Molecular characterization of a human rotavirus reveals porcine characteristics in most of the genes including VP6 and NSP4

Summary. Long electropherotype with Subgroup I specificity is a common feature of animal rotaviruses. In an epidemic of infantile gastroenteritis in Manipur, India, long but SG I strains predominated in the outbreak in the year 1987–88. One such strain isolated from that region, following the outbreak had G9P [19] specificity. As this is a rare combination, the gene sequences encoding VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 and NSP5 of this strain were analyzed. All these genes except VP7 were closely related to porcine rotaviruses (95–99% identity at amino acid level) and clustered with the porcine strains in phylogenetic analysis. In addition, it had subgroup I nature and belonged to NSP4 genotype B which is characteristic of animal rotaviruses. This is the first report of a rotavirus with VP6 and NSP4, two crucial proteins thought to be involved in host range restriction and pathogenicity, were of porcine origin and caused diarrhoea in a human host. Among the genes of this strain sequenced so far, only VP7 had highest identity to human strains at amino acid level. This study suggests reassortment may be occurring between human and other animal strains and some of the reassortant viruses may be virulent to humans.     

Molecular characterization of VP4, VP6, VP7, NSP4, and NSP5/6 genes identifies an unusual G3P[10] human rotavirus strain

An unusual strain of human rotavirus G3P[10] (CMH079/05) was detected in a stool sample of a 2‐year‐old child admitted to the hospital with severe diarrhea in Chiang Mai, Thailand. Analysis of the VP7 gene sequence revealed highest identities with unusual human rotavirus G3 strain CMH222 at 98.7% on the nucleotide and 99.6% on the amino acid levels. Phylogenetic analysis of the VP7 sequence confirmed that the CMH079/05 strain formed a cluster with G3 rotavirus reference strains and showed the closest lineage with the CMH222 strain. Analysis of partial VP4 gene of CMH079/05 revealed highest degree of sequence identities with P[10] rotavirus prototype strain 69M at nucleotide and amino acid levels of 92.9% and 94.6%, respectively. Phylogenetic analysis of the VP4 sequence revealed that CMH079/05 and 69M clustered closely together in a monophyletic branch separated from other rotavirus genotypes. To our knowledge, this is a novel G–P combination of G3 and P[10] genotypes. In addition, analyses of VP6, NSP4, and NSP5/6 genes revealed these uncommon genetic characteristics: (i) the VP6 gene differed from the four other known subgroups; (ii) the NSP4 gene was identified as NSP4 genetic group C, an uncommon group in humans; and (iii) the NSP5/6 gene was most closely related with T152, a G12P[9] rotavirus previously isolated in Thailand. The finding of uncommon G3P[10] rotavirus in this pediatric patient provided additional evidence of the genetic diversity of human group A rotaviruses in Chiang Mai, Thailand. J. Med. Virol. 81:176–182, 2009. © 2008 Wiley‐Liss, Inc.     

Detection of a porcine rotavirus strain with VP4, VP7 and NSP4 genes of different animal origins

A new rotavirus strain, sh0902, was detected in diarrheic piglets on a farm in Shanghai, China, and its genotype was characterized as G1P[7]. Analysis of the VP4, VP7 and NSP4 genes demonstrated VP4 homology to bovine and swine rotavirus strains; the nucleotide (nt) and amino acid (aa) identities were 99.7% and 99.5%, respectively. The VP7 gene was highly homologous to that of a giant panda rotavirus strain, with 98.5% similarity at the nt level and 99% similarity at the aa level. The nucleotide sequence of the NSP4 gene displayed high homology to human rotavirus strain R479, with 99.7% identity at the nt level and 99.3% identity at the aa level. This is the first report of an unusual porcine rotavirus strain with VP4, VP7 and NSP4 genes that are highly homologous to bovine, swine, giant panda and human strains isolated at geographically distant sites (South Korea, China and India). Our data indicate that rotaviruses have circulated among humans and animals and undergone genome reassortment.     

Molecular characterization of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of bovine group B rotaviruses: identification of a novel VP4 genotype

Studies on bovine group B rotaviruses (GBRs) are limited. To date, only the VP6 gene of a single bovine GBR strain and the VP7 and NSP5 genes of a few bovine GBR strains have been sequenced and analyzed. In the present study, using a single-primer amplification method, we have determined the full-length nucleotide sequences of the VP1, VP2, VP4, VP6, NSP1 and NSP2 genes of three bovine GBR strains from eastern India. In all six of these genes, the bovine GBR strains shared high genetic relatedness among themselves but exhibited high genetic diversity with cognate genes of human, murine and ovine GBRs. Interestingly, as with group A rotaviruses, the bovine GBR VP1, VP2, VP6 and NSP2 genes appeared to be more conserved than the VP4 and NSP1 genes among strains of different species. The present study provides important insights into the genetic makeup and diversity of bovine GBRs, and also identifies a novel GBR VP4 genotype.     

Molecular analysis of the VP7, VP4, VP6, NSP4, and NSP5/6 genes of a buffalo rotavirus strain: identification of the rare P[3] rhesus rotavirus-like VP4 gene allele

We report the detection and molecular characterization of a rotavirus strain, 10733, isolated from the feces of a buffalo calf affected with diarrhea in Italy. Strain 10733 was classified as a P[3] rotavirus, as the VP8* trypsin cleavage product of the VP4 protein revealed a high amino acid identity (96.2%) with that of rhesus rotavirus strain RRV (P5B[3]), used as the recipient virus in the human-simian reassortant vaccine. Analysis of the VP7 gene product revealed that strain 10733 possessed G6 serotype specificity, a type common in ruminants, with an amino acid identity to G6 rotavirus strains ranging from 88 to 98%, to Venezuelan bovine strain BRV033, and Hungarian human strain Hun4. Phylogenetic analysis based on the VP7 gene of G6 rotaviruses identified at least four lineages and an apparent linkage between each lineage and the VP4 specificity, suggesting the occurrence of repeated interspecies transmissions and genetic reassortment events between ruminant and human rotaviruses. Moreover, strain 10733 displayed a bovine-like NSP4 and NSP5/6 and a subgroup I VP6 specificity, as well as a long electropherotype pattern. The detection of the rare P[3] genotype in ruminants provides additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

Molecular characterization of the VP4, VP6, VP7, and NSP4 genes of lapine rotaviruses identified in italy: emergence of a novel VP4 genotype

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].     

Molecular characterization of the rotavirus NSP4 enterotoxin homologue from group B rotavirus

The RNA segment (Gene 10) from a human group B rotavirus which encodes the homologue of the rotavirus enterotoxin (NSP4) has been cloned and sequenced. The gene is of the same length (751 nucleotides) as its better-characterized group A rotavirus counterpart but shows minimal homology (10%) to it at the primary sequence level. Despite this low level of sequence homology, secondary structure predictions for the group B protein (ADRV-NSP4) showed a close similarity of structural features with the group A protein. Full-length ADRV-NSP4 was expressed in Escherichia coli with an amino terminal 6xHis tag that was used to purify it to homogeneity. The cytotoxicity of the purified protein was examined in a rapid dye-uptake assay that assesses membrane permeability and was found to be comparable to its group A counterpart.     

Evidence for independent segregation of the VP6- and NSP4- encoding genes in porcine group A rotavirus G6P[13] strains

Summary. Molecular characterization of two porcine group A rotavirus strains (HP113 and HP140), detected from eastern India, revealed a VP7 closely related to those of human G6P[14] strains, VP4 with a borderline P[13] genotype, and VP6 related to bovine and human SGI strains rather than porcine SGI and/or SGII group A rotaviruses. Both strains had NSP4 and NSP5 of porcine origin. Therefore, to our knowledge, the present study is the first report of detection of group A rotavirus strains with G6P[13] genotype specificities and provides evidence for independent segregation of the VP6- and NSP4-encoding genes in porcine group A rotaviruses.     

South African G4P[6] asymptomatic and symptomatic neonatal rotavirus strains differ in their NSP4, VP8*, and VP7 genes

Over the past decade, a G4P[6] strain has been found to be circulating in different neonatal wards in the Pretoria area. This endemic strain was associated with both asymptomatic and symptomatic infection, providing the opportunity to undertake a molecular study of some of the putative "virulence" genes. The genes encoding NSP4, VP8*, and VP7 of two asymptomatic and two Symptomatic strains were sequenced and compared with ST3. Within each of these genes, amino acid substitutions unique to South African strains were recorded. Four conserved amino acid differences between asymptomatic and symptomatic strains at aa 82 (serine to leucine), aa 114 (aspartic acid to glutamic acid), aa 138 (proline to threonine), and aa 169 (leucine to serine) were identified within the NSP4 gene. The hypervariable region of VP8* exhibited 10 specific amino acid differences (at aa 73, 78, 98, 111, 116, 142, 145, 167, 169, and 188) between asymptomatic and symptomatic strains, while three amino acid substitutions within VP7 were noted. These changes to VP7 occurred within the glycosylation site at aa 70 (leucine to serine), at antigenic region A (aa 96, asparagine to threonine), and at aa 318 (aspartic acid to glycine). It may be speculated that these changes are specific to G4P[6] strains. Furthermore, the observed substitutions may also be particular to South African strains. NSP4, VP8*, and VP7 have been associated with virulence and the amino acid substitutions within these genes correlate with both asymptomatic and symptomatic infection observed in neonates.     

Molecular characterization of VP4, VP6 and VP7 genes of a rare G8P[14] rotavirus strain detected in an infant with gastroenteritis in Italy

In this study, the molecular characterization of a rare G8P[14] group A rotavirus (GARV) strain detected in Northern Italy during the 2004-2005 epidemiological rotavirus season is described. Two hundred and seventy three rotavirus-like particle positive stools out of 856 stools from children (31.9%) hospitalized with gastroenteritis were analyzed using polyacrilamide gel electrophoresis and 271 GARVs were genotyped by VP7 and VP4 specific RT-PCRs. One strain (PR/1300/04) with a long electropherotype (e-type) displayed the G8 specificity and was VP4 un-typeable. The P and the subgroup (SG) specificities were determined by sequencing the VP4 and the VP6 gene, respectively. The PR/1300/04 strain exhibited P[14] and SGI specificities. By sequence and phylogenetic analyses of the VP4, VP6 and VP7 amplicons, the PR/1300/04 VP4 and VP6 genes were demonstrated to be of human rotavirus origin, with the VP4 gene closely related to the human Italian PA169 strain (G6P[14]), while the VP7 gene was of animal origin (bovine). These data suggest that the Italian PR/1300/04 strain could be a reassortant between a PA169-like Italian strain with P[14] specificity, long e-type and SGI, and a G8 animal strain. The increasing number of reports of atypical GARVs in humans suggests that interspecies transmission of genes greatly contributes to the GARV genetic evolution.     

Multiple‐gene characterization of rotavirus strains: Evidence of genetic linkage among the VP7‐, VP4‐, VP6‐, and NSP4‐encoding genes

A total of 162 rotavirus strains detected between 1996 and 2006 among individuals with diarrhea in Rio de Janeiro, Brazil, were analyzed by multiple‐gene genotyping. Characterization of strains was done by RT‐PCR assay for amplification and typing of the VP7‐, VP4‐, VP6‐, and NSP4‐encoding genes. Overall, 139 (85.8%) strains belonged to the common group A rotavirus combinations: 67 (41.4%) belonged to genotype G1‐P[8]‐I1‐E1; 18 (11.1%) were G2‐P[4]‐I2‐E2; 11 (6.8%) were G3‐P[8]‐I1‐E1; 12 (7.4%) were G4‐P[8]‐I1‐E1; and 31 (19.1%) were G9‐P[8]‐I1‐E1. Two samples presented mixed genotypes (G1 + G3‐P[8]‐I1‐E1 and G1 + G9‐P[9]‐I1‐E1) and rare combinations, such as G2‐P[6]‐I2‐E2 and G9‐P[6]‐I2‐E2, were detected in six (3.7%) strains. The results suggest a linkage among all four genes. Genotypes G1/G3/G4/G5/G9‐P[8] were correlated strongly to I1‐E1 genotypes and G2‐P[4]/P[6] were correlated to I2‐E2 genotypes. Unusual combinations of genes, such as G3‐P[9]‐I2‐E2, G9‐P[9]‐I1‐E2, and G3‐P[9]‐I3‐E3, were observed in 15 (9.3%) strains. The characterization of multiple genes allows a more complete analysis of the rotavirus isolates and provides evidence of natural reassortment of strains. J. Med. Virol. 82:1797–1802, 2010. © 2010 Wiley‐Liss, Inc.     

VP4 relationships between porcine and other rotavirus serotypes

Summary VP4 relationships of Australian porcine rotaviruses were identified using genetic reassortants and MAbs. All porcine virus isolates except BEN-144 appeared to share VP4 antigenicity with OSU virus. VP4 of BEN-144 virus (Gottfried-like virus) showed some antigenic relationships with the human neonatal viruses ST-3 and RV-3. In addition, VP4 of porcine CRW-8 showed antigenic relationships with simian SA-11, RRV and also canine K9 viruses, while that of porcine TFR-41 showed at least one way VP4 antigenic relatedness with UK bovine rotavirus. Furthermore, BMI-1 virus which is antigenically similar to an American virus SB1-A (a naturally occurring reassortant) may have arisen similarly by gene reassortment in nature in Australia.     

新的成人腹泻轮状病毒J19株NSP4和NSP5基因克隆和序列分析

目的克隆新的成人腹泻轮状病毒J19株NSP4和NSP5基因,并分析其基因序列.方法利用一种改进的非依赖核酸序列的单引物扩增方法扩增J19株NSP4和NSP5基因,克隆到pMD18-T载体中并进行测序.在此基础上,将J19株NSP4和NSP5的蛋白序列与其他轮状病毒蛋白序列进行比较分析和种系进化分析.结果J19株NSP4和NSP5基因为基因10和11,全长为739 bp和649 bp,它们分别编码213个和176个氨基酸.与J19株NSP4和NSP5蛋白序列一致性较高的分别是B组成人腹泻轮状病毒Bang373株（20.3%）和B组猪轮状病毒db101株（29.5%）.对J19株的NSP4和NSP5的遗传进化分析表明,J19株在进化树上的位置都靠近A、B和C组轮状病毒分支的根部,而且它比较偏向B组轮状病毒的分支.结论J19株的NSP4和NSP5与其他轮状病毒的相应蛋白序列存在显著差异.J19株NSP4和NSP5的蛋白序列比较和遗传进化分析表明新的成人腹泻轮状病毒与成人腹泻轮状病毒可能有共同起源;但是新的成人腹泻轮状病毒与成人腹泻轮状病毒存在显著差异.     

Detection of human rotavirus G9P[8] strains circulating in Argentina: phylogenetic analysis of VP7 and NSP4 genes

During the surveillance of rotavirus strains that were circulating in Argentinean children from 2000 to 2004, seven rotaviruses were detected bearing the genotype combination G9P[8]. The molecular characterization of the VP7 and NSP4 genes and the RNA migration patterns support the hypothesis that rotaviruses G9 could have been reintroduced into Argentina as a novel G9P[8] strain, rather than represent VP7 gene reassortants from G9P[6] strains that had been circulating previously in this country.     

中国轮状病毒非结构蛋白NSP4基因变异特征的分析

目的　研究我国轮状病毒流行株NSP4基因的变异特点。方法　对近年来从我国不同地区获得的 2 7份人轮状病毒流行株的NSP4基因用RT PCR进行扩增 ,克隆后进行全长cDNA序列分析 ,并利用Clustal× 1.8,TreeView3 2及DNAStar软件与参比株Wa、KUN、AU 1、EW及来自GenBank的OSU、SA11、Hochi、US2 44、Bristol株的NSP4序列进行分析比较。采用PCR分型方法对VP7血清型进行鉴定 ,确定轮状病毒G型与NSP4基因型的关系。结果　氨基酸同源性比较表明 ,我国轮状病毒不同流行株NSP4之间同源性为 81.7%～ 99.4% ,据此可将 2 7株RVNSP4分为 2组 ,分别以Wa株和KUN株为代表 ,其中以Wa组为主。组内同源性分别为 92 .0 %～ 99.4%和 92 .0 %～ 98.9% ,组内变异率分别为 0～ 8 5 %及 1 2 %～ 8 5 %。两组间变异率达 16 6%～ 2 1 0 %。氨基酸进化树提示在Wa组内包括 3个亚组。轮状病毒G血清型与NSP4基因型之间的联系不确定。结论　我国流行株NSP4基因主要可分为Wa组和KUN组 ,在Wa组内可形成三个亚组 ,并且在高变区有特征性的氨基酸位点。NSP4的变异与年份有关而与地域关系不密切     

VP4 serotype of the Gottfried strain of porcine rotavirus.

The antigenic relationships of the VP4 serotype of porcine rotavirus Gottfried strain with other rotaviruses were determined by using antiserum to Gottfried VP4-baculovirus recombinant. This antiserum failed to react significantly with virus of serotypes P1A, P1B, P3, and OSU; however, it reacted with P2 strains. In the reciprocal assay, antiserum to VP4 of an asymptomatic strain (P2) failed to neutralize the Gottfried strain virus to a significant level. It thus appears that the Gottfried strain should be considered a subtype of P2.