The Adaptive Research Of Duck Hepatitis Virus Type Ⅰ E₅₃ Strain In Duck Embryo Fibroblasts

Duck virus hepatitis （Duck Viral Hepatitis, DVH） is a kind of ducklings highly lethal acuteinfectious disease, characterized by hepatitis, pathogen is duck hepatitis virus （Duck HepatitisVirus, DHV）. Chinese popular type is the duck hepatitis type I DHV （DHV-I）. The mosteffective measure to prevention DVH is vaccination. At present there is no commercial vaccine,DHV chicken embryo attenuated vaccine, DHV chicken inactivated vaccine and DHVinactivated vaccine of duck embryo were mainly used to prevention for DVH. Chicken embryovaccine and duck embryo vaccine, relative to the cell vaccine, are the complex operation, lowyield. Therefore, to adapt DHV to chicken embryo fibroblast cell or duck embryo fibroblast cellis most important for improving the production of DHV vaccine.DHV-E₅₃chicken embryo attenuated strain was continuously adapted to duck embryofibroblast in this study. Clear cell lesions appeared in the sixteenth generation, and the cellularlesions of twenty-first generation showed regularly, that cells lesions could reached80%afterinoculated72h. The cellular lesion was characterized by cell gap widened, cells die off anddisintegration. The target DNA fragment were amplified from each DHV-ED adaptivegeneration by RT-PCR, which showed that the DHV-E₅₃strain could proliferated in duckembryo fibroblast. The specificity green fluorescence were observed after the duck embryofibroblast cells infected DHV-ED₁₆by indirect immunofluorescence （IFA）. The analysis of plantgenetic characteristics, using RT-PCR to amplified DHV-ED₁₆and DHV-ED₃₀of VP0, VP1andVP3, so the sequences alignment revealed that between VP1gene and reference sequences ofDHV-ED₁₆and DHV-ED₃₀nucleotide, amino acid had no difference. Gene between DHV-ED₁₆and DHV-ED₃₀VP0, VP3gene and amino acid homology was similarlier more than99%.Studyon biological characteristics: DHV-ED₁₆, DHV-ED₂₁, DHV-ED₂₆and DHV-ED₃₀TCID50were10^-3.42/0.1ml,10^-3.86/0.1ml,10^-3.93/0.1ml,10-3.46/0.1ml in duck embryo fibroblast;100TCID50DHV-ED₁₆and DHV-ED₃₀were inoculated on duck embryo fibroblast, collect thevirus in different time to determination of virus titer and to make virus growth curve, the resultsshowed that the DHV-ED₁₆, DHV-ED₂₁, DHV-ED₂₆and DHV-ED₃₀reached the peak at about72h virus titer, overtime, the virus titer was stably slow down; DHV-ED₁₆strain in chickenembryo ELD50is10-5.32/0.2mL （9day old SPF chicken embryos）, lesion characteristics are poorembryo development, embryo small, and the embryo is surface hyperemia, bleeding, abdominalswelling, but the embryo of liver bleeding spots and small necrotic foci; DHV-ED₁₆strain ofduck embryo ELD50is10^-4.78/0.2mL （9day old SPF duck embryo）, pathological characteristics and similar to chicken embryo. Safety test indicated that inoculated0.5ml of DHV-ED₁₆celladapted strain ducklings were good growth status and abnormal changes of animal autopsyfound DHV-ED₁₆weak adult duck; immune antibody level of parental strains and virus strainDHV-E₅₃in chicken embryo allantoic fluid was rising, and reached the peak in fifth weeks, thendecreased slowly, but the immune effect of DHV-ED₁₆immune group compared with the parentstrain DHV-E₅₃antibody level is slightly lower, but no have significant difference.