Identification And Pathogenicity Analysis Of Duck Hepatitis Virus Ⅰ LY0801 Isolate

Duck virus hepatitis type-Ⅰ(DVH-Ⅰ) is a fatal, rapidly spreading and widely distributed disease of young ducklings (Woolcock, 2003). It can cause mortality higher than 90% in ducklings under 3 weeks of age while DHV-Ⅱand DHV-Ⅲhave only been reported in the UK and the USA respectively. DVH-Ⅰwas first described on Long Island in 1949 (Levine and Fabricant, 1950). Subsequently, out breaks were reported from England, Canada, Germany, Japan and elsewhere. Now DHV-Ⅰis becoming a worldwide in distribution and one of the most economic importance disease.This research consists of four parts as follows:PartⅠIsolation and Identification of the pathogen of Shandong province and simplex serums preparation against the isolatesIn this study, hepatitis samples were collected from different area of Shandong provience from 2007 to 2008, which ducklings show the clinical symptom of DHV-Ⅰ. Eleven DHV-Ⅰstrains were isolated by embryonated duck inoculateing, RT-PCR detection and reference serum neutralization from those hepatitis samples. Eleven groups 7-day-old SPF chickens were immunized four times with eleven DHV-Ⅰisolates in four weeks, and chickens blood was harvested at the fifth week later and then serums against the isolates were purified. The valence of serums were tested by embryonated duck inoculateing experiment, the result of serum neutralization to isolate oneself were all good effectivenessPartⅡStudy on pathogenicity of the DHV-ⅠLY0801 isolateIn this study, the ELD50 and LD50 of the strain LY0801 DHV-Ⅰwere tested respectively by duck embryos and ducklings. The ELD50 of LY0801strain detected by duck embryos was ELD50=106.46/ 0.2ml and the LD50 detected by ducklings was LD50=106.09/ 0.2ml. It is obvious that DHV-ⅠLY0801 strain was high mortality to both duck embryo and duckling. Cross-neutralization assays about part of eleven serums displays strain LY0801 is one representative DHV-Ⅰin Shandong province during 2007 to 2008.PartⅢComplete genome sequencing to LY0801 isolate and molecular analysisThe MegAlign program (Lasergene? expert sequence analysis software Version 7.1, DNASTAR Inc., Madison, WI, USA) using the Clustal W method (Thompson et al., 1994) was used to align nucleotide and amino acid sequences of each gene region of representative virus strains from the thirty-six strains Gene Bank accession. Nine degenerate PCR primers were designed for each gene region based on conserved motifs in representative virus strains. The full-length genome of LY0801 isolate was sequenced with 9 pairs of specific primers, and the results showed that the complete nucleotide sequence of LY0801 consisted of 7690nt excluding the poly(A) tail and contained a single ORF of 6747 nt terminating in a UGA codon. The genomic sequence of LY0801 has been submitted to GenBank and its accession number is FJ436047. The MegAlign program using the ClustalW method was used to align LY0801 complete genome. It shared 93.3%～95.2% similarity at the nucleotide level with that of other 36 DHV-Ⅰstrains listed in GenBank, and show 71.8% and 71.7% similarity with N-DHV isolated form Taiwan. The large open reading frame of LY0801 encoding a polyprotein of 2249 aa. It share 97.5%～98.7% similarity with the other 36 isolates by aligneing with same method. Phylogenetic tree about polyprotein amino acid of 39 strains was constructed by Meg4.0 with 1000 bootstrap replicates (K. Tamura, J. Dudley, M. Nei, S. Kumar, 2007). And antigen index of VP1 hypervariable region amino acid was analysis by Lasergene protean.PartⅣDevelopment and utilization of high titer vitelline antibody against DHV-ⅠLY0801 isolateIn this study, vitelline antibody to aim directly at LY0801 was advanced development. And it got good therapeutic efficacy in animal experiment and clinical application.