RESUMO
A recombinant La Sota strain (KBNP-C4152R2L) in which fusion (F) and hemagglutinin-neuraminidase (HN) genes were replaced with those of a contemporary genotype VIId virus, KBNP-4152, has been developed. To attenuate the virulence of the recombinant strain, the F cleavage motif was mutated from (112)RRQKR(116) to (112)GRQAR(116), and to reduce pathogenic instability, a codon which does not allow changes to basic amino acids by single point mutation was inserted at codon 115. In addition a six-nucleotide sequence was inserted into the intergenic region between matrix protein and F genes for attenuation without breaking the "rule-of-six." The HN protein length was increased from 571 to 577 as a marker. Serological tests revealed that the antigenicity of KBNP-C4152R2L was similar to that of KBNP-4152 but distinct from that of the La Sota strain. KBNP-C4152R2L was avirulent (intracerebral pathogenicity index, 0.0; mean death time, >168 h) and stable in pathogenicity through in vivo passages. The killed oil emulsion of and live KBNP-C4152R2L were completely protective against mortality and egg drop caused by virulent strains, and KBNP-C4152R2L was applicable to in ovo vaccination. Therefore, KBNP-C4152R2L is a promising vaccine strain and viral vector in terms of antigenicity, productivity, safety, and pathogenic stability.
Assuntos
Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Substituição de Aminoácidos/genética , Animais , Galinhas , Proteína HN/genética , Dados de Sequência Molecular , Mutagênese Insercional , Doença de Newcastle/prevenção & controle , RNA Viral/genética , Análise de Sequência de DNA , Sorotipagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas Virais de Fusão/genética , VirulênciaRESUMO
The large (L) protein of vesicular stomatitis virus (VSV), catalytic subunit of RNA-dependent RNA polymerase is responsible for the transcription and replication of VSV. The L protein of the Indiana serotype of VSV (VSV(Ind)) has previously been cloned and expressed, and used in the reverse genetics of VSV(Ind). However, the cDNA clones expressing functional L proteins of the VSV(NJ) serotype were not available. It was necessary to obtain functional clones of the New Jersey serotype of VSV (VSV(NJ)) in order to study homologous viral interference. Here we report the cDNA cloning, expression, and functional analyses of L proteins from both the Hazelhurst subtype and Concan subtype of VSV(NJ). The analysis of the expressed L proteins for the transcription and replication of VSV demonstrate that both VSV(NJ) L clones express functional RNA-dependent RNA polymerase.