Isolation And Identification Of Porcine Astrovirus Complete Genome Analysis Of The Isolated Strain And Prokaryotic Expression Of Its Capsid Protein

Astroviruses are a group of enteric pathogens that could infect human and many other kinds of animals. To date, astrovirus has been detected from31mammal and6avian species, including human, pig, cattle, bat, dog, cat chicken, turkey, duck and pigeons. However, the relationship of astrovirus infection to enteric disease has not been fully elucidated in animals. Genetic variation has been found in astroviruses from many hosts. As a consequent,the antigenic diversity was only verified on human astrovirus. Furthermore, more and more evidences implied that genetic recombination may have happened among astroviruses. In can be seen that, the genetic diversity, multiple host specificity and genetic recombination, the ability to cross species barriers and become adapted to new host make astroviruses as potential candidate for emerging zoonotic infection. In order to provide theoretical and practical gist for a comprehensive understand ing of porcine astrovirus, virus growth and isolation properties of porcine astrovirus were explored; the genetic variation and evolution, and antigenic characteristics of capsid protein were also analyzed in this study.The fecal samples detected PAstV positive were used for virus isolation. With the aid of trypsin, one cytopathic porcine astrovirus strain(PAstV-GX1) was isolated in PK15cell line and propagated as long as20passages. However,the cytopathic effect of the virus decreased remarkably when trypsin wasn’t added to the culture medium. The full-length genome of the isolated strain was amplified by using PCR and rapid amplification of cDNA end (RACE) methods and sequenced subsequently. The suquence obtained was6432bp in length covering majority of the ORF1a, the complete ORF1b, ORF2,3’UTR and the poly(A) tail. The complete genomic nucleotide homology analysis revealed that PAstV-GXl shared the highest homology (83.2%) with the Shanghai strain,which was the only complete genome of genotype MAstV3. The homology between PAstV-GX1and other genotypes of porcine astrovirus was under50%. Phyogenetic analysis based on full-length ORF2gene further revealed that PAstV-GX1clustered with PAstV-Shanghai and other MAstV3strains in one branch. Comparing with other strains of porcine astrovirus, PAstV-GX1shared the closest relationship with classical human astro viruses. Moreover,3conservative nucleotide motifs (ribosomal frameshift signal, subgenomic transcription promoter and stem-loop motif at the3’ end of the genome) of astrovirus were also found in PAstV-GX1genome.Analysis based on the deduced amino acid sequences showed that5transmembrane helices were found at the5’ end of the nsp1a;2coiled-coil were also found around aa50and aa500of nspla. The analysis of the capsid amino acid sequence of the isolated strain revealed that2regions could be divided into conserved-region(aal～aa419) and hyper variable region(aa420～aa750).In consistence to the homology analysis, antigenic analysis of the capsid amino sequence found that2regions located at5’and3’terminal of the capsid sequence showed high antigenic index. A recombinant protein derived from the hyper-variable region was successfully expressed by prokaryotic expression method and the protein expressed showed good reactogenicity by western-blot analysis, which layed the basis for further immunogenicity studies of the capsid protein and the development of a serological diagnosis kit or vaccine for porcine astrovirus in Guangxi.