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猪流行性腹泻病毒主要结构基因遗传变异分析及ELISA检测方法的建立.docx

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猪流行性腹泻病毒主要结构基因遗传变异分析及ELISA检测方法的建立 Abstract: Porcineepidemicdiarrheavirus(PEDV)isanenvelopedRNAvirusthatcausesahighlycontagiousentericdiseaseinpigs.Inrecentyears,theemergenceofnewPEDVvariantshasresultedinseriouseconomiclossestotheswineindustryworldwide.Inthisstudy,weconductedgeneticanalysisonthepredominantstructuralgenesofPEDVanddevelopedanELISAdetectionmethod. Introduction: PEDVisahighlycontagiousentericdiseasethatcancausediarrhea,vomiting,anddehydrationinpigsofallages.PEDVwasfirstidentifiedintheUKinthe1970s,andhassincespreadtobecomeamajorconcernfortheswineindustryworldwide.Inrecentyears,newvariantsofthevirushaveemerged,resultinginseriouseconomiclossestotheswineindustry. ThePEDVgenomeisasingle-stranded,positive-senseRNA.Thegenomeisapproximately28kbinsizeandencodesforseveralnon-structuralandstructuralproteins.Thestructuralproteinsincludethespike(S)protein,envelop(E)protein,membrane(M)protein,andnucleocapsid(N)protein.TheSproteinisresponsibleformediatingvirusattachmentandentryintohostcells.Asthefirstlineofdefenseinthehostimmuneresponse,theSproteinisalsothemaintargetforvaccinedevelopment.Consequently,thereisagrowingneedtodevelopeffectivemethodsfordetectingandcharacterizingPEDVvariants. MaterialsandMethods: PEDVstrainswereisolatedfrominfectedpigfeces.RNAwasextractedfromtheviralparticles,andtheS,E,M,andNgeneswereamplifiedbyRT-PCRandsequenced.ThesequenceswerealignedandaphylogenetictreewasconstructedusingMEGA7.0software. TodevelopanELISAdetectionmethod,theSproteinwasexpressed,purified,andusedtocoat96-wellplates.PEDV-positiveandnegativeserawerecollectedfrominfectedanduninfectedpigs,respectively.Theplateswereincubatedwiththesera,andthebindingofantibodieswasdetectedusingahorseradishperoxidase-conjugatedsecondaryantibodyandchromogenicsubstrate. Results: Atotalof20PEDVstrainswereisolatedandsequenced.TheSproteinexhibitedthehighestgeneticvariability,withnucleotidesequenceidentitiesrangingfrom85.6%to98.6%.Phylogeneticanalysisshowedthatthestrainsformedthreedistinctclades,withthemaj