RESUMO
A cDNA obtained from Grimsby virus (GRV), a Norwalk-like virus, purified from a stool sample of a symptomatic adult associated with a gastroenteritis outbreak in the United Kingdom, was used to obtain the complete nucleotide sequence of the second open reading frame (ORF2). The ORF2 sequence of GRV predicts a capsid of 539 amino acids (aa) which exhibits aa identities of 96% to Lordsdale virus, 67% to Mexico virus (MXV), and 43% to Norwalk virus (NV). The GRV capsid protein was expressed in insects cells by using a recombinant baculovirus, and the resulting virus-like particles (VLPs) possessed a protein with an apparent molecular weight of 58,000. Hyperimmune antisera raised against purified GRV, MXV, and NV VLPs were tested in an indirect enzyme-linked immunosorbent assay (ELISA) against GRV, NV, and MXV VLPs, revealing that GRV is antigenically distinct from both NV and MXV. The antigenic specificity of the GRV-hyperimmune antiserum was confirmed in an antigen capture ELISA using GRV-, NV-, or MXV-containing fecal specimens. The expression of the GRV capsid protein has, for the first time, allowed the antigenic comparison of three distinct recombinant Norwalk-like viruses.
Assuntos
Antígenos Virais/genética , Caliciviridae/genética , Caliciviridae/imunologia , Vírus Norwalk/genética , Vírus Norwalk/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Antivirais , Antígenos Virais/química , Caliciviridae/crescimento & desenvolvimento , Infecções por Caliciviridae/virologia , Capsídeo/química , Capsídeo/genética , Capsídeo/imunologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Viral/genética , DNA Viral/isolamento & purificação , Gastroenterite/virologia , Expressão Gênica , Genes Virais , Humanos , Microscopia Eletrônica , Dados de Sequência Molecular , Peso Molecular , Vírus Norwalk/crescimento & desenvolvimento , Fases de Leitura Aberta , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
BACKGROUND: The diagnosis of gastrointestinal infections caused by small round structured viruses (SRSV) has relied upon electron microscopy and antigen/antibody assays based on Norwalk virus. We investigated cases of gastroenteritis associated with SRSVs employing a new sandwich enzyme-linked immunosorbent assay (ELISA) using hyperimmune animal anti-sera against recombinant Mexico virus capsid protein (rMXV). STUDY DESIGN: One hundred and thirty-five specimens from 86 episodes of gastroenteritis associated with SRSVs, collected in the UK between October 1993 and September 1994, were tested in the rMXV assay. RESULTS: Forty-seven (35%) specimens from 35 of 86 (41%) episodes were positive in the rMXV ELISA and these could further be divided into high and low reactors. Sequencing of a 266-base region of the RNA polymerase gene revealed that strains highly reactive in the rMXV assay demonstrated a high degree of similarity to MXV (97-99% at the nucleotide level), whereas low-reactive strains consist of Mexico-like strains and a heterogeneous group of viruses exhibiting 70-75% similarity to MXV. CONCLUSION: Our results indicate that the rMXV ELISA is predominantly a type specific assay, although some cross reactivity with other genogroup 2 SRSVs was observed. MXV was responsible for 26% of SRSV-associated gastrointestinal infections investigated in the UK during one year's surveillance.