Pathogenicity And VP1gene Structural Characteristics Of Duck Hepatitis A Virus Genotype C

A DHAV-C isolate from Sichuan Province was used to experimentally infectSPF ducklings. Pathological autopsy, tissue section hematoxylin (H) and eosin (E)staining, immuno histochemical staining and apoptosis detection were adopted tosystematically research and illustrate the pathogenesis of the DHAV-C isolate toducklings. At the same time, the VP1genes in14DHAV-C isolates from SichuanProvince were cloned, sequenced, analyzed, showing the genetic diversity ofVP1genes. The VP1gene was also and prokaryotic-expressed, which established thebases for further research of DHAV-C subunit vaccine and DVH rapid diagnostictechnologies. The outcomes are as follows:1The pathogenicity and pathologic characteristics of DHAV-C to infected SPFducklings were determinedThe pathological model was successfully established by subcutaneous injectionof DHAV-C venom into3-day-old SPF duckling neck areas. The infected ducklingswere randomly selected and autopsied,10organs including heart, liver, spleen, lung,kidney, thymus, bursa of fabricius, pancreas, intestine and cerebrum were fixed inparaformaldehyde and sectioned. The different organ tissue sections were stained byH.E staining, immno histological staining and apoptosis detection. It was observedwhen the infected ducklings were autopsied that the DHAV-C isolate mainlydisoperated liver, spleen, bursa of fabricius, kidney, thymus and pancreas, showinglight hemorrhage and swelling of different organs at the early infection stage; a largearea hemorrhage and enlarged swelling of different organs, especially liver, wereobserved as the infection period prolonged. Histopathological observation found thatall10organs showed different extent of pathological changes, liver, spleen, bursa offabricius, kidney, thymus and pancreas among10organs showed obviouslypathological changes, coincidence with autopsy observed changes. Pathologicalchanges was firstly observed in liver and spleen at1h post infection (PI), most organsshowed different extent of pathological changes at18h PI. Serious hemorrhage and necrosis of liver, spleen and bursa of fabricius may be the cause of clinical symptoms,neurosis and death of the infected ducklings. All10organs had found DHAV-Cantigen. DHAV-C antigen was firstly detected out in liver, spleen and bursa offabricius at1h PI, then in heart, kidney, intestine, and thymus at6h PI. DHAV-Cantigen was detected out in pancreas and lung at12h PI, few DHAV-C antigen wasdetected out in cerebrum till24h PI. DHAV-C antigen distributed in all detectedorgans and increased rapidly with the prolonged infection time, except not obviouslyincrease in cerebrum. The largest amount of DHAV-C antigen was in liver, spleen andkidney, then in pancreas, bursa of fabricius, thymus, intestine and heart, a few amountof DHAV-C antigen was detected out in lung and cerebrum, fewer in cerebrumcompared to lung. DHAV-C antigen distributed in the cytoplasm of infected tissuecells. Apoptosis cells was observed in all10detected organ sections. Greenfluorescence apoptosis detection was accordant with DAB apoptosis detection andthere were no or few apoptosis cells in control, so, it can be concluded that DHAV-Ccan directly or indirectly induce apoptosis. Apoptosis was firstly detected out inimmune organs spleen, bursa of fabricius and thymus at1h PI, a large amount ofapoptosis cells were found in all detected organs with the prolonged infection time.Apoptosis induced by DHAV-C plays an important role in infected ducklings fallingill and in pathological changes of the infected ducklings.2Heterigenity in VP1gene and VP1protein antigenic epitope diversity ofgenotype C Duck Hepatitis A Virus isolatesVP1gene was cloned from14DHAV-C isolates, and the VP1geneheterogenicity and the coding VP1protein epitope was analyzed. The results showedthat14DHAV-C isolates belonged the same genotype. Six genotype C DHAV isolatesfrom Huayang district in Sichuan Province were clustered together with the strain DuCH LJS090905from Heilongjiang,3genotype C DHAV isolates from Qionglaidistrict were clustered together with the strains C-GY from Beijing and FJ01fromFujian Province. One isolate from Qionglai district and4from Pixian district inSichuan province were clustered forming a separate branch. These5isolates may bethe epidemic strains endemically infecting ducklings in Sichuan district because of their VP1gene differences and obvious genetic distance to other isolates in China.DHAV-C isolates may be from different districts of China through ducklingintroduction. The antigen index of DHAV-C VP1protein was high and thedifferences of antigen index concentrated in three regions including57~69、97~149and182~223amino acids of VP1protein. These regions were the hypervariableregions. Multi-epitopes vaccine can be designed according to these regions. Theseresults provided the theoretical bases for further research of the influence of DHAV-Cgenovariation to its pathogenesis, and for the preparation of diagnostic antigen and theresearch of newtype vaccine.3Successful expression of DHAV-C VP1proteinProkaryotic expression plasmid pET-32a(+)-VP1was successfully constructedand expressed in E.coli Rosetta (DE3), the molecular weight of the recombined VP1protein was about47KDa. Western-blot analysis showed that the recombined VP1protein could be recognized by rabbit anti DHAV-C antibody and had no reaction withnormal rabbit sera. This recombinant protein had good reactiongenicity. The greatquantitive and stable expression of the recombined VP1protein provided the bases forestablishing serodiagnostic technologies using the recombined VP1protein asdiagnostic antigen, and for carrying on DHAV-C immunologic research, pathogenesisand DHAV newtype vaccine research.