Sequence Analysis of the Gene Encoding VP4 of a Bovine Group C Rotavirus: Molecular Evidence for a New P Genotype

Nucleotide sequence of the bovine group C rotavirus Shintoku strain gene 3 was determined. Segment 3 is 2253 nucleotides (nt) in length and contains a long open reading frame (ORF) beginning at nt 22 and terminating at nt 2223. This ORF encodes a polypeptide of 733 amino acids with a predicted molecular mass of 83 kDa. The deduced gene 3 amino acid sequence shares 79% and 73% identities with VP4 of the porcine Cowden and human Bristol strains, respectively. Lack of high amino acid sequence homology in VP4 of bovine, porcine, and human group C rotaviruses indicates that the Shintoku strain represents a new P genotype.     

Molecular cloning, sequence analysis and coding assignment of the major inner capsid protein gene of human group C rotavirus.

The VP6 gene of human group C rotavirus was cloned and sequenced. Hybridization to the human group C and the porcine group C/Cowden dsRNA genomes assigned this coding sequence to segment 5. The complete human VP6 sequence contained an open reading frame of 1185 nucleotides (395 amino acids; deduced Mr 44,669 Da). The protein sequence demonstrated low homology with the group A VP6 sequences (41.7 to 42.7%) and high homology (88.9%) with the porcine group C VP6 sequence. However, the protein sequence alignments revealed a region of 10 amino acids that were significantly different between the human and the porcine group C viruses.     

Sequences of the four larger proteins of a porcine group C rotavirus and comparison with the equivalent group A rotavirus proteins.

The sequences of the four larger proteins of rotavirus group C (Cowden strain) are presented and compared with the sequences of the corresponding group A proteins. They exhibit a significant level of homology, allowing gene coding assignment for the group C rotavirus. The coding strategy of the group C virus RNA segment is the same as that for the group A large segments as one long open reading frame is present in each segment. The genome segment 1 encodes the structural protein VP1 which presents the RNA-dependent RNA polymerase consensus motifs. The VP1 protein is the most highly conserved between the rotaviruses of groups A and C. The genome segment 2 encodes the VP2 protein. The deduced protein sequence does not present the putative leucine zippers identified in the group A protein but its amino terminal is hydrophilic and highly charged as previously noted for the group A protein. The genome segment 3 encodes for a protein homologous to the group A outer capsid protein VP4. As observed among the various group A sequences, the amino terminal is the region presenting the fewest similarities. A cleavage region and a putative fusion motif similar to those present in the group A viruses have been identified. For this protein the comparison has been extended to the IDIRV [corrected] VP3 previously sequenced and indicates that groups A and C VP4 proteins are much more related to each other than to the group B equivalent. The genome segment 4 encodes for a protein showing an approximate 40% sequence identity to the minor core protein, VP3, of the group A rotavirus. This remarkable conservation of primary structures argues for severe functional constraint on the evolution of these proteins.     

Expression of the major inner capsid protein of the group B rotavirus ADRV: primary characterization of genome segment 5.

A complete cDNA copy of the fifth RNA segment of the human group B rotavirus, ADRV, has been cloned into plasmid AD512. Gene segment 5 contains 1269 bases and encodes one long open reading frame of 391 amino acids beginning at base 31 and terminating at base 1203. The gene 5 polypeptide, expressed in vitro in a rabbit reticulocyte lysate, comigrates with the 44-kDa major inner capsid protein present on EDTA treated ADRV virions. The gene 5 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent serum and by a group B-specific monoclonal antibody. In addition, this protein shares amino acid identity and similarity with the VP6 proteins from group C and group A rotavirus strains. The ADRV VP6 equivalent protein appears to be more closely related to the group C VP6 than the Group A VP6 polypeptide and a common ancestral rotavirus VP6 precursor protein is suggested. As a result, the fifth RNA segment of ADRV defines the major inner capsid protein, or VP6 equivalent, in the group B rotavirus. Expression of the ADRV VP6 equivalent protein is potentially useful for evaluating the prevalence of serum antibodies to group B rotavirus in human and animal populations as well as for generating antibodies for the direct detection of group B rotavirus antigen.     

The VP3 gene of human group C rotavirus

The complete nucleotide sequence of genome segment 4 from the human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequences of the genome termini with the consensus 5 and 3 terminal non-coding sequences of the human group C rotavirus genome revealed characteristic 5 and 3 sequence motifs. Human group C rotavirus genome segment 4 is 2, 166bp long and encodes a single open reading frame of 2,082 nucleotides (693 amino acids) starting at nucleotide 55 and terminating at nucleotide 2,136 giving a 3 untranslated region of 30 nucleotides. Alignment with the porcine group C VP3 equivalent gene showed the human gene is one amino acid longer, and that the proteins have 84.1% amino acid sequence identity. A conserved potential nucleotide binding motif shared with the porcine VP3 sequence was identified. Analogy with the group A rotaviruses suggested that the genome segment 4 encodes the group C rotavirus guanylyltransferase.     

Sequence analysis of genes encoding structural and nonstructural proteins of a human group B rotavirus detected in Calcutta, India

Nucleotide sequences of RNA segments encoding structural proteins(VP4, VP6, and VP7) and nonstructural proteins(NSP1 and NSP3) of a human group B rotavirus CAL-1, which was detected in Calcutta, India, were determined and their relatedness with cognate genes of other group B rotaviruses was analyzed. The CAL-1 genes showed generally high sequence identities (more than 90%) to those of human group B rotavirus, adult diarrheal rotavirus (ADRV) in China, while identities with bovine, murine, and ovine viruses were considerably lower (58-73%). Among RNA segments analyzed, sequence identity of the VP6 gene was relatively high compared with other gene segments. In the CAL-1 VP7 sequence, many characteristics were shared by ADRV, but not by other animal group B rotaviruses. In contrast, VP4 and NSP3 of CAL-1 were single amino acid and 23 amino acids longer than those of ADRV strain, respectively, due to differences of a few nucleotides. These findings suggested that human group B rotaviruses CAL-1 and ADRV might have originated from a common ancestral virus distinct from animal group B rotaviruses reported so far, while some notable sequence differences indicated the distinct nature of these viruses.     

Structural homologies between RNA gene segments 10 and 11 from UK bovine, simian SA11, and human Wa rotaviruses

The nucleotide sequences of gene segments 10 and 11 from UK bovine rotavirus have been determined. Gene 10 is 751 nucleotides long and contains a single long open reading frame capable of coding for a protein of 175 amino acids. When compared with the published data for gene 10 of the simian rotavirus SA11 and human Wa strains it was found to be more closely related to the SA11 structure (92% nucleotide sequence homology; 97% amino acid sequence homology) than to the human Wa structure (84% nucleotide, 86% amino acid sequence homology). All three strains have two potential N-glycosylation sites in the hydrophobic N terminus of the gene 10 protein. Gene 11 from UK bovine rotavirus is 667 nucleotides long with a single long open reading frame capable of coding for a protein of 198 amino acids. When compared with the published sequence of gene 11 from the human rotavirus Wa, the UK bovine rotavirus gene 11 was found to be one nucleotide longer in the 5'-noncoding region and three nucleotides longer in the coding region. The nucleotide sequence homology was 86%. The predicted proteins coded by segment 11 in UK and Wa rotaviruses are both rich in serine and threonine (23%) and very hydrophilic, but differ appreciably in amino acid sequence (83% homology).     

Whole genomic characterization of a human rotavirus strain B219 belonging to a novel group of the genus Rotavirus

Novel rotavirus strains B219 and ADRV-N derived from adult diarrheal cases in Bangladesh and China, respectively, are considered to belong to a novel rotavirus group (species) distinct from groups A, B, and C, by genetic analysis of five viral genes encoding VP6, VP7, NSP1, NSP2, and NSP3. In this study, the nucleotide sequences of the remaining six B219 gene segments encoding VP1, VP2, VP3, VP4, NSP4, and NSP5 were determined. The nucleotide sequences of the group B human rotavirus VP1 and VP3 genes were also determined in order to compare the whole genome of B219 with those of group A, B, and C rotavirus genomes. The nucleotide and deduced amino acid sequences of all B219 gene segments showed considerable identity to the ADRV-N (strain J19) sequences (87.7-94.3% and 88.7-98.7%, respectively). In contrast, sequence identity to groups A-C rotavirus genes was less than 61%. However, functionally important domains and structural characteristics in VP1-VP4, NSP4, and NSP5, which are conserved in group A, B, or C rotaviruses, were also found in the deduced amino acid sequences of the B219 proteins. Hence, the basic structures of all B219 viral proteins are considered to be similar to those of the known rotavirus groups.     

Genetic characterization of group C rotavirus isolated from a child hospitalized with acute gastroenteritis in Chiang Mai,Thailand

During an epidemiological survey of human rotavirus infection in Chiang Mai, Thailand, from 2002 to 2004, in which 263 stool specimens tested, one isolate of group C rotavirus was detected from a two-year-old child admitted to hospital with acute gastroenteritis. The human group C rotavirus, named CMH004/03, was characterized further by molecular analyses of its VP4, VP6, and VP7 gene segments as well as determination of RNA pattern by polyacrylamide gel electrophoresis (PAGE). Molecular characterization of VP4, VP6, and VP7 genes by sequence analyses showed high levels of sequence identities with those of human group C rotavirus reference strains isolated worldwide at 95.2% to 99.4% on nucleotide and 97.5% to 100% on amino acid levels. In contrast, the CMH004/03 strain exhibited far lesser nucleotide and amino acid sequence identities at 67.7% to 84.1% and 68.7% to 91.3%, respectively, when compared with those of porcine and bovine group C rotaviruses. Phylogenetic analyses of VP4, VP6, and VP7 genes clearly confirmed that the CMH004/03 strain clustered in a monophyletic branch with other human group C rotavirus reference strains and distantly related to the clusters of animal group C rotavirus strains. In addition, the RNA electrophoretic migration pattern of CMH004/03 showed a typical pattern (4-3-2-2) of group C rotavirus. To our knowledge, this study is the second report of group C rotavirus infection in pediatric patients in Thailand after it was reported for the first time about two decades ago.     

Completion of the genomic sequence of the simian rotavirus SA11: nucleotide sequences of segments 1, 2, and 3

The nucleotide sequences for gene segments 1, 2, and 3 of the simian rotavirus SA11 genome, coding for the structural polypeptides VP1, VP2, and VP3, respectively, have been determined. Comparison of the VP1 and VP2 amino acid sequences with those determined for other strains indicates that certain features of these proteins are conserved. The possible functions of the viral polypeptides VP1, VP2, and VP3 are discussed in the light of enzyme functions known to be present in the rotavirus particle. The complete sequence of the entire SA11 genome, which consists of 11 segments of dsRNA totaling 18,555 nucleotides, has now been determined. This is the first complete sequence available for a rotavirus genome. Each genome segment appears to code for only one primary product; there are no significant, alternative open reading frames which are conserved between strains. Relevant data for each genome segment are tabulated.     

Genomic characterization of human rotavirus G8 strains from the African rotavirus network: Relationship to animal rotaviruses

Global rotavirus surveillance has led to the detection of many unusual human rotavirus (HRV) genotypes. During 1996–2004 surveillance within the African Rotavirus Network (ARN), six P[8],G8 and two P[6],G8 human rotavirus strains were identified. Gene fragments (RT‐PCR amplicons) of all 11‐gene segments of these G8 strains were sequenced in order to elucidate their genetic and evolutionary relationships. Phylogenetic and sequence analyses of each gene segment revealed high similarities (88–100% nt and 91–100% aa) for all segments except for gene 4 encoding VP4 proteins P[8] and P[6]. For most strains, almost all of the genes of the ARN strains other than neutralizing antigens are related to typical human strains of Wa genogroup. The VP7, NSP2, and NSP5 genes were closely related to cognate genes of animal strains (83–99% and 97–99% aa identity). This study suggests that the ARN G8 strains might have arisen through VP7 or VP4 gene reassortment events since most of the other gene segments resemble those of common human rotaviruses. However, VP7, NSP2 (likely), and NSP5 (likely) genes are derived potentially from animals consistent with a zoonotic introduction. Although these findings help elucidate rotavirus evolution, sequence studies of cognate animal rotavirus genes are needed to conclusively determine the specific origin of those genes relative to both human and animal rotavirus strains. J. Med. Virol. 81:937–951, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of human group C rotavirus in Argentina

A survey was conducted for identification of human group C rotaviruses in stool specimens taken from children suffering diarrhea in suburban Buenos Aires regions. Among 90 true negative group A samples as defined by ELISA, RT-PCR and PAGE, five were positive by group C specific RT-PCR (VP7 and VP6 genes) and three of these samples exhibited the characteristic 4-3-2-2 dsRNA pattern of group C rotavirus. These results were further confirmed by electron microscopy and by ELISA for detection of group C VP6 specific antigens. Sequence analysis of the VP7 gene from one of these isolates revealed a 97.3-98.6% nucleotide identity and up to 99.1% protein homology with human group C rotavirus strains found scattered throughout the last ten years in other countries. Conversely, similar analysis performed with porcine strains showed a much lower homology degree both at the nucleotide (75.5% nucleotide identity) and amino acid level (85.5% protein homology). Detection of group C rotavirus in children with acute diarrhea in Argentina extends the identification range of this agent in the region and is consistent with previous reported data that demonstrate a global distribution of this virus.     

Molecular characterization of a rare G3P[3] human rotavirus reassortant strain reveals evidence for multiple human-animal interspecies transmissions

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian-like VP7 and caprine-like VP4 genes, was isolated from a 2-year-old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000-2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4 gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4 and VP7 genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4 genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Nucleotide sequence of gene segment 9 encoding a nonstructural protein of UK bovine rotavirus

A full-length ds cDNA copy of UK bovine rotavirus gene segment 9, which codes for a nonstructural protein, has been cloned into the PstI site of pBR322, and its sequence has been determined by cloning into bacteriophage M13mp8. Gene 9 is 1076 nucleotides long and contains a single, long, open-reading frame capable of coding for a protein of 313 amino acid residues. The possible function of this nonstructural protein in virus replication is discussed.     

The correct sequence of the porcine group C/Cowden rotavirus major inner capsid protein shows close homology with human isolates from Brazil and the U.K.

Amino acid sequence alignments between the human group C/Bristol and the published porcine group C/Cowden VP6 proteins have revealed a region of extreme sequence divergence. We have been unable to confirm the nucleotide sequence of the Cowden VP6 gene corresponding to this region of divergence. Direct sequencing of a PCR-amplified cDNA pool has revealed a frame shift, and three nucleotide changes, within the published sequence of the porcine (Cowden) VP6 gene. The corrected sequence of the porcine protein revealed a closer homology with VP6 from the Bristol strain and two new human group C rotavirus isolates. Atypical rotaviruses have been detected in the feces of children living in Belém, Brazil, and Preston, U.K. Direct sequencing of PCR-amplified cDNA corresponding to the VP6 gene of one isolate from each location confirmed the presence of a group C rotavirus. The complete nucleotide sequences of the VP6 genes from the group C/Belém and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belém VP6 gene demonstrated 97.9% nucleotide homology with the human group C/Bristol VP6 gene and 83.4% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. The Preston VP6 gene demonstrated 99.6% nucleotide homology with the human group C/Bristol VP6 gene and 84.0% nucleotide homology (91.6% deduced amino acid homology) with the corrected porcine group C/Cowden sequence. Remarkably, the deduced amino acid sequence of the Brazilian strain was identical to that of the U.K. isolates.     

New P serotype of group A human rotavirus closely related to that of a porcine rotavirus

The VP7 and VP4 genes of two human group A rotavirus strains Mc323 and Mc345 with unique serologic and genomic properties, and isolated in Chiang Mai, Thailand, in 1989 [Urasawa et al. (1992) Journal of Infectious Diseases 166:227-234] were further characterized. The nucleotide and deduced amino acid sequences of the VP7 genes allowed the classification of both strains as serotype G9. The VP4 genes of both strains are 2,359 nucleotides in length and encode a protein of 775 amino acids like in most human rotaviruses. A comparison of the VP4 amino acid sequence of strain Mc323 with those of strain Mc345 and 24 human and animal rotaviruses representing 20 distinct VP4 genotypes reported to date showed that VP4 of Mc323 and Mc345 belong to genotype 19 previously reported for porcine rotavirus [Burke et al. (1994) Journal of General Virology 75:2205-2212]. To investigate the serological type (P serotype) of these VP4s, six reassortant viruses each containing a distinct VP4 gene characteristic of human rotaviruses and the VP7 gene of porcine rotavirus strain Gottfried (G4) were prepared, and antisera to these reassortants produced in rabbits. In neutralization tests, the P serotype of Mc323 was clearly differentiated from the five major P serotypes reported previously for human rotaviruses, suggesting that Mc323 and Mc345 represent a new human rotavirus P serotype tentatively called P11.