| Duck virus hepatitis (DVH) is an acute and fatal disease in young ducklings characterized by its rapid transmission. It was first described on Long Island in 1949. The major pathologic change in infected ducklings is hepatitis.Three distinct serotypes of duck hepatitis virus (DHV; types 1-3) have been described and all were originally classified as picornaviruses, with DHV-1 considered most like viruses of the genus Enterovirus. Duck hepatitis virus type 2 (DHV-2) has since been re-classified within the Astroviridae and has been renamed duck astrovirus 1 (genus Avastrovirus). No antigenic relationships have been found between DHV-1 and DHV-3 by the serum neutralization test. Among the three types of DHV, DHV-1 is the most widely distributed and most virulent and can cause mortality higher than 80% in ducklings under 3 weeks of age, while DHV-2 has only reported in the UK and DHV-3 has only found in the USA, both occur sporadically and cause a low mortality. To date, no molecular sequence data have been reported for DHV-1 and this has greatly hindered diagnosis and research.In this research, the DHV-1 was purified through by ultrafiltration—concentration method, PEG 6000 entrapment—concentration and differential centrifugation and then the purification effects was assessed. The Structure proteins of complete virion of DHV-1 was detected by SDS-PAGE and antigene-associated protein was analyzed by Western—bloting after Rbt-anti-DHV-1 IgG was prepared by purified DHV-1 antigen. Bioinformation analysis was performed on the basis of knowledge of Picornaviridae for development of genetically engineering vaccine of DHV-1 and establishment of conveniently diagnostic method. It contained genomic construct feature of DHV-1,phylogeny analysis of DHV-1 and Picornaviridae and prediction of construct protein molecular of DHV-1 polyprotein.The results were given as follow:1. The purification of DHV-1 and the assessment of purified methodsThe H strain of DHV-1 (ELD50 was 106.5/0.2ml) was amplified in duck embryo allantoic cavity and the duck embryo allantoic fluid and fetal membrane with virus were collected. They were purified by different methods in order to choose the best one. After performance of ultrafiltration—concentration method, PEG 6000—entrapment—concentration,differential centrifugation and PEG 6000 entrapment—concentration+differential centrifugation simultaneously, the last method was considered to obtain pure DHV-1virion.The concentration of virus protein was 13.257 mg/ml and the OD260/OD280 value was 1.051. It showed that the virus protein could be used to produce immune serum and carry out SDS-PAGE detection.The most pure virion of DHV-1 were obtained by the method of PEG 6000 entrapment—concentration+differential centrifugation during the methods given above. When detected by transmission electron microscope,there were a litter virion of DHV-1 and more foreign substance in sample pured by ultrafiltration—concentration method. There were also more virion of DHV-1 and foreign substance in sample pured by PEG 6000 entrapment—concentration,while a litter virion of DHV-1 and foreign substance in sample pured by differential centrifugation.It showed that only the sample ured by PEG 6000 entrapment—concentration+differential centrifugation contained the most virion of DHV-1 and the lest foreign substance.2. Preparation, purification and detection of Rbt-anti-DHV-1 IgGThe rabbits were processed by normal duck embryo allantoic fluid and Cyclophosphamide (CY) through subtractive immunization techniques.Then the rabbits were immunized after DHV-1 antibody was deactivated and emulsified. The blood was collected and tested by agar diffusion reaction and blood serum of Rbt-anti-DHV-1 IgG was gathered when the serum titer exceeded 1:64.The Rbt-anti-DHV-1 serum was extracted by caprylic acid—saturated ammonium sulfate (SAS) and Rbt-anti-DHV-1 IgG was purify by affinity chromatography with HiTrap rProtein A FF in order to purify IgG. After purification, SDS-PAGE was performed. It showed that there was one 160kDa band in 8% un-reduction SDS—PAGE patterns,one 55kDa and one 25kDa band in 12%reduction SDS—PAGE patterns.The virus neutralizing antibody titer of purified Rbt-anti-DHV-1 IgG was 1:2000. The results indicated that the Rbt-anti-DHV-1 IgG was purified and specific enough to perform Western—bloting test of DHV-1 antigene-associated protein afterward.3. Detection and analysis of structural protein and antigene-associated protein of DHV-1Parallel SDS—PAGE were performed on purified DHV-1 and normal duck embryo allantoic fluid to degrade the disparity. Two structural protein bands of 20-30 kDa molecular mass were found in 12% SDS-PAGE patterns and four structural protein bands of 7-30 kDa molecular mass were found in 15% SDS-PAGE patterns. Molecular mass of structural protein was calculated by QuantityOne on the basis of protein Maker. The molecular mass of structural protein bands in 12% SDS-PAGE patterns were 27.43kDa and 20.56kDa respectively and those in 15% SDS-PAGE patterns were 27.43kDa,25.59kDa,20.56kDa and 7.72kDa respectively. The 27.43kDa structural protein bands was proved to be the DHV-1 antigene-associated protein by Western—bloting test.The pair-wise amino acid sequence identities of DHV-1 genomic in NCBI until March 2007 showed that they possessed a typical picornavirus genome organization. The length of single-stranded positive-sense RNA genomes of DHV-1 was from 7582 to 7712bp(containing 3'poly A). The sequence of genome was 5' UTR, COS and 3' UTR. The length of 5'UTR was from 600nt to 626 nt without coat while that of 3'UTR was from 269 nt to 341 nt with 3'poly A(6-21 nt). The ORF which encoded 2249 AA and expressed polyprotein of DHV-1 was 6750bp.The homology of nucleotide sequences during 9 DHV-1 strains was 94.4%～99.5% and that of acid sequence of polyprotein was 97.1%～99%. Translation in all ORF started from ATG at the site of 600-626 nt and ended up with TGA at the site of 7350-7376 nt. Comparing polyprotein sequences in 9 DHV-1 strains, there were 138 differences in 2249 aa which meant 6.1% variation. During the concentrated variant domains that 346～542,673～727 and 845～924 aa, the most variations were at the site of 347,496,674,677,679,680,686,706,712,717 and 870. Absolutely conservative sequences were found in the domains of 1245～1295,1319～1432,1720～1800 and 1931～1991 aa. The motif that Gly-Ser-Cys-Gly-Gly (G-X-C-G-G) found in polyprotein sequences... |
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