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.     

Genetic diversity and classification of the outer capsid glycoprotein VP7 of porcine group B rotaviruses

We determined the nucleotide sequences of the outer capsid glycoprotein (VP7) genes of 38 porcine group B rotaviruses (GBRs) from feces of pigs at 27 farms in Japan between 2000 and 2007. Substantial diversity among porcine GBR VP7 genes was observed, with up to 42.4% difference in nucleotides and 49.8% in amino acids. On comparison of VP7 genes, porcine GBRs were clearly distinct from the published corresponding genes from human, bovine and murine GBRs (53.7–70.8% identity in nucleotides and 45.8–73.4% identity in amino acids). Phylogenetic analysis showed that the VP7s of GBRs could be divided into five genotypes: the murine strain was genotype 1, human strains were genotype 2, bovine and some porcine strains were genotype 3, and other porcine strains belonged to genotype 4 or 5. In addition, GBR VP7s in genotypes 3 and 5 were further divided into four and five clusters, respectively. No relationship between VP7 genotype and double-stranded RNA migration patterns of porcine GBRs in polyacrylamide gel electrophoresis were observed. However, an antigen enzyme-linked immunosorbent assay using antiserum to recombinant bovine GBR VP6 did not react with fecal samples containing one cluster of genotype 5 of porcine GBRs. The abundant divergence of porcine GBR VP7 genes suggests that porcine species might be an original natural host of GBR infection and that different serotypes might exist among porcine GBRs. To our knowledge, this is the first report to describe the gene sequences and typing of porcine GBR VP7s.     

Genetic heterogeneity in the VP7 of group C rotaviruses

Evidence for a possible zoonotic role of group C rotaviruses (GCRVs) has been recently provided. To gain information on the genetic relationships between human and animal GCRVs, we sequenced the VP7 gene of 10 porcine strains detected during a large surveillance study from different outbreaks of gastroenteritis in piglets. Four GCRV strains were genetically related to the prototype GCRV porcine Cowden strain. A completely new VP7 genotype included 4 strains (344/04-7-like) that shared 92.5% to 97.0% aa identity to each other, but <83% to human GCRVs and <79% to other porcine and bovine GCRVs. A unique 4-aa insertion (SSSV or SSTI), within a variable region at the carboxy-terminus of VP7, represented a distinctive feature for these 4 unique strains. An additional strain, 134/04-18, was clearly different from all human and animal GCRVs (<85% aa identity) and likely accounts for a distinct VP7 genotype. The VP7 of a unique strain, 42/05-21, shared similar ranges of aa sequence identities with porcine and human strains (88.0-90.7% to porcine GCRVs and 85.2-88.2% to human GCRVs). Plotting the VP7 gene of strain 42/05-21 against the VP7 of human and porcine strains revealed discontinuous evolution rates throughout the VP7 molecule, suggesting different mutational pressure or a remote intragenic recombination event. These findings provide the need for future epidemiological surveys and warrant studies to investigate the pathogenic potential of these novel GCRVs in pigs.     

Molecular analysis of outer capsid glycoprotein (VP7) genes from two isolates of human group C rotavirus with different genome electropherotypes.

Nucleotide sequences for the VP7 gene of human group C rotavirus were determined for two strains isolated in Okayama, Japan, during a 1988-1990 epidemic. These isolates, OK118 and OK450, were selected as prototypes of two different electropherotypes, patterns I and II, respectively. The genes were identical in size (1,063 bp), and both contained singled open reading frames encoding 332 amino acids. The alignment of two sequences revealed 46 nucleotide substitutions, 11 of which were predicted to give amino acid changes. The deduced amino acid sequence of VP7 from OK118 was similar to published sequences of a Japanese isolate and three foreign isolates (more than 98.4% identity), whereas the VP7 sequence of OK450 revealed around 96% identity with these isolates and had nine unique amino acid substitutions. The VP7 genes of nine Okayama isolated were than analyzed by dot blot hybridization with the VP7 probes of OK118 and OK450. Under highly stringent conditions, the OK118 probe produced strong hybridization signals with the genes of five pattern I strains and one pattern II strain, while the OK450 probe strongly reacted only with those of three pattern II strains. Our results concluded that relative sequence diversity in the VP7 gene was observed between two different electropherotypes prevalent in a limited area.     

Detection of human group C rotaviruses in Nigeria and sequence analysis of their genes encoding VP4, VP6, and VP7 proteins.

In a survey on the etiology of acute gastroenteritis in infants and young children in Nigeria, group C human rotaviruses were detected in two of 112 rotavirus positive stool specimens collected between 1999 and 2000. The VP7, VP6, and VP4 genes of the two Nigerian human group C rotavirus strains (Jajeri and Moduganari) were sequenced in this study. Comparative sequence analysis with other published human group C rotaviruses showed that the genes encoding the three structural proteins were remarkably conserved in primary structure with few mutations. The VP4 and VP7 genes from the two Nigerian strains were related more closely to each other than to those of other published strains, and formed a separate cluster on the phylogenetic tree. In contrast, it was of note that VP6 gene of strain Moduganari was related more closely to the Brazilian strain Belem than to the other Nigerian strain Jajeri. This is the first report of identification of human group C rotavirus in Nigeria and constitutes the first sequence data of human group C rotaviruses in the African continent.     

Sequence analysis of the gene encoding the serotype-specific glycoprotein (VP7) of two new human rotavirus serotypes

Human rotavirus strains 69M and WI61 are distinct from human rotavirus serotypes 1, 2, 3, and 4 and from each other by plaque reduction neutralization and have been proposed as new human rotavirus serotypes (serotype 8 and serotype 9, respectively). The nucleotide sequence of the gene encoding the serotype-specific capsid glycoprotein, VP7, of strains 69M and WI61 was determined. In addition, the sequence of the VP7 gene of strain F45 (serotypically indistinguishable from WI61) was determined. Comparative analyses of the nucleotide and deduced amino acid sequences with those of reference strains from serotypes 1,2,3,4,5, and 6 demonstrated that WI61 and F45 share a high degree of sequence similarity with each other and that strains 69M, WI61, and F45 are distinct from established serotypes 1,2,3,4,5, and 6 in nine defined regions of the VP7 which are variable across rotavirus serotypes.     

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).     

Nucleotide sequence of the structural glycoprotein VP7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses

A full-size cloned cDNA copy of the rotavirus gene encoding the structural neutralization glycoprotein (VP7) of Nebraska calf diarrhea virus (NCDV), a strain recently shown to be effective as a vaccine in children, has been sequenced. Comparison of the deduced amino acid sequence of NCDV (serotype 6) VP7 with that of four other rotavirus strains (human WA serotype 1, human HU-5 serotype 2, simian SA-11 serotype 3, and bovine UK serotype 6) indicates that the degree of amino acid homology among VP7 neutralization proteins of these serotypes ranges from 75 to 86%. Four hydrophilic regions at amino acid residues 174-183, 248-256, 287-294, and 310-317 exhibit significant homology and hence may represent common antigenic determinants, while one hydrophilic area at amino acid residues 83-102 exhibits sufficient divergence to suggest it may be involved in serotype specificity.     

Sequence analysis of VP4 and VP7 genes of nontypeable strains identifies a new pair of outer capsid proteins representing novel P and G genotypes in bovine rotaviruses

During a limited epidemiological study, the serotype specificities of several isolates of bovine rotavirus, exhibiting identical electropherotypes, from a single cattle farm near Bangalore, India, could not be determined using a panel of serotyping monoclonal antibodies (MAbs) specific for G serotypes 1-6 and 10. To determine the genotypes of these isolates, the nucleotide sequences of the genes encoding the outer capsid proteins VP4 and VP7 of two representative isolates, Hg18 and Hg23, were determined. The corresponding gene sequences from the two isolates were identical, indicating that these isolates represented a single strain of bovine rotavirus. Comparison of the VP4 nucleotide (nt) and the deduced amino acid (aa) sequences with those of several human and animal rotavirus strains representing all of the currently recognized 20 different VP4 (P) genotypes revealed low nt and aa sequence identities of 61.0 to 74.2% and 57.9 to 78.2% for VP4. The percentages of amino acid homology for the VP8* and VP5* regions of VP4 were 37.7 to 67.9 and 68.1 to 84.2%, respectively. The nt and aa sequences of the VP7 gene were also distinct from those of human and animal strains belonging to the previously established 14 VP7(G) serotypes (65.9 to 75.5% nt and 59.5 to 77.6% aa identities). These findings suggest the classification of the VP4 and VP7 genes of the bovine isolates represented by Hg18 as new P and G genotypes and provide further evidence for the vast genetic/antigenic diversity of group A rotaviruses.     

Molecular characterization of VP7 gene of human rotaviruses from Bangladesh

This study was carried out during July 2005–June 2006, to characterize rotaviruses circulating in Bangladeshi children less than 5 years attended a peri-urban hospital. The proportion of rotavirus diarrhea was 39.5%. Genotype G2 was dominant (45.5%) followed by G1 (24.8%), G12 (9.6%), G9 (8.5%), and G4 (2.1%). G2 were mainly in combination with P[4], G1 and G9 with P[8], and G12 with P[6]. Phylogenetically Bangladeshi G1, G2, and G12 were closely related with the respective types from India, whereas Bangladeshi G9s of lineage III were with strains from Belgium and Australia. A G9 strain of lineage IV was clustered with strains from Sri Lanka and Turkey. Compared with prototype rotaviruses, Bangladeshi strains showed several amino acid substitutions at the antigenic sites of VP7. This study showed that the generation of diverse strains continued as evidenced by long G2, short G1 and G9 strains, and various combinations of G and P types.     

Sequence analysis of genome segment 10 encoding the major outer capsid protein (VP7) of genogroup B aquareovirus and its relationship with the VP7 protein of genogroup A aquareovirus

Summary.  Genome segment 10 of the coho salmon (CSR) strain of genogroup B aquareovirus has been cloned and sequenced. The gene is at least 936 nucleotides in length and has a major open reading frame encoding a protein of 293 amino acids with a calculated molecular mass of 31.7 kDa. Comparison of nucleotide and deduced amino acid sequences of genome segment 10 of genogroup A and genogroup B strains indicated 55.7 and 36.5% identity at the nucleotide and amino acid level, respectively. Baculovirus expression and serological identification of the genome segment 10 product of the CSR strain indicated that this gene encodes the major outer capsid protein of genogroup B aquareovirus. Accepted July 18, 1997 Received April 3, 1997     

Genetic characterization of VP3 gene of group A rotaviruses

We sequenced VP3 gene of four rotavirus strains and examined the diversity of VP3 and its other genetic characteristics in a total of 22 Group A rotaviruses. The 22 rotavirus strains were derived from six different host species (human, avian, bovine, equine, simian and porcine). Comparison of the partial VP3 peptide sequences (aa 141–294) showed identities ranging from 49 to 99% among different species. The phylogenetic analysis of VP3 sequences revealed segregation according to the species origin. This was further confirmed by identification of three host specific domains within the VP3 peptide. Thus, the host range restriction or attenuation previously shown to associate with VP3 may be attributed to the species-specific nature of the gene.     

Isolation and serotyping of animal rotaviruses and antigenic comparison with human rotaviruses

Summary Rotaviruses were isolated from Australian farm animals (calf, pig and foal) with diarrhoea. Reciprocal cross neutralisation studies showed antigenic similarities with overseas animal strains and with human strains including a newly defined fifth human serotype.With 2 Figures     

Molecular characterization and genetic variation of the VP7 gene of human rotaviruses isolated in Paraguay

Rotavirus is the main cause of acute diarrhea in infants and young children worldwide. In Paraguay, acute diarrhea ranks fourth among the causes of mortality in children under 4 years of age. Rotavirus was detected in 93 out of 410 patients admitted to three main hospitals in Asunción, Paraguay from August 1998 to August 2000. Samples from 64 patients were analyzed by RT-PCR for G and P typing. G4P[8] (46.9%; 30/64) was the most common strain detected, followed by G9P[8] (17.2%; 11/64) and G1P[8] (10,9%; 7/64). Since G4 was predominant during the epidemiological peaks of 1998 and 1999, four samples were sequenced and all grouped into sublineage Ic. This sublineage was reported for the first time in 1998 in Argentina, southern border of Paraguay, and it was shown to be responsible for the increased prevalence of G4 during the epidemiological season of 1998 in that country. In addition, Paraguayan G1 strains grouped in different lineages (I and II). However, G9 was predominant during the rotavirus epidemiological peak of 2000, and phylogenetic analysis of five samples grouped into a common emergent/reemergent lineage that circulates worldwide. Since vaccination could reduce the severity and the number of cases of rotavirus disease, this study suggests that a vaccine containing recently isolated variants of the most prevalent types (G1-G4) together with the emerging G9 type, might be sufficient to elicit a protective immune response against the rotavirus types circulating currently in Paraguay.     

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 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.     

Sequence analysis of the gene (6) encoding the major capsid protein (VP6) of group C rotavirus: higher than expected homology to the corresponding protein from group A virus

Two overlapping c-DNA clones, which hybridized in Northern blots to RNA segment 6 of the prototype strain (Cowden) of group C rotavirus, were selected from a c-DNA library in pBR322 and sequenced. The gene 6 sequence obtained was 1349 nucleotides and contained a single long open reading frame encoding a protein of 394 amino acids (total MW, 44,479) which is in line with the size of the major capsid protein VP6. Comparison of the group C sequence with that of the corresponding group A rotavirus gene revealed homology levels of 55 and 42% for nucleotides and amino acids, respectively. These values were surprisingly high in view of previous immunological and nucleic acid hybridization data which failed to show any cross-reaction between group A and non-group A rotaviruses. The epidemiological implications of these observations are discussed.     

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.