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The gene coding for the major inner capsid protein VP6 of human group C rotavirus was cloned into baculovirus using the pBlueBac2 vector and expressed in insect cells. When cultured in High Five cells, VP6 was expressed at a high level and exported to the cell culture medium. Purified VP6 was used to immunise rabbits. Hyperimmune rabbit serum, which reacted with native human group C rotavirus in infected cells, was used to develop and optimise an EIA for the detection of antibodies to group C rotavirus using the recombinant VP6 as a source of antigen. In a local epidemiological survey of 1000 sera grouped by age, an average of 43% of samples were found to have antibodies to human group C rotavirus with the highest proportion (66%) in the 71-75 year age group. In comparison, 97% of adults and 85% of children had antibodies to recombinant VP6 from the bovine RF strain of group A rotavirus. These results suggest that infection with human group C rotavirus is a common occurrence despite the apparent rarity of reports of human group C rotavirus in clinical samples from patients with gastroenteritis.

A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.

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.

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 Belem, 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/Belem and C/Preston rotaviruses contained an open reading frame of 1185 nucleotides (395 amino acids; deduced M(r) 44,669 Da). The Belem 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.