Epidemiological Investigation,isolation And Identification Of Porcine Astrovirus In Some Areas Of Shandong Province

Astrovirus（Ast V）is a single-stranded positive-stranded RNA virus,which is a zoonotic pathogen that can cause diarrhea in humans and other animals.The virus has high genetic variation and genetic variability,and is prone to gene recombination,which makes it have the ability to adapt to new hosts and interspecific transmission.Porcine astrovirus（PAst V）has a high prevalence in pigs,and it is easy to be co-infected with other diarrhea viruses,such as coronavirus and rotavirus,causing serious losses to pig farms.Therefore,this study will investigate and analyze the epidemic situation of porcine astrovirus in Shandong area,and isolate and identify porcine astrovirus,in order to provide basis for the epidemic characteristics and pathogenic mechanism of the disease.In this study,RT-PCR was used to detect porcine astrovirus in 205 samples of porcine diarrhea cases in some areas of Shandong Province from January 2021 to December 2021.The results showed that the total positive rate of PAst V was 34.6%（71/205）,of which the proportion of co-infection with porcine epidemic diarrhea virus（PEDV）and rotavirus（RV）was 46%（13/28）and 43%（6/14）,respectively.In addition,the survey results show that there is PAst V infection in almost all areas,pig farms of different sizes have the disease,there is no significant seasonal difference,and the pig farm environment will affect porcine astrovirus infection.The genotypes of the above 71 positive samples were tested,and the results showed that all four genotypes that could cause porcine diarrhea could be detected.The proportions of PAst V 1,PAst V 2,PAst V 4 and PAst V 5 were 25.4%（18/71）,28.2%（20/71）,35.2%（25/71）and 22.5%（16/71）,respectively,and mixed infection existed.28 of them were selected for genetic evolution analysis,and it was found that the homology among 8 PAst V 1 sequencing sequences were 73.4%-95.2%,and that with reference strains were 77.2%-94.4%,among which the genetic relationship with KF787112 in Guangxi was relatively close.The homology among the 7 PAst V 2 sequencing sequences were 60.8%-94.7%,and the homology with reference strains were 52.4%-86.2%.Among them,625-7-2 and 719-5-2 were closely related to the reference sequence JF713712 from America and LC201586 from Japan.1015-1-2 had the shortest genetic distance with the reference sequence LC201585 from Japan,and 719-6-2,1112-19-2,1112-22-2 and 1112-24-2 had close relationship with the reference sequence KY940077 from Uganda in 2012.The homology of 13 PAst V 4 sequencing sequences were56.2%-95.5%,and with reference strains were 59.8%-94.6%.The homology among the sequencing sequences were quite different,and there were also great differences in homology with reference strains in different countries and regions.The homology between one PAst V 5sequencing sequences and 8 reference sequences were 83.8%-86.5%.At the same time,it was found that Past V 1 was in the same branch with cat and tiger Ast V,and PAst V 2 was in the same branch with roe deer and bovine Ast V.To sum up,the detection rate of PAst V4 was the highest in Shandong province,and there were great genetic differences among sequences.In this study,by optimizing the isolation conditions of porcine astrovirus,a strain of PAst V 4,named GRF-1,was successfully isolated in PK-15 cells with 5 μg/m L trypsin.The strain could proliferate stably in PK-15 cells and produce obvious cytopathic effect.In order to further verify that the obtained strain was porcine astrovirus,the samples of GRF were sequenced by macrovirus transcriptome.The RNA of the sample was sequenced by Illumina second generation sequencing platform Novaseq 6000.After quality control,37333610 Clean Reads were obtained,and 70787 transcripts were assembled by splicing software Trinity.70787 viral sequences were screened by comparing the spliced sequences with Gen Bank Virus Ref Seq protein database by DIAMOND software,and then CDS annotations were made by comparing them with NR,NT,CDD,WGS database by Blast.Finally,it was found that 88.30% of the GRF samples were mammalian astroviruses,while by species classification,PAst V 4 and PAst V 2 accounted for 21.79% and 0.32%,respectively,and there was no other porcine diarrhea virus,indicating that porcine astrovirus does have the possibility of interspecies transmission with other species,and there is a mixed infection of porcine astrovirus with different genotypes in pigs.After splicing the obtained fragments,the results showed that the c15050 fragment was the same as the GRF-1 sequencing fragment of the isolated strain,and had the highest homology with the Hunan PAst V 4 sequence MK460231 in China.It was further verified that the strain GRF-1 isolated from PK-15 cells was PAst V 4.In summary,this study investigated the prevalence of PAst V in Shandong Province for the first time and mainly analyzed the genetic evolution of PAst V 1,PAst V 2,PAst V 4 and PAst V 5,which enriched the content of epidemiological investigation of porcine astrovirus,and isolated a strain of PAst V 4,named GRF-1,which provided a basis for further research and scientific basis for the prevention and control of the disease in Shandong Province.