| Porcine respiratory disease complex(PRDC)in pigs is mixed respiratory infections of multiple etiologies.The sickness is characterized by dyspnea,emaciation and lethargy in PRDC-infected pigs which increased mortality and mobility,and thus resulting in huge economic losses to pig industry.There are a variety of viral,bacterial and mycoplasma pathogens commonly associated with PRDC,of which viruses play an important role in the development and outcome of PRDC as a major pathogen.In this study,the viral communities in serum and nasal swabs from PRDC-affected piglets were investigated using metagenomics,and analyzed molecular characteristics of new viruses associated with PRDC.The following results were obtained: 1.Viral metagenomics identification of virus species in PRDC pigletsTo identify the virus specie,we collected the serum,nasal swab and lung tissue from PRDC piglets with obvious symptoms from 11 pig farms in Sichuan Province in 2016.After processing samples and doing viral nucleic acid extraction and reverse transcription,two pool serum and nasal swab samples were obtained.We used the double-stranded cDNA to construct a library and rloaded on a HiSeq 4000(Illumina)for sequencing.Then viral nucleotide sequences in database were organized and analyzed.The results showed that 16 viruses were identified in serum pool sample included Porcine parvovirus 6(PPV-6),PPV-3,PPV-5,PPV-4,PPV-2,porcine pseudorabies virus(PRV),torque teno sus virus 1b(TTSuV-1b),TTSuV-1a,porcine kobuvirus(PKV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus 2(PCV-2),porcine bocavirus 5(PBoV-5),ungulate bocaparvovirus 2(PBoV-1),ungulate bocaparvovirus 5(PBoV-3),pig stool associated circular ssDNA virus(PigSCV)and porcine cytomegalovirus(PCMV,0.01%).Nine distinct viruses were identifed from the swabs pool sample,were PRV,PPV-6,PPV-3,PBoV-3,TTSuV-1b,porcine astrovirus(PAstV),PCV2,PBoV-1 and PPV-2.The 17 different viruses associated with PRDC were identified in the serum and nasal swab samples from the piglets.The contigs from these viruses were de novo assembled using SOAP assembly software,from which six complete or near full-length genome sequences included PPV-2,PPV-3,PPV-4,PPV-5,PPV-6,and PCV-2 were obtained.The assembled PPV-2,PPV-3,PPV-4,PPV-5,PPV-6,and PCV-2 shared 94.8~97.3%,96.8~99.1%,98.1~99.6%,97.0~99.5%,97.0~99.5% and 95.9~99.4% nucleotide sequence identities with reference strains,respectively.Phylogenetic analysis of the six viruses showed a close genetic distance between the assembled PPV-3,PPV-5,PPV-4 and PPV-6 and the reference strains.The unique evolutionary relationship of assembled PPV-2 and PCV-2 strains indicated that the assembly of PPV-2 was a new genotype and the assembly of PCV-2 was a new PCV-2e.To verify the existence and distribution of 17 viruses identified in diseased piglets,RT-PCR and PCR were used to detect 26 lung tissue samples.The results showed that the detection rate of 17 viruses was as follows: PCV-2 in 96.2%,PRRSV in 84.6%,PAstV in 84.6%,PPV-2 in 73.1%,PKV in 69.2%,PCMV in 57.7%,PPV-3 in 53.8%,PBoV-1 in 46.2%,TTSuV-1b in 42.3%,PPV-5 in 34.6%,PPV-6 in 34.6%,TTSuV-1a in 19.2%,PBoV-3 in 15.4%,PRV in 7.69%,PBoV-5 in 7.69%,PigSCV in 7.69% and PPV-4 in 3.85%.In the individual lung samples,the mean number of virus categories for each sample(co-infections)was 7.4 for piglets with PRDC,with a maximum of 11 different viruses shed in one sample.Furthermore,PCV-2 and PRRSV were the viruses most frequently detected in the individual lung samples from the PRDC-affected piglets.The above results indicate that co-infections with multiple viruses were common in the diseased piglets with PRDC in Sichuan Province.To better understand which viruses are associated with PRDC,the previously determined 17-virus detection rate was investigated in an additional 74 serum samples from 36 PRDC-infected piglets and 38 location-matched clinically healthy control animals between 1 to 2 months of age,which were collected from five farms from 2016 to 2017.RT-PCR and PCR methods were used to investigate the distribution of viruses in 74 serum samples,and Fisher’s exact test was used to investigate the relationship between 17 viruses and PRDC.The results showed that 6.81 different viruses were shed by the 36 PRDC-infected piglets.In contrast,the mean value for the 38 clinically healthy controls showed that 4.09 distinct viruses were shed into the serum samples.The statistical analysis showed both PCV-2(OR=3.22,OR 95% CI=1.22-8.47,P =0.01)and PRRSV(OR=6.5,OR 95% CI=2.34-18.0,P<0.001)had statistically significant different detection rates in the serum samples between the PRDC cases and controls,indicating the two viruses are important pathogens associated with PRDC.According to statistics found that PPV-2(OR = 4.5,OR 95% CI = 1.52-13.3,P = 0.005),PPV-3(OR = 7.38,OR 95% CI = 2.35-23.2,P <0.001),PPV-6(OR = 8.57,or 95% CI = 2.86-25.67,P <0.001)and TTSuV-1a(OR = 3.96,OR 95% CI = 1.39-11.31,P = 0.008).Hence,these results suggest that PPV-2,PPV-3,PPV-6 and TTSuV-1a are significantly correlated with the probability of animals displaying the clinical signs of PRDC.2.Molecular Characterization of PPV-2,PPV-3,PPV-6 and PCV-2To further analyze molecular characterization of novel viruses identified in PRDC-infected pigs,we amplified the complete genome of PPV-2 and PCV-2,VP1 gene of PPV-3 and PPV-6 from 26 lung samples.Six PPV-2 full-length genome were obtained from 19 positive samples collected from different pig farms,with a length of 5361-5432 bp.The homology analysis showed they shared 96.3%-99.0% nucleotide sequence identities with each other,and 94.0%-98.4% nucleotide sequence identities with other reference sequences.The phylogenetic analysis showed that S1,S16 and S23 strains displayed unique genetic evolution.Moreover,the S1 and S23 strains were from different pig farms and gathered a single cluster in the phylogenetic tree,indicating that a novel PPV-2 genotype has existed in PRDC-affected pig farms in Sichuan,and the same new genotype PPV-2 was circulated in different pig farms.The six PPV-3 VP1 genomes were amplified from 14 positive samples collected from five pig farms.The length of VP1 genomes were 2518 bp.The homology analysis showed they shared 99.5%~100% nucleotide sequence identities with each other,and 97.3%~99.5% nucleotide sequence identities with other reference sequences.Phylogenetic analysis showed that the VP1 genomes of six PPV-3 strains was clustered into a large branch.This genetic evolution relationship indicated that the evolutionary trend of PPV-3 strains were consistent in Sichuan.Three partial VP1 genomic sequences were obtained from nine PPV-6 positive samples with a length of 1129 bp.The homology analysis showed three strains shared 99.6%~100% nucleotide sequence identities with each other,and 95.5%~99.8% nucleotide sequence identities with other reference sequences.Phylogenetic analysis showed that PPV-6 from Tibetan pigs fall into a cluster with American strains KSU1-AZ-2014 and KSU3-KS-2014,which indicated that PPV-6 of this study were related to PPV-6 identified in pigs affected withrespiratory diseases in USA.The nine PCV-2 strains were obtained from 25 PCV-2 positive samples collected from 9 pig farms,with length of 1767 bp~1768 bp.The homology analysis showed nine strains shared 96.3%~99.0% nucleotide sequence identities with each other,and 94.0%~98.4% nucleotide sequence identities with other reference sequences.Phylogenetic analysis showed that five PCV-2 strains belonged to PCV-2b,and remaining four belonged to PCV-2d.In addition,the genetic evolution showed that S21 strain was a new PCV-2b.These results indicated the genetic and evolutionary of three new viruses associated with PRDC were diversity but their pathogenicity needs further study.In addition,the prevalence of PCV-2 in Sichuan is basically consistent with the prevalence of PCV-2 in China. |
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