| Diarrhea is a common disease in pig herds,and one of the main diseases affectedpig health and economic profit in pig industry.Since 2010,severe diarrhea in swine has ocurred in China and many other pig-raising countries.The disease in neonatal piglets was characterized by vomiting,watery diarrhea and dehydration with morbidities and mortalities as high as 80%to 100%.These diarrhea outbreaks have led to substancial economic losses in pig industry.Piglets in Jiangxi province,a leading pork-producing province in China,have also suffered from this wave of diarrhea outbreak during the same period.However,the epidemiology and molecular characterization of main diarrheal associated virus circulating in Jiangxi province and the potential new pathogens and the alternations of gut microflora havn't been studied yet.Hence,we proposed to elucidate the profiles of several important re-emerged and potentially emerged novel virus(es)which might associate with the diarrhea in pigs in Jiangxi,China.For this purpose,the identification,molecular chacterization and epidemiology of diarrheal viruses responsible for the outbreak was performed by using conventional RT-PCR.Subsequently,the profiles of the gut virome and microbiota of both diarrheal and healthy pigs was explored by metagenomic technologies,and meanwhile potential new enteric pathogens were also hunted,and thus to reveal the relationships between diarrhea and the structure,diversity,abundance and function of gut virome and microbiome.The main project as well as the results presented in this thesis are as follows:1.Epidemiology investigation of several main diarrheal viruses in clinical samples and complete genome sequence analysis of PEDVA total of 1552 fecal and/or intestinal samples were collected from diarrheal pigs in95 premises suffered diarrhea in Jiangxi during 2012 to 2016.Epidemiology investigation was conducted on these diarrheal samples to determine the frequency of infection with PEDV,TGEV,and PoRV by conventional RT-PCR.The results showed that the detection rates of the three viruses were PEDV 58.38%(906/1,552),TGEV 1.74%(15/861)and PoRV 2.32%(20/861),respectively.The co-infection rates of PEDV with TGEV,PEDV with PoRV and PEDV with TGEV and PoRV were 0.46%and 0 was observed.In the context of the sampling ages of pigs,suckling piglets with diarrhea had the highest infection rate of PEDV(66.41%,698/1,051),and then followed by nursery pigs with positive rate of 55.22%(74/134);and diarrheal sows and finishing pigs had37.55%(98/261)and 33.96%(36/106)positive rate for PEDV.The intestinal samples presented a higher infection rate of PEDV than that of fecal samples(66.74%vs.47.04%;ANOVA,p<0.05).In the present study,the full-length genome sequences of two PEDV strains from Jiangxi province were determined.Sequence analyses indicated that the two Jiangxi PEDV strains has 99.9%similarity with each other,and showed homologies varies from96.3%to 99.7%with the reference strains.Compared to the reference strains,the two Jiangxi PEDV strains showed 26 nucleotide(nt)mutation during the whole genome,mostly located in the Rep and S gene,and 14 of them led to the alternation of amino acids(aa),the aa changes(~H2579~Y and ~H3232~Y)in ORF1a resulted in the transformation of polarity.Phylogenetic analysis based on the complete genome and structure genes of PEDVs showed that the two Jiangxi strains have higher homology with the strain determined post 2010 from China,United Stated and North Korea,while distiguished with the vaccine strain CV777 used in China.Multiple mutations at the nucleotide and amino acid level were observed between PEDV Jiangxi strains and the vaccine strain CV777,which would lead to the dysfunction of the vaccine present used in China.2.Complete genome sequencing and analyze of newly emerged porcine deltacoronavirusTo detect the emergence and infection of porcine deltacoronavirus(PDCoV)in pigs in Jiangxi,a RT-PCR assay was established,and PDCoV was first discovered in mainland China by using the established assay.Retrospective study indicated PDCoV could be detected in diarrheal samples in Jiangxi province as early as 2012.Molecular epidemiology investigation on 1216 diarrheal samples from 82 premises in Jiangxi province during 2012 to 2016.The results showed 62.20%of these pig farms were positive for PDCoV,and 30.26%of the diarrheal samples were positive.The combination infection rate of PEDV and PDCoV was 14.97%(182/1216)and one sample was co-infected with PEDV,PDCoV and PoRV.Sixteen pairs of primers targeting the genome of PDCoV were designed based on the PDCoV sequences from GenBank.The full-length genome sequence of a PDCoV strain from Jiangxi was amplified and sequenced,and was desinated as PDCoV/CHJXNI2/2015.The entire genome of PDCoV/CHJXNI2/2015 was 25,419 nt in size,3 nt longer than a HongKong strain HKU 15-44 due to 3 nt insertion in 3'UTR,and3 nt shorter than another HongKong strain HKU 15-155 as a result of 3 nt deletion in spike gene.Phylogenetic analysis incdicated that the current determined strain PDCoV/CHJXNI2/2015,together with deltacoronaviruses from swine and bird,were clustered together,distiguished from the alphacoronaviruses,betacoronaviruses and gammacoronaviruses.The sequences of E and M genes of PDCoV/CHJXNI2/2015showed 100%identities with the PDCoVs confirmed since 2014,and the complete genome and strutural genes all have>97%identities,which indicated high homologies of PDCoVs.3.Comparative analyses of viral metagenomics between healthy and diarrheal pigsIn present study,the feces of 3 healthy sows,4 diarrheal sows and 4 sows recoved from diarrhea,and the intestinal contents of 4 healthy neonatal piglets,15 diarrheal piglets(DP)and 15 non-diarrheal piglets farrowed in the same pens with DPs were selected.By using the method of viral metagenomics,comparison and analyses of the gut virome of healthy,asymptomatic and diarrheal pigs were conducted.A total of 28species of viruses belonged to 15 viral families and one unclassified familly were observed from the metagenomic sequences.An average of 8.5 species of viruses was detected per sample,with the fewest of 3 species and the most of 17 species.Among these observed viruses,porcine kobuvirus(PKoV)showed the highest detected rate and viral reads in all samples(91.1%,41/45),followed with PEDV(88.89%,40/45)and the newly emerged PDCoV(88.67%,39/45),porcine stool associated circulate DNA virus(PoSCV)(71.11%,32/45),(55.56%,25/45),and(35.56%,16/45).Statistical analysis indicated that the diarrheal pigs contained significant higher abundances of PEDV,while significant lower abundances of PKoV;reads assigned to PoSCV were predominant in samples from healthy and asymptomatic sows than other groups.Although the asymptomatic sows recovered from dirrrhea and asymptomatic piglets keeped in the sample pens with diarrheal piglets have not showed any dirrhea,but there still were high abundances of PEDV,PDCoV and other diarrhea associated viruses in these samples.4.Comparative study of gut microflora between diarrheal and healthy pigsIn order to study the influence of diarrhea on the gut microflora of swine,the structure and diversity of gut microflora among healthy,asymptomatic and diarrheal sow and piglets were analyzed and compared by using Illumina MiSeq sequencing of the V1-V3 region of the 16S ribosomal RNA(16S rRNA)gene.By using UniFrac pricipal component analysis(PCoA)and Unweighted Pair Group Method with Arithmetic Mean(UPGMA),we observed the fecal microbial communities of the healthy,asymptomatic and diarrheal sows were distinct;the gut bacterial communities of PEDV infected piglets were significantly different with those of the healthy piglets under 10 days old,while the difference of bacterial communities between diarrheal and asymptomatic piglets was not significant.PEDV infection could caused alternations of gut microbiota from levels of phylum to genus,or even species.The abundances of inflammatary or diarrhea associated bacteria from Desulfovibrionaceae,Desulfovibrio,and Clostridium perfringens,Clostridium difficile were significantly increased(p<0.05)while the butyrate-producing bacterium,Clostridium butyricum etc.,were reduced in diarrheal sows when compared those with the healthy sows.For piglets within 10-day-old,the abundances of short-chain fatty acid producing bacteria of Prevotella,Bacteroides,Coprococcus and Bacteroides,and the butyrate-producing bacterium Clostridium butyricum were remarkably decreased in diarrheal and asymptomatic piglets when compared those to the healthy piglets.However,the abundances of pathogenic bacteria Enterococcaceae,Enterococcus,Peptostreptococcus,and Clostridium perfringens were significantly increased in diarrheal piglets.The results revealed the diarrhea caused by PEDV casued diminish of probiotic bacteria while enrich of pathogenic bacteria,which could intensify the diarrhea.5.Comparative metagenomic study on the gut micribiome of diarrheal and healthy neonatal pigletsBy using the Illumina HiSeq sequencing and bacterial metagenomics technologies,the similarities and differences of gut microbial struture and function among healthy,asymptomatic and diarrheal neonatal piglets were analyzed and compared.At the level of microbial struture,the most abundant phylum in healthy and asymptomatic piglets was Bacteroidetes,and then followed by Firmicutes and Proteobacteria;but for diarrheal piglets,the abundances of Bacteroidetes were sharply decreased,and the abundances of Proteobacteria were increased.Functional potential analysis of metagenomics found that the relative gene abundance targeted the CAZy database was significantly lower in diarrheal piglets when compared that to the healthy piglets,indicated a lower level of carbohydrate-active enzymes in diarrheal piglets.When compared to KEGG database,the metagenomics of diarrheal piglets were remarkably enriched in relative abundances of catagories of L1 transposable element,Transposase,Endonuclease/Exonuclease/phosphatase family,RNA-dependent DNA polymerase and unknown functions.To sum up,we found that the diarrheal associated with PEDV did either alter the microbial stucture and abundances,or changed the functions of gut bacteria. |
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