Infection And Molecular Characteristics Of Porcine Epidemic Diarrhea Virus And Application Of Next Generation Sequencing In New Pathogen Discovery

The main pathogens of porcine enteric coronavirus diseases are porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine deletacoronavirus(PDCo V),recombinant porcine enteric coronavirus(Se Co V)and the recently reported,porcine enteric alphacoronavirus(PEACo V).The infections of these pathogens lead to varying degrees of intestinal problems in pigs,causing great economic losses to the global pig industry.Currently,the worldwide porcine enteric coronavirus diseases show emerged and re-emerged characteristics.Traditionally,TGEV and group A rotavirus caused the diarrhea of piglets of different age.Since late 2010,variant PEDV caused the worldwide,including China and United States,PED outbreaks,resulting in great economic losses.This study focused on the study of porcine epidemic diarrhea virus and the application of next generation sequencing on new pathogen discovery.The main contents of this study are as followings: 1.The prevalence of PED and molecular characteristics of PEDV strains in ChinaSince late 2010,the diarrhea disease,characterized by outbreak at immunized pig herds,rapidly spread,outbreak without apparent seasonality,high morbidity and mortality,outbreak at main pig raising regions in our country.To study the infection characteristics of three main porcine diarrhea pathogens,a triplex(PEDV,TGEV and RV)RT-PCR detection method was used to detect a number of 2643 clinical samples collected during 2012 and 2015.The results showed that the detection rate of PEDV,TGEV and Po RV were 61.1%(1615/2643)、0.6%(12/2076)and 5.2%(95/1828),respectively.To get more knowledge of the PEDV strains and unveil the unique characteristics of the emerging PEDV strains,the complete S and complete genome of variant PEDV strains were sequenced and analyzed.To further study the complicated infection status of different types of PEDV strains in China,the partial S gene(1-1323 bp of N terminal of S gene)of 91 PEDV strains and the ORF3 gene of 46 PEDV strains were sequenced and analyzed.The results showed that the variant PEDV is the dominant PEDV type in China.The genetic markers were identified for differentiating different types PEDV strains and could lay the foundation for the establishment PEDV differentiation method.The unveil the molecular epidemiology of Chinese PEDV strains,a total of 405 Chinese PEDV strains were obtained and analyzed.2.The prevalence of PED and molecular characteristics of PEDV strains in the United StatesThe real time RT-PCR detection results of a total of 13,269 samples collected from 24 US states during December 2016 to May 2017 were provided by UMVDL.The detection results of PEDV,PDCo V and TGEV were 5.8%(763/13,269),1.0%(137/13,269),and ~0%(2/13,269),respectively.The co-infection ratio of PEDV and PDCo V were 0.1%(13/13,269).The results showed that the PEDV was the main porcine enteric coronavirus pathogen.The US PEDV strains were mainly clustered into two groups: emerged virulent PEDV group and emerged INDELs PEDV group.To better understand the complicated PEDV infection status in the US,a duplex PEDV differentiation RT-PCR method with good repeatability,specificity,and sensitivity was established and evaluated in the clinical.The detection results showed that the singlet virulent PEDV and INDELs PEDV detection rate in 287 PEDV positive samples were 80.8%(232/287)and 14.6%(42/287),respectively.The co-infection rate of these two PEDV strains was 1.0%(3/287).The coincidence rates the established method with previously reported real time RT-PCR were 91.3%(42/46)for INDELs PEDV strains,96.6%(229/237)for virulent PEDV strains and 75% for co-infection of INDELs and virulent PEDV strains.Using statistical analysis,there is not significant difference between the detection rate of these two methods.3.Characterization of novel PEDV isolates and etiology and genetic characteristics of different passages PEDV strainsA variant PEDV strain(designated as YN1)that has high nucleotide homology with epidemic PEDV strains in complete S gene and complete genome level,was isolated from the pig farm with severe diarrhea problems(the mortality of piglets within one-week is as high as 80.0%).The successful isolation of variant PEDV could help us understand the characteristics of variant PEDV strains and lay the foundation for the development of attenuated vaccine.The YN1 was propagated at Vero cell culture for 200 times.The complete genome of different passages YN1 strains were sequenced to study the genetic variation characteristics of different passages of YN1.These results could help us study the relationships between PEDV genetic variation and PEDV cell culture adaption and/or virulence.The complete genome of PEDV YN1 and its different passages counterparts showed that the nucleotides sequences of non-structural protein(NSP)2,NSP4-7,NSP10,NSP12 and NSP13 genes did not change during the Vero cell adaptation process.The comparison of the complete genome sequences of virulent and its attenuated PEDV strains revealed that the 9-26 amino acid(aa)changes in ORF1a/b and S protein,early termination and 8 amino acid changes in ORF3 gene,1-3 aa changes in the E,M and N protein and some synonymous nucleotide sequences’ mutations were associated with PEDV attenuation.The amino acid deletion at about 144 aa of S protein could be the attenuation marker for the PEDV.The pig infection study showed that the early termination in ORF3 was more important for virus cell adaptation than virus attenuation.The pig infection study showed that the YN144 was attenuated variant PEDV strains.Some clinical trials have demonstrated that YN144 is safe for piglets and pregnant sow and could stimulate the production of milk PEDV Ig A.The YN144 could be an oral vaccine candidate that can lay the foundation for further attenuation vaccine development.4.The preliminary application of next generation sequencing in new pathogen discoveryThrough next generation sequencing,a novel TGEV strain was identified in the US.The real time RT-PCR detection results of TGEV from porcine enteric cases(n=29,397)distributed across 41 states in the US collected between January 2008 and November 2016 were provided by UMVDL.The study showed that TGE occurred periodically in the US,especially in the cold seasons.Since 2013,the detection rate of TGEV were lower than 0.4%.Using de novo assembly and mapping algorithms of next generation sequencing,the complete genome nucleotide sequences of 18 US TGEV strains and 1 US PRCV strain were obtained.In those 18 TGEV strains,16 variant TGEV strains share 8 unique deletions and 119 unique amino acid changes were identified in this study.The phylogenetic tree analysis showed that these 16 TGEV strains constituted a unique phylogenetic cluster.The variant TGEV seems to be the dominant TGEV strains in US,while the clinical importance of the variant TGEV strains need to be further investigated.The recombination event was detected between variant PRCV and the variant TGEV strains.In conclusion,through investigation the prevalence of porcine enteric coronavirus diseases in both China and the US,it is found that the primary pathogen is PEDV.The study of genetic epidemiology and complete genome sequences of porcine enteric coronaviruses in both China and the US showed the complicated infection status of PED.The isolation and cell culture attenuation of variant PEDV strains in China could unveil the biological characteristics of variant PEDV in China and lay the foundation for attenuation vaccine development.The study of the application of next generation sequencing on new pathogen discovery could help us better cope with emerging and re-emerging veterinary clinical diseases(such as novel PED).