Virome Study Of Clinical Classical Swine Fever Samples

Classical swine fever is a highly contagious and severe infectious disease caused by classical swine fever virus（CSFV）,which has seriously endangered the development of my country’s pig industry.Clinically,co-infection of CSFV with other viruses is very common.Multi-virus co-infection is closely related to the occurrence and development of the disease.The interaction between pathogens will affect the immune protection efficacy of the vaccine,resulting in complicated clinical symptoms of diseased pigs.These have brought challenges to the diagnosis and control of swine diseases.At present,traditional detection methods for co-infection research only target several potential related pathogens,and cannot reflect the full picture of coinfection virus spectrum.Therefore,this study used the metatranscriptome method（MTT）and the multiple displacement amplification method（MDA）to analyze samples collected from 19 provinces,municipalities and autonomous regions in the country from 1990 s to 2020.These samples include 82 CSF clinically afflicted and 17CSFV-negative samples which share similar clinical symptoms with CSF.The samples were subjected to viromic analysis to resolve their virome structure.MTT and MDA sequencing data were annotated to 56 viruses belonging to 32 genera of 20 Eukaryotic virus families,including 27 DNA viruses and 29 RNA viruses.All 82 CSF clinical samples had reads annotated to CSFV,with an average of1,042,486 CSFV reads per sample,while 17 CSFV-negative samples with clinical symptoms similar to CSF were not annotated to CSFV.This is completely consistent with the RT-PCR results.PCV2,TTsu V 1a,TTsu V k2 a,TTsu V 1b and PRRSV obtained the highest number of reads,all exceeding 100 million.The amino acid homology between the annotated eukaryotic DNA and RNA viruses and known reference viruses was 97.03%±4.57% and 92.86%±11.51%.Statistical analysis of virus annotation showed different types of viruses in different CSFV.There were 14（14/82）samples with more than 10 viruses,and the highest with 26 viruses.There was no significant difference in the structure of virome of the CSFV clinically afflicted samples and CSFV-negative samples with clinical symptoms similar with CSF.Eight viruses,PCV2,TTsu V 1b,TTsu V k2 a,PRRSV,PPV2,TTsu V 1a,PSV and EV G,were highly annotated（more than 20%）in the virome of CSF clinical disease samples and PCV2（67.1%）was the dominant virus species in the CSFV virome.Through de novo assembly,a total of 114 near-full-length genome sequences of 17 viruses were obtained,which provided materials for the genetic evolution analysis of these viruses.In this study,51 CSFV complete genome sequences and 12 complete ORF gene sequences were obtained.Based on the genotyping of the CSFV complete E2 sequence,13 of the 63 CSFV strains belonged to the 2.2 subgenotype,and 8 strains belonged to the 2.3 subgenotype.4 strains belonged to the 2.1a sub-subgenotype,13 strains belonged to the 2.1b sub-subgenotype,18 strains belonged to the 2.1c subsubgenotype,1 strain belonged to the 2.1g sub-subgenotype,2 strains belonged to the2.1h sub-subgenotype,and 4 belonged to the 2.1i sub-subgenotype.Compared with the HCLV strains,the strains obtained in this study had amino acid substitutions at 18 positions in E2 protein.The results of the virus group showed that the infection rate of PCV2 in the CSFV virus group（67.1%）was significantly higher than that of other viruses.In this study,the whole genome sequences of 60 PCV2 strains were obtained,and the nucleotide homology between the strains was 94.6～100%.Based on the phylogenetic analysis of the Cap gene and the whole genome,they were divided into 5 genotypes:PCV2a（43.3%）,PCV2b（13.3%）,PCV2d（30.0%）,PCV2f（11.7%）and PCV2h（1.7%）.And for the first time,a PCV2 h strain PCV2-GD50-2011 was found in Guangdong,which was closely related to two PCV2 strains form Vietnamese.Viromics analysis identified a novel birnavirus in CSFV-positive samples,named porcine birnavirus（PBRV）.Follow-up RT-PCR screening of archived tissues of diseased pigs identified 16 PBRV representatives from 28 provinces/autonomous regions in China spanning 21 years（1990～2019）,and all PBRVs were detected only from CSFV positive samples.Genome-based sequence comparison showed that PBRVs are genetically distant from existing members within birnaviridae family.The nucleotide sequence similarities in segment A and B is 45.8～61.6% and 46.2～63.2%,respectively.Phylogenetic analysis based on the pre VP2 and VP1 genes showed that PBRV is independent of the other 4 birnavirus genera.PBRVs may form a new genus of birnavirus,named Mambirnavirus.This study revealed the compositional characteristics of the swine fever virus coinfection virus,confirmed the complexity and universality of clinical virus mixed infection,and discovered a variety of novel viruses.The results provide important epidemiological data for the prevention and control of related swine viral infectious diseases,and help to formulate effective strategies to prevent and control the infections.