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.     

Analysis of the genetic diversity of genes 5 and 6 among group C rotaviruses using cDNA probes

Summary Two partial cDNA clones of genes 5 (encoding the major inner capsid protein VP 6) and 6 (encoding a nonstructural protein) of the porcine group (Gp) C rotavirus (Cowden strain) were radiolabeled with³²P and used individually as probes in Northern and dot blot hybridization assays. The specificity of each probe was tested against genomic dsRNA from: (1) porcine Gp A, B, and C rotaviruses; (2) Gp C rotaviruses from different species; and (3) porcine Gp C rotavirus field strains with varying electropherotype patterns. Neither probe hybridized with ds RNA from the porcine Gp A and B strains under the stringency conditions employed in the study. However, the gene 5 probe hybridized with the corresponding gene from the homologous porcine and the heterologous human and bovine Gp C rotaviruses tested. The gene 6 probe hybridized with the corresponding gene from the homologous Cowden strain, but hybridized weakly with gene 6 from the human and bovine Gp C rotaviruses. Both probes recognized all six different porcine Gp C field strains, although with varying intensities. Our results demonstrate that the gene 5 and 6 probes used in this study are specific for Gp C rotaviruses. However, evidence for greater genetic variation in the gene 6 among porcine, bovine and human Gp C strains suggested that the gene 5 probe may prove more broadly reactive among Gp C strains from different species. cDNA probes used in our study should prove useful for the detection of Gp C rotaviruses in feces and facilitate epidemiologic studies.     

Analysis of the gene encoding the outer capsid glycoprotein (VP7) of group C rotaviruses by northern and dot blot hybridization.

The genetic diversity of gene 8 (encoding the outer capsid glycoprotein VP7) among group (Gp) C rotaviruses was examined by Northern and dot blot hybridization. A cDNA clone of the porcine Gp C Cowden strain gene 8 was labeled with 32P by nick translation and used as a probe. The gene 8 probe hybridized with the corresponding gene of one human (88-196) and four porcine (Cowden, NB, WH, and Wi) strains of Gp C rotaviruses under both moderate (50% formamide, 5X SSC, and 42 degrees) and high (50% formamide, 5X SSC, and 52 degrees) stringency conditions. However, under high stringency conditions little or no hybridization was detected with the corresponding gene of one bovine (Shintoku) and three other porcine (Ah, HF, and KH) strains of Gp C rotaviruses. In control experiments, the Cowden gene 8 probe did not hybridize with Gp A (Gottfried strain) or Gp B (Ohio strain) rotaviruses. These data demonstrate that the Cowden gene 8 probe is Gp C rotavirus-specific and that genetic diversity exists among Gp C rotaviruses in the gene encoding the outer capsid glycoprotein VP7. Our gene 8 probe may be useful in hybridization assays for serotyping Gp C rotaviruses, analogous to the use of VP7 probes for serotyping Gp A rotaviruses. However, final confirmation of our genetic approach to serotype Gp C rotaviruses awaits the serologic analysis of these viruses.     

Nucleotide sequence of gene 5 encoding the inner capsid protein (VP6) of bovine group C rotavirus: comparison with corresponding genes of group C, A, and B rotaviruses.

To further study the molecular characteristics of group (gp) C rotaviruses, we produced, cloned, and sequenced cDNA to gene 5 of the Shintoku strain of bovine gp C rotavirus. The resulting clone was specific for gene 5 and was genetically related to the human and porcine gp C rotaviruses, as demonstrated by Northern blot hybridization analysis. The Shintoku gene 5 is 1352 nucleotides in length and has one open reading frame encoding a polypeptide of 395 amino acids with a predicted molecular mass of 44.5 kDa. Comparative sequence analysis indicated that: (i) the Shintoku gene 5 protein shared 88.4 to 90.6% homology with the VP6 of the human (Bristol and 88-220) and porcine (Cowden) strains of gp C rotaviruses, but only low homology with the VP6 of bovine gp A (RF) and human gp B (ADRV) rotaviruses (41.3 and 16.3%, respectively); (ii) the predicted secondary structure was highly conserved among the gene 5 proteins of the bovine, porcine, and human gp C rotaviruses; and (iii) seven highly conserved regions were identified for the first time in the deduced primary amino acid sequences of gene 5 of gp C and gene 6 of gp A rotaviruses. However, only three of these highly conserved areas were present in the regions of VP6, where the secondary structure was predicted to be similar for the rotavirus strains examined. These three regions may contribute to common epitopes between the two groups of rotaviruses. Our results, in comparison with data for other rotaviruses, indicate that gene 5 of the bovine gp C rotavirus codes for the major inner capsid protein (VP6).     

Sequence comparison of the VP7 gene encoding the outer capsid glycoprotein among animal and human group C rotaviruses

Summary The nucleotide sequences of the outer capsid glycoprotein (VP7) genes from the Shintoku bovine and the HF and WH porcine group C rotaviruses were determined and compared with those of the published corresponding genes from the Cowden porcine and Ehime human group C rotaviruses. The VP7 genes of all 5 strains were 1063 nucleotides in length and possess one open reading frame encoding a polypeptide of 332 amino acids. Comparative analysis of the deduced amino acid sequences indicated that 85.2–97.0% identity was observed for the VP7 of the serotypically related strains of group C rotaviruses (Cowden, WH and Ehime) whereas 69.9–74.7% identity was observed among the serotypically distinct strains (Shintoku; Cowden, WH and Ehime; and HF). At least 8 variable regions in the VP7 were recognized among serotypically distinct strains, and these locations were similar to those of the variable regions in the VP7 of group A rotaviruses.     

Production and characterization of monoclonal antibodies to porcine group C rotaviruses cross-reactive with group A rotaviruses.

Five monoclonal antibodies (MAbs) to porcine group (gp) C rotaviruses (Cowden and Ah strains) reactive with both gp A and C rotaviruses in cell culture immunofluorescence (CCIF) tests were produced and characterized. These MAbs reacted with three strains of gp A and two strains of gp C rotaviruses in a CCIF test and were classified into two groups based on their CCIF titers. The MAbs also reacted to various degrees with cell-culture-propagated porcine gp C rotavirus (Cowden) and bovine gp A rotavirus (NCDV) in an enzyme-linked immunosorbent assay by using the MAbs as capture antibodies. Fecal samples containing human, bovine, and porcine strains of gp A and C rotaviruses were positive when tested using one of the MAbs in this assay. The MAbs recognized VP6 of gp A rotavirus and the VP6 counterpart (41-kDa protein) of gp C rotavirus in a Western blot assay. Results of competitive binding assays on four MAbs indicated that gp A and gp C rotaviruses share three overlapping epitopes within a single antigenic domain. These results suggest that gp A and C rotaviruses share a common antigen located on the VP6 protein, which is recognized by certain MAbs in various serologic assays.     

Evidence of serologic diversity within group C rotaviruses.

The Cowden strain of porcine group C rotavirus and the Shintoku strain of bovine group C rotavirus were classified as different serotypes by two-way cross-neutralization tests. Two neutralization patterns against the Cowden and Shintoku strains were observed when hyperimmune or convalescent-phase antisera to three noncultivatable porcine group C rotaviruses and a human group C rotavirus were used in one-way cross-neutralization tests. Antisera to two porcine group C rotaviruses and the human group C rotavirus neutralized the Cowden strain at high titers but did not neutralize the Shintoku strain, suggesting that these three strains are serotypically related to the Cowden strain. The remaining antisera to a porcine group C rotavirus (HF strain) reacted with the Cowden and Shintoku group C rotaviruses in cell culture immunofluorescence tests but did not neutralize either virus in one-way cross-neutralization, suggesting that the HF strain belongs to a third serotype. However, confirmation of these findings requires additional analysis by two-way cross-neutralization. Our findings support the existence of at least two distinct serotypes of group C rotaviruses, and possibly a third, among animals and humans. The serotypic similarity observed between the Cowden strain and a human group C rotavirus suggests that the cultivatable Cowden strain and antiserum to this virus may provide important reagents for the diagnosis of group C rotaviruses in humans.     

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.     

Sequence analysis of cDNA for the VP6 protein of group A avian rotavirus: a comparison with group A mammalian rotaviruses

Summary cDNA corresponding to the genomic segment 6 of avian rotavirus strain PO-13, which has group A common and subgroup I antigens, but does not hybridize in Northern blots with RNA probes from group A mammalian rotaviruses, was cloned and sequenced. When the deduced amino acid sequence was compared between strain PO-13 and eight group A mammalian rotaviruses, the extent of homology ranged from 73–75%. An alignment of the amino acid sequences allowed us to identify three amino acids (Positions 120, 317 and 350) that may contribute to determining the subgroup epitopes. A phylogenetic tree constructed on the basis of nucleotide substitutions in the VP6 gene of nine rotaviruses strongly suggests that the avian rotavirus is an ancestral prototype of mammalian rotaviruses.     

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.     

Comparative nucleotide and deduced amino acid sequence analysis of VP7 gene of the NCDV Cody (I-801) strain of group A bovine rotavirus

Summary The prevalent G serotypes of group A bovine rotavirus (BRV) reported are G6, G10, and less commonly, G8. Neonatal Calf Diarrhea Virus (NCDV), Lincoln and Cody strains were first isolated from diarrheic calves in Nebraska. The NCDV Lincoln strain is the currently used U.S. vaccine strain and has a G6 serotype. In this study, the complete nucleotide sequence of the VP7 gene of NCDV Cody (I-801 strain) was determined using the primer extension method. The VP7 gene nucleotide sequence homologies between Cody I-801 and established G8 rotaviruses, A5 (Thailand BRV), 678 (UK BRV), B37 (human RV) and 69M (human RV) were 84.7%, 86.4%, 84.7% and 85.9%, respectively. The deduced VP7 amino acid sequence of Cody I-801 was similar to that of A5, 678, B37 and 69M (93.6%, 95.7%, 92.6% and 95.1%, respectively). The VP7 gene nucleic acid sequence homologies between NCDV Cody (I-801) and NCDV Lincoln or B223 (G10) was 76.2% and the deduced VP7 amino acid sequence homologies between Cody I-801 and NCDV Lincoln or B223 were 82.5% and 81.3%, respectively. Thus, our sequence data suggests that the VP7 gene of Cody I-801 strain of BRV is genetically most similar to G8 rotaviruses and unrelated to the NCDV Lincoln G6 rotavirus strain.     

Comparative sequence analysis of VP7 genes from five Australian porcine rotaviruses

Summary The genes coding for the rotavirus major neutralizing protein, VP7, from 5 Australian porcine rotaviruses representing glycoprotein (i.e. VP7 or G) serotypes 3, 4, and 5, were sequenced. The genes were each 1,062 nucleotides long with two long open reading frames for proteins of either 326 or 297 amino acids and containing only one potential glycosylation site at amino acid position 69. When compared to the corresponding genes of human viruses, the porcine genes showed very high nucleotide and deduced amino acid homology. Sequence comparison also revealed that Australian porcine rotaviruses of G serotype 4 and 5 were similar to the corresponding porcine strains found in the U.S.A. and U.K., while G serotype 3 and 4 porcine rotaviruses were closely related to human G serotype 3 strain, RV-3 and serotype 4 strain, ST-3, respectively. These Australian rotavirus VP7 sequences were found to correlate with serological data we reported previously.     

Complete nucleotide sequence of a group A avian rotavirus genome and a comparison with its counterparts of mammalian rotaviruses

The nucleotide sequences encoding four structural proteins (VP1-4) and six nonstructural proteins (NSP1-6) of avian rotavirus PO-13 were determined. Based on the results of earlier sequencing studies [Ito et al., 1995, Sequence analysis of cDNA for the VP6 protein of group A avian rota viruses. Arch. Vriol. 140, 605-612; Rohwedder et al., 1997, Chicken rotavirus Ch-1 shows a second type of avian VP6 gene, Virus Genes 15, 65-71; Rohwedder et al., 1997, Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein, Virus Genes 14, 147-151], determination of PO-13 genome sequence has been completed. The PO-13 genome is 18845 nucleotides in length. It is 290 nucleotides longer than the genome of SA11. The amino acid sequence homology between PO-13 and mammalian rotaviruses ranged from 76-77% (VP1) to 16-18% (NSP1). The features of gene and amino acid sequence were compared with those of the corresponding protein of mammalian rotaviruses. Based on results of the phylogenetic analyses of NSP1, we speculate that an ancestral rotavirus could have separated into groups A, B and C rotaviruses at an early evolutionary stage and that group A rotavirus separated into mammalian and avian rotaviruses with host evolution.     

Sequence analysis of the VP6-encoding genome segment of avian group F and G rotaviruses

Rotavirus groups A to E are mainly defined by antibody reactivity to the capsid protein VP6. Additionally, two putative rotavirus groups (F and G) have been identified in birds. Here, the first nucleotide sequences of the VP6-encoding genome segment of group F (strain 03V0568) and group G (strain 03V0567) rotaviruses, both derived from chickens, are presented. The group F rotavirus is most closely related to avian group A and D rotaviruses, with 49.9-52.3% nucleotide and 36.5-39.0% amino acid sequence identity. The group G rotavirus is most closely related to mammalian group B rotaviruses, with 55.3-57.5% nucleotide and 48.2-49-9% amino acid sequence identity. The terminal sequences of the genome segment were similar in groups A, D and F, and in groups B and G. The findings indicate a long-term evolution of rotavirus groups in two separated clades and support the development of a sequence-based classification system for rotavirus groups.