Identification of an epitope common to genogroup 1 "norwalk-like viruses".

A panel of 10 monoclonal antibodies (MAbs) to recombinant Norwalk virus (NV) capsid protein were tested in competition enzyme-linked immunosorbent assays. Patterns of competition indicated that these MAbs recognize six to eight epitopes covering five nonoverlapping regions of the capsid protein. A single epitope, recognized by NV MAbs NV3901, NV3912, and NV2461 was found to occur in the majority of genogroup 1 (G1) but not genogroup 2 (G2) "Norwalk-like viruses" (NLVs). This observation supports the subdivision of human NLVs into two genogroups and provides an assay for the rapid identification of G1 NLVs in fecal specimens.

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.

Antigenic mapping of the recombinant Norwalk virus capsid protein using monoclonal antibodies.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Rotavirus gene detection with biotinylated single-stranded RNA probes.

Biotinylated single-stranded RNA probes from two of the eleven genome segments of the simian rotavirus SA11 were synthesized from cloned DNA and used in dot-blot and Northern-blot hybridization assays. Different types of membranes and conditions to prepare and use synthetic non-radioactive transcript probes were evaluated to obtain optimal test results. Nytran membranes showed the highest sensitivity and lowest backgrounds for hybridization with biotinylated RNA probes. When a gene 6 single-stranded biotinylated probe was used in a dot-blot format, test sensitivity was 0.1 ng for detection of homologous RNA and 0.4-1.5 micrograms for detection of RNA from heterologous rotavirus strains. When used in Northern blots, detection with this gene 6 probe required 1 ng of total SA11RNA or 50 ng of heterologous RNA to be applied to the gels for transfer. Simultaneous hybridization with probes from two different genes on one membrane showed a detection level similar to that seen with single probes alone. The advantages of using biotinylated single-stranded RNA probes to detect or characterize the genes of viruses with double-stranded RNA genomes are shown.

Molecular characterization of three rabbit rotavirus strains.

We report biochemical (RNA and protein patterns and gene-coding assignments) and serologic (serotype and subgroup) properties of three strains of rabbit rotaviruses--Ala C11 and R2. The RNA electropherotypes were a standard "short" pattern for R2, an unusual "short" pattern for Ala, and an unusual "long" pattern for C11. Serologic studies indicated that these viruses were all group A serotype 3 rotaviruses. In addition, the Ala and C11 viruses were found to possess subgroup I specificity, whereas the R2 virus possessed subgroup II specificity. In contrast to their distinctive RNA patterns, the polypeptide patterns of the rabbit viruses were similar to those of SA11. To identify cognate genes and determine gene-coding assignments for the rabbit rotaviruses, cDNA probes of individual SA11 genes were hybridized to rabbit rotaviral genomic RNA segments that had been separated by polyacrylamide gel electrophoresis and transferred to filters (Northern blots). The order of genome segments 7-11 for each of the rabbit rotaviruses was unique and differed from that of SA11 genes. These differences were possibly due to rearrangements of the RNA sequences within these individual genome segments. Sequence analysis of the individual RNA segments will confirm whether genome rearrangements are the molecular basis for these novel migration patterns.