Genetic Variation Analysis,Isolation And Identificaiton Of Porcine Astrovirus In Hunan Province

Astroviruses(AstV)are associated with enteric and systemic disease in mammals and birds.Astroviruses have received increased attention recently as they have been found to be associated with sporadic neurologic disease in mammals including humans.In pigs,porcine astrovirus(PoAstV)can be widely detected and has been grouped in five genotypes(PoAstV1 to PoAstV5).Porcine astrovirus can cause swine viral diarrhea,neurological and respiratory diseases,and bring certain economic losses to the pig industry.Astrovirus hosts have a wide range,rapid genetic variation,have the ability to spread across species and adapt to new hosts,and are increasingly important to public health.However,they are still one of the least studied enteroviruses,mainly focused on the study of human astrovirus,and there are few studies on the molecular biological characteristics of the astrovirus capsid protein and the interaction between the virus and the host.In this study,37 serum samples,20 nasal swabs and 43 fecal swabs were collected from 43 pigs from two pig farms in Hunan Province.And 17 pigs were detected by conventional PCR as PoAstV positive(detection rate 39.5%),among which PoAstV 1 accounted for 16.3%(7/43),PoAstV2 for 18.6%(8/43),PoAstV4 for 9.3%(4/43),and PoAstV5 for 14.0%(6/43).The distribution of viruses in different samples or tissues is different.The positive rate of each sample is:serum sample 24.3%(9/37),nasal swab 20%(4/20),fecal swab 20.9%(9/43).28 PoAstV partial RdRp sequences and two complete genomes of PoAstV2-WG-R2/2017 and PoAstV4-WG-R2/2017 were obtained,and the obtained gene sequence was compared with the existing PoAstV gene sequence in the database using genetic analysis software.Through genome analysis,it is found that the current PoAstV2 and PoAstV4 are quite different from other strains within the same genotype.The PoAstV2-WG-R2/2017 genome showed identities of 67.2-77.4%to other known PoAstV2 genomes,and the PoAstV4-WG-R2/2017 genome showed identities of 72.8-80.5%to other known PoAstV4 genomes.The predicted Spike domain of open reading frame 2(ORF2)of these strains showed the highest genetic variation,with amino acid identities of 13.7-70.9%for PoAstV2-WG-R2/2017 to other known PoAstV2 strains,and identities of 24.4-63.3%for the PoAstV4-WG-R2/2017 to other known PoAstV4 strains.In addition,RDP4 recombination analysis software was used to detect possible recombination events in both strains.The low similarity of the viral capsid protein amino acids may cause pathogenic differences between virus strains.Moreover,this study further found that the new porcine astrovirus has extensive tissue tropism,and the data obtained provide further evidence for the intestinal infectivity of PoAstVs,and that a single pig can be co-infected with different types of PoAstV and infectious Isotype PoAstV with large genetic differences.In addition,under culture conditions without adding pancreatin,PK15 cells can be used to stably culture and propagate the PoAstV5 isolate.The capsid proteins Cpl and Spike of PoAstV5 were successfully expressed using the pHUE prokaryotic expression system,and immunized Balb/C mice obtained polyclonal antibodies with good effects.Immuno fluorescence Assay(IFA)showed that these two multi-antibodies recognized the virus in infected cells,and thus determined the location of PoAstV5 Cpl and Spike in PK15 cells,for further study of PoAstV5 replication and signaling in cells The study of transduction mechanism provides a reliable antibody tool.