Baculovirus expression and antigenic characterization of the capsid proteins of three Norwalk-like viruses

Abstract

Human caliciviruses (HuCVs) are antigenically diverse. The antigenic relationships among different HuCVs have been difficult to study because HuCVs cannot be passaged in the laboratory. In this study, we describe cloning, sequencing and expression of the viral capsid proteins of three HuCVs that were identified in outbreaks of acute gastroenteritis in Virginia in 1997-1998. Yields of the capsid proteins similar to previously expressed recombinant Norwalk virus were obtained using the baculovirus expression system. Recombinant VA97207 capsid protein (rVA97207) and rVA98387, but not rVA98115, formed virus-like particles (VLPs). All three recombinant capsid antigens detected seroresponses in patients involved in outbreaks of acute gastroenteritis associated with genetically homologous or related HuCVs. The antigenic relationships of the three strains were further characterized using hyperimmune antisera against the three capsid antigens as well as four previously characterized recombinant capsid antigens of Norwalk (rNV), Mexico (rMxV), Hawaii (rHV), and Grimsby viruses (rGrV). VA98387 shared 98% aa identity with GrV; rVA98387 was detected by antisera to GrV. VA98115 shared 87% aa identity with Desert Storm virus and 65% aa identity with prototype Norwalk virus (NV); rVA98115 reacted weakly with NV antisera. VA97207 shared 80% aa identity with Amsterdam and 75% aa identity with Leeds strains and rVA97207 was not detected by any of the heterologous antibodies. In conclusion, VA97207 and VA98115 may belong to CV antigenic types not previously expressed, while VA98387 is a GrV-like virus. Low levels of cross-reactive antibodies were detected between types. Further studies to characterize these antigens and to develop enzyme immune assays (EIAs) for these strains are in progress.

Development of an Enzyme Immunoassay for Detection of Sapovirus-Specific Antibodies and Its Application in a Study of Seroprevalence in Children

“…The virus strains used in this study are listed in Table 1 and were described in our previous studies (7,20). cDNA clones covering the C-terminal part of the viral genomes (2.3 to 3.2 kb) were stored at Ϫ70°C and used as templates for PCR amplification of virus-specific sequences in this study, using high-fidelity Pfu DNA polymerase (Promega, Madison, WI).…”

“…Baculovirus expression of VA387 has been described previously (20). For baculovirus expression of Mex340 and Mex14917 capsid proteins, virus-specific sequences from the capsid protein initiation codon to the poly(A) tail were cloned into pFastBac1 vector (Invitrogen, Carlsbad, CA), utilizing restriction enzyme sites (BamHI and NotI) added to the PCR primers.…”

“…For large-scale production of recombinant SV capsid proteins, insect cell cultures were inoculated with a multiplicity of infection of 5 to 10 of recombinant baculoviruses. Cultures were harvested at 5 days postinoculation, and recombinant SV VLPs were purified on 10 to 50% sucrose gradients as previously described for NV VLPs (20). Sucrose gradient fractions were analyzed on 10% polyacrylamide gels containing sodium dodecyl sulfate (SDS) and/or by Western blotting, using hyperimmune sera generated against SV capsid fusion proteins or human sera collected during previous gastroenteritis outbreak investigations.…”

“…The sucrose gradient analysis also indicated that Mex340 capsid proteins formed VLPs, and EM examination of positive fractions revealed scattered virus-like particles (data not shown). Furthermore, hyperimmune sera produced against E. coli-ex- a Virus strains and cDNA clones were obtained in our previous studies (7,20).…”

Norovirus and Histo-Blood Group Antigens: Demonstration of a Wide Spectrum of Strain Specificities and Classification of Two Major Binding Groups among Multiple Binding Patterns

“…VLPs of 14 strains representing 13 genetic clusters of norovirus produced from baculovirus were used in this study, as follows: in genogroup I, Boxer (AF538679), C59 (AF435807) (2), Norwalk virus (M87661) (12,13), Desert Shield virus (DSV; U044469), and VA115 (AY038598), and in genogroup II, BUDS (AY660568), Grimsby (GrV; AJ004864) (4), Hawaii (HV; U07611) (16), Mexico (MxV; U22498) (11), MOH (AF397156) (1), Parris Island (PiV; AY652979) (22), VA387 (AY038600) (14), VA207 (AY038599) (14), and Operation Iraqi Freedom 031998 (OIF, AY675554) (22). Among them, Norwalk, VA387, MOH, and VA207 represent the four previously published receptor binding patterns (8).…”