Identification And Complete Genome Sequence Analysis Of Porcine Epidemic Diarrhea Virus Shaanxi Strain

Porcine epidemic diarrhea(PED),caused by porcine epidemic diarrhea virus(PEDV),is an acute and highly contagious enteric disease in swine.PEDV is the major cause of viral enteritis and fetal diarrhea in swine neonates.The disease is characterized by vomiting,severe enteritis and watery diarrhea,leading to death with a mortality rate of up to 100% in5-day old piglets.Recently,the outbreaks of the PED in many countries resulted in significant economic losses to the swine industry.The accumulated mutation in PEDV is one of the mainly reason for the occurrences of PED.Therefore,PEDV has become a hot spot in current swine disease research.Since 2010,outbreaks of viral diarrhea have been observed on most swine breeding farms in Shaanxi province.This study aimed to isolate and identify of PEDV from Shaanxi region,clone and sequence analyze the complete genome.The results can help to provide research data for epidemiology of PED,and the data will provide a theoretical basis for the effective prevention and control of PED.In this study,the PEDV Shaanxi isolated(named PEDV-SX)from a certain pig farm in Shaanxi was used to identify.After the virus was propagated,the S gene was firstly amplified by RT-PCR and confirmed by the following sequencing.Sequence analysis results show that the S gene of PEDV-SX strain has four insertion regions(nt 44 to 46,nt 57 to 60,nt 61 to 65,and nt 418 to 420)and one deletion region(nt 479 to 481).Large number of nucleotide differences is clustered in the region of the S gene of PEDV-SX strain compared with CV777 strain.Taken the above-mentioned results together,PEDV-SX strain is confirmed to be a PEDV variant.Multiple alignments and phylogenetic trees were constructed using the Meg Align program from the DNAStar software package.Phylogenetic tree constructed using the S protein sequences of 35 strains showed that the 16 reference strains were grouped with PEDV-SX strain.PEDV-SX strain formed a cluster with the strains SH,GD3,and AH2012.Nucleotides sequence analysis showed that PEDV-SX strain was more closely related to the Chinese strains SH,GD3,and AH2012.Protein sequences analysis showed that PEDV-SX strain shared higher amino acid identity to the American strains ISU13-19338E-IN-homogenate,and USA/Colorado/2013,and Chinese strain GD3 among allthe 35 PEDV reference strains.Based on the full sequence of the PEDV strains CV777 and AH2012 published on the Gen Bank,The full genome sequences were generated by 9 overlapping c DNA fragments to encompass the entire genome and were determined by genome walking sequencing.The complete genome sequence of PEDV-SX strain is 28,038 nucleotides(nt)in length [excluding the Poly(A)tail].Phylogenetic analysis was performed on the genome sequences of PEDV-SX strain and the available 25 PEDV strains in the Gen Bank.PEDV-SX strain formed a cluster with the strains AH2012,MEX/124/2014,ISU13-19338E-IN-homogenat,USA/Colorado/2013,NPL-PEDv/2013/p10.1,and 13-019349.The complete genome sequence of PEDV-SX strain has higher nucleotide sequence identity with those of strains NPL-PEDv/2013/p10.1,LZC,AH2012,13-019349,and USA/Colorado/2013.Taken together these results,it appears that PEDV-SX strain is more closely related to the Chinese strain named AH2012.It is speculated that the PEDV-SX strain is likely to be of early Chinese origin.The sequencing results would help us to determine the genetic diversities and molecular characterizations of the PEDV strains in Shaanxi province.The above analysis would provide a reasonable data support for control of PED in Shaanxi province.