| Atypical porcine pestivirus(APPV)which is a novel pathogen of swine A-II congenital tremor(CT)in newborn piglets with geographical scope that has caused huge economic losses for the pig industry.Historically,APPV was first identified in the United States in 2015.Since then,the virus has been reported in newborn piglets in southern provinces of China which had proved the popularity of the trend in China.The results showed that the APPV Taq Man real-time RT-PCR detection method was established in this study had rapid and accurate for the detection of APPV,which laid the foundation to investigate the prevalent trend of APPV in the serum of newborn pigs in southwest China and to analyze its molecular characteristics:1?Establishment and Application of Taq Man real-time RT-PCR assayEight APPV whole genome sequences published in Gen Bank were compared and analyzed,and the primers and probes had designed in the relatively conservative region of 5'non-coding region(5'-UTR)of APPV genome in 2017.Using q PCR optimization,q PCR reactions were performed with annealing temperature of 54°C,0.5 ?L each of forward and reverse primers(final concentration: 250 n M)and 0.8 ?L of probe(final concentration: 400 n M).The minimum detection limit was 130 copies per reaction,the sensitivity of the method was at least 20 times higher than that of the reported real-time RT-PCR assay.The Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Pseudorabies virus(PRV),Bovine viral diarrhea virus 1(BVDV-1),Porcine circovirus 2(PCV-2),Escherichia coli(E.coli)and Salmonella positive strains were identified specifically by this method.The results showed the superior specificity by this method which could amplify APPV genes.The intra-and inter-group coefficients of variation were both less than 2.5%,which shows that the method has a good repeatability.At the same time,the result was consistent with those obtained using the previously reported real-time RT-PCR method,and the positive coincidence rate was 100%,thus a high sensitivity,good specificity,good repeatability and easy to operate and fast of Taq Man real-time RT-PCR method of targeting the 5'-UTR was successfully established in thisstudy.Based on the q RT-PCR method in this study,a total of 18 out of 166(10.84%,95% CI: 6.6%~16.6%)serum samples tested were positive for APPV RNA by q RT-PCR in 22 farms in southwest China,and there were 8 positive farms in 22 farms(36.36%,95% CI: 17.2%~59.3%).Interestingly,APPV had significantly different detection rates in CT symptoms samples(18/40)compared with the clinically healthy controls(0/126)(OR:45,95% CI: 29.3%~61.5%,P<0.001),suggesting that APPV was significantly associated with the probability of CT in newborn piglets in southwest China.At the same time,the results were compared with those of the reported methods.The results showed that the negative and positive reexamination rate was 100% compared with the real-time RT-PCR method.2?Analysis of Molecular characteristics of APPVIn this study,we obtained 15 APPV NS3 genomes(439 bp)from 18 positive samples by RT-PCR in southwest China.Sequence analysis showed that the 15APPV-NS3 strains shared 81%~100% nucleotide sequence identities with each other,and have 80.1%~94.2% and 47.1%~56.7% identities with other 13 APPV strains in Gen Bank and other representative hosts of Pestiviruses,respectively.The seven representative APPV strains were chose from seven positive farms and one strains was from every farms.We successfully assembled seven complete genomic sequences from the seven APPVs in this study,namely SWU-DY/2018 ? SWU-KZ/2018 ?SWU-MY/2018 ? SWU-QL/2018 ? SWU-XC/2017 ? SWU-YB/2018 and SWU-ZH/2017.The genomes were 11,269~11,462 bp in lengths,the G/C ratio of the polyprotein gene was 46.11%~46.48%.The seven APPV strains shared 83.1%~99.5%nucleotide sequence identities and 91.3%~99.6% amino acid sequence identity with each other,they shared 82.8%~98% nucleotide sequence identity and 91.3%~98.8%amino acid sequence identity with the 13 APPV reference strains available in Gen Bank.A phylogenetic tree was constructed based on the seven ORF genomic sequences of APPV and other 13 APPVs from Gen Bank.The result indicated that the APPV strains separated into two clusters and the seven APPVs were divided into two atypical porcine pestivirus clusters and form three small branches,which suggested that rich genetic diversity.Five of these strains(SWU-DY/2018,SWU-MY/2018,SWU-QL/2018 and SWU-XC/2017,SWU-YB/2018)were divided into two small branches formed by Chinese sequences.It is suggested that these two strains may be the potential dominant strains in China.The SWU-KZ/2018 and SWU-ZH/2017 strains show that the two strains were closer to the foreign strain of APPV which were formed gradually from the evolved gradually after introduction into China or the large genetic variation of the epidemic APPV strain in southwest China.E2 protein is the most antigenic protein being respensible for eliciting neutralizing antibodies and conferring protective immunity in Pestiviruses.In this study,the antigenic determinants of E2 proteins of seven APPVs were predicted and compared to the PPV1-000515 strain.The results demonstrated that the predicted antigenic determinants in the E2 proteins exhibited obvious genetic and antigenic differences among the seven APPV strains identified in this study.At present,mainly based on Nrpo and E2 genes of APPV for genotyping,and divided into four genotypes(A/B/C/D).To further understand the seven APPVs of evolutionary relationships,phylogenetic trees constructed using full-length E2 and Npro gene sequences showed that the seven APPVs sequenced in this study divided into three distinct subgroups.Strains SWU-XC/ 2017 and SWU-YB/2018 both belonged to Type D,SWU-KZ/2018 and SWU-ZH/2017 both belonged to Type A.It is worth noting that the genetic distance between the branches of SWU-DY/2018,SWU-MY/2018 and SWU-QL/2018 and other branches in E2 and Nrpo genes were 16.42%~19.25%,21.26%~24.60%,respectively.Suggesting the existence of a potential novel APPV genotype.We have given this novel genotype the preliminary designation of Type E.The results of study showed that APPV was significantly prevalence in newborn piglets with CT,and at least one novel APPV genotype to be circulating in swine farms in China.This is of great significance for further understanding of the genetic evolution of APPV. |
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