Strains that Have Emerged in Thailand: Reassortment of Bovine and Human Rotavirus Gene Segments in Emerging DS-1-Like Intergenogroup Reassortant Strains

The emergence and rapid spread of unusual DS-1-like intergenogroup reassortant rotavirus strains have been recently reported in Asia, Australia, and Europe. During rotavirus surveillance in Thailand in 2013–2014, novel DS-1-like intergenogroup reassortant strains having G8P[8] genotypes (i.e., strains KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, SWL-12, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55) were identified in stool samples from hospitalized children with severe diarrhea. In this study, we determined and characterized the complete genomes of these 12 strains (seven strains, KKL-17, PCB-79, PCB-84, PCB-85, PCB-103, SKT-107, and SWL-12, found in 2013 (2013 strains), and five, NP-130, PCB-656, SKT-457, SSKT-269, and SSL-55, in 2014 (2014 strains)). On full genomic analysis, all 12 strains showed a unique genotype constellation comprising a mixture of genogroup 1 and 2 genes: G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. With the exception of the G genotype, the unique genotype constellation of the 12 strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) was found to be shared with DS-1-like intergenogroup reassortant strains. On phylogenetic analysis, six of the 11 genes of the 2013 strains (VP4, VP2, VP3, NSP1, NSP3, and NSP5) appeared to have originated from DS-1-like intergenogroup reassortant strains, while the remaining four (VP7, VP6, VP1, and NSP2) and one (NSP4) gene appeared to be of bovine and human origin, respectively. Thus, the 2013 strains appeared to be reassortant strains as to DS-1-like intergenogroup reassortant, bovine, bovine-like human, and/or human rotaviruses. On the other hand, five of the 11 genes of the 2014 strains (VP4, VP2, VP3, NSP1, and NSP3) appeared to have originated from DS-1-like intergenogroup reassortant strains, while three (VP7, VP1, and NSP2) and one (NSP4) were assumed to be of bovine and human origin, respectively. Notably, the remaining two genes, VP6 and NSP5, of the 2014 strains appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses. Thus, the 2014 strains were assumed to be multiple reassortment strains as to DS-1-like intergenogroup reassortant, bovine, bovine-like human, human, and/or locally circulating DS-1-like G2P[4] human rotaviruses. Overall, the great genomic diversity among the DS-1-like intergenogroup reassortant strains seemed to have been generated through additional reassortment events involving animal and human strains. Moreover, all the 11 genes of three of the 2014 strains, NP-130, PCB-656, and SSL-55, were very closely related to those of Vietnamese DS-1-like G8P[8] strains that emerged in 2014–2015, indicating the derivation of these DS-1-like G8P[8] strains from a common ancestor. To our knowledge, this is the first report on full genome-based characterization of DS-1-like G8P[8] strains that have emerged in Thailand. Our observations will add to our growing understanding of the evolutionary patterns of emerging DS-1-like intergenogroup reassortant strains.     

Evolutionary history and global spread of the emerging g12 human rotaviruses

G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.     

G5型人A组轮状病毒LL36755株的VP4、VP6和NSP4编码基因的分子特征

最近在亚洲首次发现并报道了感染人的G5型人A组轮状病毒LL36755株,为进一步探讨其进化来源,克隆了G5型人A组轮状病毒LL36755株的VP4、VP6、NSP4编码基因,并分析其基因序列的分子特征。结果发现卢龙株LL36755为罕见的G5P[6]型,其VP6的亚群为SGⅡ型,NSP4的基因型为B型。系统进化树分析表明,卢龙株LL36755的VP7、VP4编码基因与猪来源的毒株关系密切,而VP6、NSP4编码基因与人来源的毒株紧密相联系。可以推断新的人腹泻A组轮状病毒LL36755株是猪的VP7,VP4编码基因与人的VP6,NSP4编码基因的自然重组;而且该毒株不是G5的原型,很可能是人类轮状病毒与猪轮状病毒毒株的自然重组后逐步进化而来。     

Complete nucleotide sequence of the gene encoding VP4 of a human rotavirus (strain K8) which has unique VP4 neutralization epitopes.

In our previous study (K. Taniguchi, Y. Morita, T. Urasawa, and S. Urasawa, J. Virol. 62:2421-2426, 1987) in which the cross-reactive neutralization epitopes on VP4 of human rotaviruses were analyzed, one strain, K8, was found to bear unique VP4 neutralization epitopes. This strain, which belongs to subgroup II and serotype 1, was not neutralized by any of six anti-VP4 neutralizing monoclonal antibodies which reacted with human rotavirus strains of serotypes 1, 3, and 4 or serotypes 1 through 4. We determined the complete nucleotide sequence of the gene encoding VP4 of strain K8 by primer extension. The VP4 gene is 2,359 base pairs in length, with 5' and 3' noncoding regions of 9 and 25 nucleotides, respectively. The gene contains a long open reading frame of 2,325 bases capable of coding for a protein of 775 amino acids. When compared with those of other human rotaviruses, VP4 of strain K8 had an insertion of one amino acid after residue 135, as found in simian rotavirus strains, and in addition, it had a deletion of one amino acid (residue 575). The amino acid homology of VP4 of strain K8 and those of other virulent human rotaviruses was only 60 to 70%. This was unusual, since over 90% VP4 homology has been found among the other virulent human rotavirus strains. In contrast, the VP7 amino acid sequence of the K8 strain was quite similar (over 98% homology) to those of other serotype 1 human rotaviruses. Thus, the K8 strain appears to have a unique VP4 gene previously not described.