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.     

Avian-to-mammal transmission of an avian rotavirus: analysis of its pathogenicity in a heterologous mouse model.

It has been suggested that group A avian rotaviruses can be transmitted to mammals, but there is no direct evidence that such viruses induce disease in mammals. Suckling mice were orally inoculated with two avian rotaviruses. A pigeon rotavirus, PO-13, was found to induce diarrhea, but a turkey rotavirus, Ty-3, did not. The diarrhea induced by PO-13 was dependent on the age of the mouse. In histopathological examinations, antigens of PO-13 were sporadically detected in absorptive cells in the ileum, and lesions were observed as ballooning degenerations of absorptive cells in a region from the duodenum to the ileum. However, the rotavirus antigen was not detected in the majority of these degenerative cells. These results indicated that PO-13 could infect and induce diarrhea in suckling mice. This is the first evidence of an avian rotavirus being experimentally transmissible to a mammal.     

Bovine Rotavirus 993/83 Shows a Third Subtype of Avian VP7 Protein

VP7 genes of rotavirus (RV) 993/83 isolated from a German calf with diarrhea and of RV PO-13 isolated from a Japanese pigeon were sequenced. Alignment of the deduced VP7 amino acid sequence showed 98.8% sequence identity, while only 70% and 84% identity was seen with VP7 from chicken RV Ch-2 and turkey RV Ty-1, respectively. Over the antigenic regions A, B, and C mammalian RV 993/83 showed more aa identity with mammalian G3 RVs than with chicken RV Ch-2, which could explain the strong one-way cross-neutralization observed between RV 993/83 and G3 RVs. Despite marked VP7 sequence diversity avian RVs could not be differentiated into distinct G types. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

The avian rotavirus Ty-1 Vp7 nucleotide and deduced amino acid sequences differ significantly from those of Ch-2 rotavirus

Summary The Vp7 gene of the avian strain Ty-1, which is classified as G7 serotype, was sequenced and the amino acid sequence deduced. The gene is 1065 nucleotides long with a long open reading frame of 987 nucleotides producing a protein 329 amino acids in length. The amino acid homology of the Ty-1 Vp7 protein to that of the avian Ch-2 Vp7 was 70%. The A, B, and C variable epitope regions of Ty-1 were unique compared to those of Ch-2 and other strains representing the 14 G serotypes. The low 53% homology of the A and C regions of Ty-1 and Ch-2 would suggest that Ty-1 may be of a different serotype to the G7 reference strain Ch-2.     

Chicken Rotavirus Ch-1 Shows a Second Type of Avian VP6 Gene

VP6 protein from chicken rotavirus Ch-1 showed more than 13% amino acid differences in comparison with pigeon and turkey VP6 sequences. This difference is greater than that observed between subgroup I and II mammalian rotavirus VP6 sequences. Phylogenetic tree analysis demonstrated that RV Ch-1 VP6 is not a link between avian and mammalian VP6 sequences. RV Ch-1 showed variant aa in 17 positions which were otherwise absolutely conserved in mammalian and avian group A RVs. The 17 replacements were scattered through the entire VP6 sequence except the C-terminal part implicated in the assembly of subviral particles. In RV Ch-1 the proline residue 309, reported to be critical for the trimerization of VP6, was replaced by leucine, but VP6 trimers were still observed. The sequence and hydrophilicity analysis of avian RV VP6 do not explain the anomalous electrophoretic migration behavior of avian VP6 proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of interspecies transmission and reassortment among avian group A rotaviruses

Avian rotaviruses are broadly distributed among birds, but only scarcely characterized on the molecular level. The VP4-, VP6-, VP7- and NSP5-encoding sequences of eight group A rotaviruses from chickens and turkeys determined here indicate a low degree of sequence similarity with mammalian rotaviruses. An NSP6-encoding region was missing in all chicken isolates except for isolate Ch2. Four novel genotypes (P[30], P[31], G22 and H8) were assigned by the Rotavirus Classification Working Group. Generally, chicken and turkey isolates clustered into separate branches of phylogenetic trees. However, chicken isolate Ch2 consistently clustered together with turkey isolates. Chicken isolate 06V0661G1 has a VP4-encoding sequence of unknown origin, but possesses VP6, VP7 and NSP5 genotypes typical for chicken isolates. These results might indicate interspecies transmission and reassortment among avian group A rotaviruses under field conditions. PCR protocols enabling amplification of avian and mammalian group A rotaviruses were developed for use in further epidemiological studies.     

Detailed Computational Analysis of a Comprehensive Set of Group A Rotavirus NSP4 Proteins

Rotavirus infection causes diarrhea to humans, animals and birds. The NSP4 protein of Group A rotavirus has been recognized as a viral enterotoxin. This single protein plays important roles in viral pathogenesis and morphogenesis. Domains involved in structure and biologic functions have been proposed mainly based on the SA11 strain, a prototype of group A rotavirus. NSP4 has been classified into different genotypes based on sequence homology. These analyses are based on representative strains selected but not comprehensive. In this paper, we collected all NSP4 sequences in the GenBank and performed a detailed computational analysis. Our analysis of 176 NSP4 proteins in Groups A, B and C rotaviruses confirms that the recently published avian NSP4 sequences belong to a new genotype (Mori Y., Borgan M.A., Ito N., Sugiyama M. and Minamoto N., Virus Res 89, 145–151, 2002), besides the four known NSP4 genotypes of Group A mammalian rotaviruses. Significant differences were discovered in the physicochemical properties between the avian and mammalian NSP4 proteins. In particular, lack of a highly probable coiled-coil region in the avian sequences implies a diversion of the NSP4 quaternary structure from the latter, although the secondary and tertiary structures may be similar. Fourteen amino acids are found absolutely conserved in the Group A NSP4 sequences, regardless of genotype. Of the conserved residues, two are glycosylation sites, one is in the middle of the transmembrane segment, seven span the VP4 binding domain, and five are clustered in the middle of the toxic peptide region, indicating the functional importance of the conservation.     

Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies

Summary Monoclonal antibodies (MAbs) were prepared to analyze antigens on the major inner capsid protein, VP 6 of avian group A rotavirus. Based on the results of a competitive binding assay using 15 MAbs directed against VP 6 of the PO-13 rotavirus strain, isolated from a pigeon in Japan, it was found that VP 6 of avian rotavirus possesses at least four spatially distinct antigenic sites. Two antigenic sites (I and II) were topologically distinct from the other two (III and IV), which were in close proximity. From the reaction of MAbs in indirect immunofluorescent antibody tests to a series of known rotaviruses, epitopes representing common antigens of all group A rotavirus including avian rotavirus were localized in sites II and III. One epitope in site IV appeared to have a subgroup antigenic specificity that reacted only with rotaviruses belonging to subgroup I. Interestingly, avian rotaviruses isolated from turkeys and chickens in Northern Ireland also reacted only with these subgroup I specific MAbs, but not with subgroup II specific MAb. This indicates that avian rotavirus has subgroup I specific antigen, which is antigenically similar to that of other mammalian rotavirus strains.     

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.     

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.     

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.     

A Human Vaccine Strain of Lamb Rotavirus (Chinese) NSP4 Gene: Complete Nucleotide Sequence and Phylogenetic Analyses

A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Comparative analyses was performed to identify differences from human rotavirus strains, that might be associated with attenuation, and to ascertain whether the lamb rotavirus gene fits among the NSP4 of other sequenced rotavirus strains. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. A group A-specific NSP4 monoclonal antibody recognized the glycosylated and unglycosylated forms of the protein from virus-infected lysates, suggesting a well-conserved group-specificity of the lamb NSP4. Phylogenetic analysis of the lamb rotavirus gene, with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.     

Mapping of antigenic sites on the major inner capsid protein of avian rotavirus using anEscherichia coli expression system

Summary The cDNA encoding the VP6 gene of avian rotavirus PO-13 strain was inserted into the bacterial expression vector pET-3a. Upon isopropyl-l-thio--D-galactoside induction, theE. coli BL21 (DE3) harboring the vector containing cDNA of the VP6 gene produced an approximately 45-kDa polypeptide, which reacted with rabbit serum against PO-13 strain in Western blotting. To study the antigenic sites on VP6, various deletion mutants were constructed, expressed inE. coli and the reactivity with antigenic site I- and II-specific MAbs analyzed by Western blotting. Site I, which is shared with all group A mammalian and avian rotaviruses except for chicken rotavirus, was found to be located at amino acid positions 45 to 65, and site II, which probably contributes to an authentic group A antigen common to both mammalian and avian rotaviruses, at amino acid positions 134 to 142.     

Genetic analysis of group B human rotaviruses detected in Bangladesh in 2000 and 2001

Group B rotaviruses detected in Bangladesh in 2000 and 2001 were analyzed genetically to clarify relatedness to human group B rotaviruses reported previously in China and India, and to animal group B rotaviruses. VP7 gene sequences of the Bangladeshi group B rotaviruses (Bang373, Bang544, Bang334, and Bang402) were almost identical to each other and also showed high sequence identity to the Indian strain CAL-1 (98%) and Chinese strain adult diarrhea rotavirus (ADRV) (92%), while identities to bovine and murine viruses were considerably low (60-63%). Other genes of Bang373 and Bang544 encoding VP2, VP4, VP6, and NSP1 through NSP5 also showed much higher sequence identities to those of CAL-1 (97.7-99.4%) than to those of ADRV (89.9-93.9%). Characterization of nucleotide substitutions among Bang373, CAL-1, and ADRV suggested that all the gene segments might have evolved neutrally at similar mutation rates, while some of the gene segments (e.g., VP2 gene) were suggested to be more conserved than others. In conclusion, group B rotaviruses detected in Bangladesh represented by Bang373 and the Indian virus CAL-1 were considered as virtually identical viruses which are distinct genetically from ADRV, and it was suggested that Bang373 (CAL-1)-like group B rotavirus (Bengali strains) might be distributed primarily in an area around the Bay of Bengal.     

Species Specificity and Interspecies Relatedness in VP4 Genotypes Demonstrated by VP4 Sequence Analysis of Equine, Feline, and Canine Rotavirus Strains

We determined the nucleotide and deduced amino acid sequences of the VP4 genes of five equine, two feline, and two canine rotavirus strains. A high degree of homology (>97.0%) was found among the VP4 amino acid sequences of the equine strains H2, FI-14, and FI23. Equine strain L338 has a distinct VP4 amino acid sequence from those of the other equine strains (78.1% or less homology), and the L338 VP4 exhibited more than 17.0% divergence at the amino acid level from those of rotavirus strains published so far. The VP4 amino acid sequence of equine strain H1, which showed low homology with those of other equine strains, shares >95.4% homology to those of porcine strains OSU and YM. VP4 amino acid sequences of feline strain Cat97 and canine strains CU-1 and K9 showed a high degree of homology (96.8 to 97.2%) to one another, and were found to be quite similar (96.0-97.0% homology) to that of a human HCR3 strain recently characterized. Feline strain Cat2, whose VP4 sequence is distinct from that of strain Cat97, has a VP4 similar to those of human strains K8 and AU-1 (97.8 and 97.5% homologies at amino acid level, respectively). Thus, the VP4 sequences of rotaviruses showed species specificity and interspecies relatedness.     

Diarrhea induction by rotavirus NSP4 in the homologous mouse model system.

Comparison of the NSP4 amino acid sequences from 31 strains of mammalian rotaviruses revealed the presence of four distinct NSP4 alleles; i.e., the Wa, KUN, AU-1, and EW alleles. The EW allele consists only of NSP4s from murine rotavirus strains and is divergent from other NSP4 alleles from the evolutionary perspective. There have been conflicting reports regarding the enterotoxigenic activity of NSP4 in the mouse model system; heterologous simian and porcine rotavirus NSP4s function as an enterotoxin in mice, while a homologous EC NSP4 does not play a dominant role as an enterotoxin in the cystic fibrosis conductance regulator knockout mice. To further examine the enterotoxigenic activity of NSP4, we expressed in Escherichia coli a recombinant protein consisting of glutathione S-transferase and amino acid residues 86-175 of the EW NSP4. We found that this fusion protein caused diarrhea in the majority (8/14) of 5- to 6-day-old CD1 mice. This study confirmed and extended that group A rotavirus NSP4s were able to induce diarrhea in neonatal mice and had an enterotoxigenic activity.     

VP6 capsid protein of chicken rotavirus strain CH2: sequence, phylogeny and in silico antigenic analyses

The inner capsid protein VP6 of group A rotavirus possesses group and subgroup epitope specificities. Avian rotaviruses have a unique VP6 that is antigenically different from its mammalian counterpart. The lack of information on the VP6 protein of chicken rotavirus strain, CH2, at the genetic and antigenic level was a major motivation for this work. Sequencing of the complete cDNA of the VP6 gene of CH2, revealed a nucleotide (amino acid) identity that varied from 78.3 to 98.5% (86.4-98.2%) when compared with other avian rotaviruses. Regardless of its host origin dissimilarity, CH2 VP6 showed a close sequence homology (97.4-98.2%) with turkey and pigeon rotaviruses. Homology-based modeling of the CH2 VP6 from the corresponding crystal structure of the bovine rotavirus, RF strain, demonstrated that the hypervariable region (residue 228-240) does have a critical role in strain specific antigenic characteristics of avian and mammalian rotaviruses. A predicted conformational epitope encompasses experimentally characterized group and subgroup epitopes suggesting that it is a major antibody binding site on the VP6 protein. The VP6 structure modeling and conformational epitope prediction together with enzyme immuno assay of SG MAbs placed CH2 in SGI/II. The study may be helpful in designing peptides for group A rotavirus diagnostic assays and to achieve heterotypic protection against rotavirus serotypes.     

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.