Detection ofHogCholera VirusandDifferentiation from OtherPestiviruses byPolymerase ChainReaction

Reverse transcription coupled withthepolymerase chain reaction (RT-PCR) wasusedforthedetection and differentiation ofpestiviruses. Forthis purpose, oneprimer pairwasselected fromahighly conserved region ofthegenomeofpestiviruses. Using these primers (PEST1-PEST 2),DNAfragments ofbetween 72and74bp could beamplified fromallpestivirus isolates tested. Inorder todifferentiate hogcholera virus (HCV)from bovine viral diarrhea virus (BVDV)andborder disease virus (BDV),weselected aprimer pairfroma conserved region inthegenomeofHCV strains thatdiffered fromthatsequenced inthegenomeofBVDV strains. Byusing these primers (HCV1-HCV2),aDNAfragment of478bpcould bespecifically amplified from HCV isolates. Bythese means, viral RNAwasdetected inextracts oflymphnode, spleen, tonsil, andlung. Such extracts wereuseddirectly forRT-PCRwithout prior RNA isolation. We also performed multiplex PCRby using boththePEST1-PEST2andHCV 1-HCV2primerpairs inasingle reaction. Thisallowed the differentiation ofHCV fromBVDVandBDVinonestep. Toassess thesensitivity ofthemethod, RT-PCRwas compared withvirus propagation intissue culture andsubsequent detection byimmunofluorescence staining. Theresults showthatRT-PCRisuseful fortherapid detection anddifferentiation ofpestiviruses. Pestiviruses, whichhaverecently beenclassified asa genusofthefamily Flaviviridae (28), areimportant pathogensinveterinary medicine. Theyareresponsible forsubstantial economic losses incattle, swine, andsheep farming. Thethree membersofthegenus Pestivirus arehogcholera virus(HCV),bovineviral diarrhea virus(BVDV),and border disease virus(BDV), whichcausehogcholera or classical swinefeverinpigsandwildboar, bovine viral diarrhea indomestic andwildruminants, andborder disease insheep, respectively. Onthebasis oftheir behaviors incell culture, twobiotypes eachofBVDV andBDV arerecognized, cytopathogenic andnoncytopathogenic