Genetic Diversity And Evolution Of RNA Viruses Co-infected With Classical Swine Fever Virus

Classical swine fever is a kind of contagious disease caused by CSF virus(CSFV).Due to immunization with attenuated vaccines,such as lapinized C-strain vaccine,the pandemic of the disease has been brought under control in China,but sporadic cases and persistent infection still exist in the clinic.And co-infection with other viruses probably is a factor which affects the immune effect of vaccines.So,it is necessary to understand the current situation of CSFV co-infection and the genetic and evolutionary characteristics of the co-infected viruses which is important for making scientific and accurate disease prevention and control strategies.Previously,82 CSFV-positive samples collected from 1990 to 2020 had been studied using high throughput sequencing and many viruses co-infected with CSFV were found in these samples.In order to get more knowledge of the co-infected viruses,the present study focused on some new and clinical important RNA viruses for viral metagenomics data analysis and RT-PCR verification.After amplifying and sequencing the viral certain gene or full genome sequences,phylogenetic analysis was conducted.In order to expand the scope of epidemiological investigation,another 388 pig tissue samples were screened for some important RNA viruses.In this study,the genome segments of two ephemeroviruses were found in two samples which were named as PoEV-HeN10 and PoEV-GDMM7.After amplification,the nearly fulllength genome sequence was obtained.And the two strains shared an aa similarity of 43.460.7%and 47.6-58.5%with other ephemeroviruses for N and L proteins,respectively,and that between PoEV-HeN10 and PoEV-GDMM7 was 78.1%and 70.7%.The diversity among the two new strains and other ephemeroviruses is beyond the criteria for demarcation of viruses assigned to different ephemerovirus species,which indicates PoEV-HeN10 and PoEVGDMM7 are two new species.Because this was the first time identifying ephemerovirus in pig tissues,the new species were named as porcine ephemerovirus(PoEV).The epidemiological investigation of 388 samples showed that no other positive samples were found.The nearly whole genome of 7 PSV strains and full-length VP1 sequences of 22 PSV strains were obtained by RT-PCR.Based on the sequence similarity and phylogenetic analysis,PSV-FJ1-1999 and PSV-GDFS9-2018 was identified as PSV-2 genotypic strains which showed similarity of 76.7-78.1%and 85.1-85.4%with 103 PSV-1 and 1 PSV-2 reference strains in ORF nucleotide sequence,respectively.And other strains were PSV-1.This is the first time to report that PSV-2 has been found circulating in China.Phylogenetic analysis showed that PSV-1 strains prevalent in China in the 1990s were mainly closer to Japanese strains,while the strains prevalent from 2010 to 2020 showed more diversity and were closely related to Japanese,German and Italian strains.The epidemiological investigation of 388 samples showed that all strains in 41 positive samples are PSV-1.Five genotypes of porcine astrovirus(PAstV)were found co-infecting with CSFV.And 14 strains sequences of PAstVs were obtained including 1 strain of PAstV1,6 strains of PAstV2,3 strains of PAstV3,3 strains of PAstV4 and 1 strain of PAstV5 by amplification and sequencing.This is the first report showed the genome sequence of PAstV3 strains in China which could cause neurological disease.After analysis of 11 strains registered in GenBank,it was found that PAstV3 strains can be divided into two subtypes:PAstV3a and PAstV3b which can be distinguished by the similarity of ORF2 of a cut-off value of 80.0%.The analysis of 90 PAstV strains in the world showed that there were great differences among the five genotypes which indicated the virus is still evolving rapidly.And compared with other 3 genotypes PAstV,PAstV1 and PAstV3 are more closely related to other mammal astroviruses.In this study,the genome sequences of 4 strains of porcine sapovirus were obtained including two G? strains(G?-GD6-1998 and G?-CQWX4-2020),one strain of GX(GXGD32-1996)and one strain of G?(G?-GD16-1998).The genome sequence identity of G?GD6-1998 and G?-CQWX4-2020 was 90.2%and that with G? reference strains was 76.888.9%and 77.0-90.1%,respectively,and they were closely related to Chinese strain G?-p2 and American strain G?-IL31538,respectively.The identity of G?-GD32-1996 and G?GD16-1998 with relevant reference strains were 78.7-79.8%and 74.9-75.6%.This is the first time detected G? and G? virus in our country.In addition,the nearly full-length genome sequences of 11 PRRSV strains and the full-length ORF5 gene sequences of 9 PRRSV strains were obtained including 4 European strains(PRRSV1)and 16 North American strains(PRRSV2)of which Lineage 8.7 was dominant(9 strains),and other strains belonged to sublineage 1.9(2 strains),Lineage 3(4 strains)and Sublineage 5.1(1 strain).At the same time,this study also traced the earliest epidemic time of PRRSV2 Lineage 3 in mainland China back to 1999.This study analyzed the genetic diversity and evolutionary characteristics of a variety of RNA viruses co-infected with classical swine fever virus,which provided important epidemiological data for the prevention and control of related porcine viral infectious diseases.At the same time,the results of this study showed that viral metagenomics technology is a powerful tool for studying viral co-infection in clinical cases,especially in the discovery of new viruses and mutated "old viruses",which can be used for accurate diagnosis and monitoring clinical swine diseases.