Analysis Of Virus Diversity And Discovery Of Potential Pathogenic Viruses In Healthy And Diseased Pig Samples

In recent years,large-scale and intensive pig breeding had gradually replaced rural free-range farming.The occurrence and prevalence of swine diseases had also undergone great changes,such as the increasing variety and constant variation of pathogens.With the increasing complexity of swine diseases and the emergence of various re-emerging or newly emerging viral infectious diseases,it was becoming more and more difficult to control swine diseases.Therefore,it was particularly important to quickly discover and identify pathogenic pathogens in swine populations.In recent years,recurrent or newly emerged epidemic diseases caused by virus infection were common in pigs reported in China.As an important source of infection,there were many kinds of viruses carried by pigs,some of which directly caused swine disease,while others might infect domestic animals and people,thus causing new zoonotic diseases.In addition to a large number of known porcine viruses,there were also a large number of unknown new viruses or recombinant viruses lurking in porcine bodies.The infection of these viruses was difficult to predict and detect,so the health hazards to animals and people might be greater.However,for the identification of pathogens of unknown new viral diseases,traditional virus detection methods had many inevitable limitations.Traditional methods can only detect known viruses or viruses with small variation,which was inevitably difficult to detect unknown viruses or viruses with large variation.In recent years,the viral metagenomics technology directly to all virus nucleic acid in the samples as the research object,and did not depend on predict virus genetic information and culture,not only the known virus can be detected effectively and quickly,but also a wide range variation degree of virus and various unknown new viruses can be found quickly.It was widely used in the diagnosis of recurrence or new virus infection and research.In this study,viral metagenomics was used to construct viral metagenomic library from fecal and tissue samples of healthy and diseased pigs.Illumina Mi Seq sequencing was used to analyze the viral genome,analyze the variation of virus diversity and discover the potential new pathogenic viruses and recombinant virus strains,which was of great significance for the prevention and control of swine infectious diseases and some zoonotic infectious diseases to accumulate viral genetic data and technical support for the prevention and control of new viral diseases in China’s pig population.The main contents of this research include:1.Analysis of virus community in fecal samples of healthy pigs and discovery of potential pathogenic virusesVirus community analysis was performed on 215 fecal samples collected from healthy pigs.With pre-screening for redundant data such as plant viruses,insect and other invertebrate viruses,and bacteriophages,the virus community in the feces of healthy pigs at the overall level of the family was as follows: Picornaviridae accounted for 57.10%,Caliciviridae accounted for 10.19%,Parvoviridae accounted for 2.63%,Astroviridae accounted for 18.12%,Coronaviridae accounted for 1.02%,Circoviridae accounted for 1.01%,Unclassified viruses accounted for 9.44% and other virus families accounted for 0.49%.The results showed that :(1)The composition of virus community in fecal samples of healthy pigs showed the diversity of virus species.(2)The virus community was mainly composed of Picornaviridae and Astroviridae,which accounted for 75.22% of the total.These two families mainly caused diarrhea and other symptoms.(3)Two Porcine Astrovirus strains,three Enterovirus G strains and one Sapelovirus A strains were obtained from the samples,and their genomic structure and phylogenetic analysis were performed.2.Analysis of virus community in tissue samples of diseased pigs and discovery of potential pathogenic virusesA total of 309 samples from different tissue parts of 94 diseased pigs were collected for virus community analysis.With pre-screening for redundant data such as plant viruses,insect and other invertebrate viruses,and bacteriophages,the virus community in the tissue of diseased pigs at the overall level of the family was as follows:Poxviridae accounted for 54.14%,Parvoviridae accounted for 30.63%,Circoviridae accounted for 6.40%,Flaviviridae accounted for 4.77%,Arteriviridae accounted for0.98%,Retroviridae accounted for 0.25%,Anelloviridae accounted for 0.23%,Iridoviridae accounted for 0.21%,None accounted for 1.74% and other families accounted for 0.65%.The results showed that :(1)Poxviridae was the main component of virus community in the tissue samples of diseased pigs,accounting for up to 54.14%.After analysis,it was found that reads sequences were highly reproducible and most of them were integrated host genes,so they were of little significance.(2)Except for Poxviridae,the remaining virus communities were mainly composed of Parvoviridae,Circoviridae and Flaviviridae.(3)A total of 20 Parvovirus,10 Porcine Circovirus and9 Classic Swine Fever Virus(CSFV)isolates were isolated,and their genomic structure and phylogenetic analysis were performed.(3)Identification of novel porcine astrovirusIn this study,a new PASTV4 strain,named ahast and Gen Bank number MT470220,was obtained from pig fecal samples collected in Anhui Province by means of viral metagenomics.According to the consensus,two sets of primers were designed for nested PCR to obtain the complete genome,and genetic evolution and recombination analysis were performed.Phylogenetic analysis showed that ahast clade with other 24 reference strains of Porcine Astrovirus Type 4,so the new strain of Porcine Astrovirus was identified as Porcine Astrovirus Type 4.RDP5.0 recombinant analysis showed that ahast was a novel recombinant strain formed by two recombinant events.In these two recombination events,PASTV4 /JPN/ MOI2-1-1/2015(Gen Bank No.LC201609)was the main parent.The strains of PASTV4 /CHN/WG-R2/2017(Gen Bank No.MK460231)and JXJA(Gen Bank No.KX060808)were secondary parents.Epidemiological investigation showed that the prevalence rate of AHAST in this pig farm in Anhui Province was low(10.70%).Conclusion :(1)The composition of virus community in feces of healthy pigs and tissues of diseased pigs were analyzed by viral metagenomics.(2)The virus community in the feces of healthy pigs and tissues of diseased pigs was diverse,and the virus community composition in tissue samples was more abundant at the family level.(3)A novel recombinant strain of porcine astrovirus type 4 and some enteroviruses were found in fecal samples of healthy pigs.Potential pathogenic strains causing typical pig clinical symptoms,such as five subtypes of porcine parvovirus,were found in tissue samples of diseased pigs.