RESUMO
The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.
Assuntos
Proteína HN/fisiologia , Vírus da Doença de Newcastle/fisiologia , Vírus da Doença de Newcastle/patogenicidade , Animais , Sequência de Bases , Linhagem Celular , Embrião de Galinha , Quimera/genética , Chlorocebus aethiops , Efeito Citopatogênico Viral/genética , Efeito Citopatogênico Viral/fisiologia , DNA Viral/genética , Genes Virais , Proteína HN/genética , Humanos , Mutação , Doença de Newcastle/etiologia , Vírus da Doença de Newcastle/genética , Células Vero , Virulência/genética , Virulência/fisiologiaRESUMO
Newcastle disease virus (NDV) causes a highly contagious and economically important disease in poultry. Viral determinants of NDV virulence are not completely understood. The amino acid sequence at the protease cleavage site of the fusion (F) protein has been postulated as a major determinant of NDV virulence. In this study, we have examined the role of F protein cleavage site sequence in NDV virulence using reverse genetics technology. The sequence G-R-Q-G-R present at the cleavage site of the F protein of avirulent strain LaSota was mutated to R-R-Q-K-R, which is present in the F cleavage site of neurovirulent strain Beaudette C (BC). The resultant mutated LaSota V.F. virus did not require exogenous protease for infectivity in cell culture, indicating that the F protein was cleaved by intracellular proteases. The virulence of the mutant and parental viruses was evaluated in vivo by intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) tests in chickens. Our results showed that the modification of the F protein cleavage site resulted in a dramatic increase in virulence from an ICPI value of 0.00 for LaSota to a value of 1.12 for LaSota V.F. However, the ICPI value of LaSota V.F. was lower than that of BC, which had a value of 1.58. Interestingly, the IVPI tests showed values of 0.00 for both LaSota and LaSota V.F. viruses, compared to the IVPI value of 1.45 of BC. In vitro characteristics of the viruses were also studied. Our results demonstrate that the efficiency of cleavage of the F protein plays an important role if the NDV is delivered directly into the brains of chicks, but there could be other viral factors that probably affect peripheral replication, viremia, or entry into the central nervous system.
Assuntos
Vírus da Doença de Newcastle/patogenicidade , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Embrião de Galinha , Galinhas , DNA Viral/genética , Mutagênese Sítio-Dirigida , Doença de Newcastle/etiologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/crescimento & desenvolvimento , Vírus da Doença de Newcastle/fisiologia , Recombinação Genética , Temperatura , Proteínas Virais de Fusão/genética , Virulência/genética , Virulência/fisiologiaRESUMO
Three aquareovirus strains isolated from grass carp (Ctenopharyngodon idellus), geoduck clams (Panope abrupta) and herring (Clupea harengus) in North America and Asia were examined by RNA-RNA blot hybridization to determine their genogroup. The isolates from clams and herring were identified as members of genogroup A, but the isolate from grass carp did not hybridize to any of the known genogroups, suggesting that this virus probably represents a new, seventh genogroup.
