| Owing to viral infectious diseases in China is now characterized by the emergence of new epidemics in succession,and the old and importantones have not yet been eradicated,which seriously restrict the healthy and rapid development of pig industry.Virus tracking plays an important role in epidemic prevention and control.The epidemiological investigation of pig infectious diseases has always relied on traditional technology and methods,with which only single pathogen detection was allowed and limited data was acquired.With the continuous development and application of new technologies,large-scale detection of known and unknown viruses existing in host animals becomes possibele with metagenomics,which has been widely used in the survailence of viral infectious diseases.In the 1990 s,a nationwide epidemiological investigation of porcine viral infectious diseases was carried out in China,such as classical swine fever virus.However,the prevalence background of other viruses in the clinical samples is not fully known because of the unavailability of molecular biology techniques.Thus,it remains unknown whether the newly discovered virus existed at that time or not and evolutionary dynamics of the long-term epidemic viruses within the past 20 years?To clarify these issues,viral metagenomics analysis of 100 tissue samples in 1990-2017 was performed.As a result,16,646 reads were annotated to viruses within 14 families,consisting of 4 families of phages and 11 families of mammalian viruses,such as Circoviridae,Parvoviridae,Flaviviridae,Picornaviridae,Anelloviridae,Retroviridae,and Arteriviridae.Among them,Family Circoviridae obtained the largest number of reads,and the variety of reads in family Parvoviridae is the richest.Porcine circovirus 3(PCV3)belonging to the genus Circovirus within the Family of Circoviridae was first detected in the United States in 2015 in piglets with porcine dermatitis and nephropathy syndrome(PDNS),reproductive failure,cardiac and multi-systemic inflammation.PCV3 infections in China was first reported in 2015,while the prevalence of PCV3 before that remained unknown,In present study,307 clinical tissue samples collected between 1990 and 2017(1990-1999,200;2011-2017,107)from 20 provinces,4 municipalities and 5 autonomous regions in China were screened for PCV3 by specific PCR.The results showed that 7.8% of the total,collected in 8 provinces and 1 autonomous region were found positive and the earliest PCV3 case to 1996.Nucleotide sequence analysis showed that PCV3 strains obtained in this study shared 96.6%-99.7% and 97.1%-99.4% sequence identity at the ORF2 gene and genome levels with all available reference strains from China and other countries,indicating the high genetic stability of PCV3 over the past 20 years.Porcine parvoviruses(PPV)is a general term for viruses that cause porcine reproductive failure,digestive and respiratory diseases,including classical PPV1,and new porcine parvoviruses,such as PPV2,PPV3,PPV4,PPV5,PPV6,and PPV7.Based on the results of viral metagenomics,437 clinical tissue samples collected between 1990 and 2017(1990-1999,200;2011-2017,237)from 21 provinces,4 municipalities and 5 autonomous regions in China were screened for PPV1-7 by specific PCR.Results showed that 57.9%(254/437)of the porcine samples had at least one parvovirus infection,and the positive detection rates from high to low were as follows,PPV6(31.1%),PPV2(28.4%),PPV3(22.2%),PPV7(13.5%),PPV4(11.4%),PPV5(5.9%)and PPV1(5.0%).We found that the earliest cases of PPV2-PPV7 occurring in 1990,1993,1994,1999,1993 and 1997 respectively.Nucleotide sequence analysis showed that PPV2 strains obtained in this study shared 92.8%-99.2% sequence identity at the ORF2 gene with reference strains,which could be divided into eight genotypes: 2a-2h,the first was dominant in 1990 s,and 2a-2d and 2g-2h were prevalent after 2011.Recombination analysis revealed that 21 out of 26 recombinant isolates belong to PPV2c-e with PPV2 b,2g and 2h viruses as the parental strains.The remaining five recombinant isolates belong to PPV2a-b and 2g.PPV3 strains obtained in this study shared 95.5%-99.5% and 96.1%-99.4% sequence identity at the ORF1 and ORF2 gene with reference strains respectively.PPV4 strains obtained in this study shared 96.2%-99.9%,98.4%-99.8% and 96.0%-100% sequence identity at the ORF1,ORF2 and ORF3 gene with reference strains respectively.PPV5 strains obtained in this study shared 97.4%-99.9% and 98.8%-99.8% sequence identity at the ORF1 and ORF2 gene with reference strains respectively.These results showed that PPV3-5 had high genetic stability in the past 20 years.PPV6 strains obtained in this study shared 98.4%-99.8% and 94.8%-99.8% sequence identity at the ORF1 and ORF2 gene with reference strains respectively,which could be divided into 6a and 6b with 6a predominant in 1990 s,and both genotypes were prevalent after 2014.PPV7 strains obtained in this study shared 92.9%-99.2% and 84.0%-98.9% sequence identity at the ORF1 and ORF2 gene with all available reference strains respectively.PPV7 shows abundant genetic diversity and can be divided into 6 genotypes: 7a-f.In 1990 s,PPV7a,7d and 7e circulated in China and the first was dominant.Since 2011,five genotypes were prevalent in China,of which all 7b genotypes came from neighboring Guangdong and Guangxi provinces.In addition,PPV7 was formed by only one strain from Guangxi,and gene deletion and gene recombination were found in 5 PPV7 strains obtained in the present study.In conclusion,we have analyzed the prevalence of viral infection in swine herds in China in 1990 s,the epidemiological situations of PCV3 and PPV1-7 infection in China in 1990-2017,and the genetic diversity and evolutionary characteristics of these viruses.The obtained results significantly improved our understanding the prevalence of PCV3 and PPV1-7 infection in China and play a key role in epidemic surveillance,early warning,diagnosis,and prevention of these diseases. |
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