The International Committee on Taxonomy of Viruses (ICTV) has recently approved several proposals submitted by the present Caliciviridae Study Group. These proposals include the division of the family into 4 new genera designated Lagovirus, Vesivirus, "Norwalk-like viruses (NLVs), and "Sapporo-like viruses (SLVs); the latter 2 genera were assigned temporary names until acceptable names can be determined by the scientific community. The genera have been further divided into the following species: Feline calicivirus and Vesicular exanthema of swine virus (genus Vesivirus), Rabbit hemorrhagic disease virus and European brown hare syndrome virus (genus Lagovirus), Norwalk virus (genus NLV), and Sapporo virus (genus SLV). In addition, the ICTV approved a proposal to remove the hepatitis E virus from the Caliciviridae into an "unassigned classification status.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotypespecific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.

We have developed a hybridization assay that permits distinction of rotavirus serotypes 1, 2, 3, and 4. The serotype of rotaviruses from stool samples or tissue culture was recognized by hybridization of specific probes to (i) blots of viral double-stranded RNAs electrophoresed in agarose gels (Northern blots) or (ii) heatdenatured double-stranded RNAs directly dotted on nylon membranes. The probes consisted of 32P-labeled cDNA synthesized by reverse transcription of in vitro derived rotavirus mRNA from rotavirus serotypes 1 to 4. To prepare these probes, mRNAs were primed with a 17-mer nucleotide common to all four serotypes whose sequence is complementary to bases 375 to 391 of the rotavirus gene encoding the VP7 glycoprotein (gene 8 or 9 depending on the rotavirus strain). The resulting downstream transcripts encompassed areas of major sequence divergence among the four serotypes. Hybridization at high stringency (50°C, 50% formamide, 4x SSC [lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) was performed for 16 to 48 h. Autoradiograms of the washed membranes allowed recognition of the rotavirus serotype present in the blotted or dotted specimens since each of them hybridized preferentially to one of the four probes. Twenty-four laboratory specimens and 103 clinical specimens from Washington, D.C., Venezuela, and Chile were "serotyped" with this assay. The results were similar to those obtained with a monoclonal antibody serotyping assay.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.