Characteristics Of H9N2Avian Influenza Viruses And Duck Tembusu Viruses And Primary Development Of Vaccines Against The Diseases Caused By These Viruses

H9N2avian influenza virus (AIV) was widely spread worldwide, and caused great losses to poultry industry. Normally, H9N2AIV was low pathogenicity to poultry, and can’t cause death, but in the last few years, we found H9N2AIV was involved in the epidemic with poultry death. Using polymerse chain reaction (PCR), both H9N2AIV and Tembusu virus were identified in the lungs of dead ducks. So in this study we focused on biological characteristics of H9N2AIV and Tembusu viruses separately. In addition, we developed primarily the vaccines against the diseases caused by these two viruses.In order to know the biological characteristics of H9N2AIV,27H9N2AIVs isolated during2003to2010were selected. The genetic evolution analysis reveal the viruses posed multiple genotypes and diversity of the biological properties, PA and NS genes may recombine from other influenza virus subtypes, at the same time, we also found Ck/Shanghai/F/98(H9N2) is prevalent gene donator. The cross HI test and neutralization test indicated that the antigenic drift happened in the H9N2AIVs circulated in China. In the mouse experiments, the H9N2AIVs show a decreasing trend of pathogenicity on mice. After inoculated intranasally four-week-old SPF chicken with H9N2viruses, the viruses mainly amplified in tracheas and lungs.Based on the antigenic analysis result, Ck/Shanghai/441/09(H9N2)(abbr. Ck441) was selected as candidate strain for vaccine developement. Four-week-old SPF chickens were injected intramuscularly with inactivated vaccine, and then challenged with Ck441and a recent H9N2AIV isolates. the vaccinated chickens didn’t shed H9N2AIVs after challenge, while100%negative control chickens shed the viruses. In order to obtain high titer of H9N2virus in cell culture, the HA and NA genes of Ck441were inserted into pBD vector, and several reassortant viruses were rescure based on12plasmids system.441HANA/PR8-mutNS was selected as vaccine candidate for its high titers in both embryonated eggs and MDCK cell (HA-2) culture. The inactive vaccine based on the allantoic fluid or the cell culture supernatants containing441HANA/PR8-mutNS provided complete protection against H9N2AIV challenge.Duck Tembusu virus was a newly emerged virus which caused egg production decline、 retarded growth, and even death among ducks. In order to know whether the virus could infect chickens or mammalians, BALB/c mouse and SPF chicken were infected artificially with Tembusu virus Fengxian2010isolate (FX2010). No virus replication was detected in the organs of the mice injected FX2010intramuscular or intraperitoneal injection. Virus replication was detected in the lungs and nasal tissue of the mice injected----FX2010intranasally. When inoculated intracerebral with FX2010, the mice got sick and lost their weight significantly, the virus replication was detected in the brains and lungs. The more viruses inoculated, the more serious the symptoms appeared. Moreover, although the virus titers decreased significantly in lungs8days after mice inoculated intracerebral, the virus titers were still high in brains. Two-week-old SPF chickens were injected with FX2010intramuscularly and intranasally. The chickens injected intranasally grew slowly, while the chickens injected intramuscularly grew normally when compared with the control chickens. When intranasally inoculated with FX2010, the laying hens excreted green feces, and egg production declined. No chicken died, indicated that FX2010was low pathogenic to chicken.To develop a DNA vaccine against duck Tembusu disease, recombinant plasmids pCAGGS-E and pCAGGS-OptiE were constructed by inserting E gene and coden-optimized E genes into pCAGGS vector. E protein was expressed in293T cells transfected with pCAGGS-E and pCAGGS-OptiE and proved by IFA and Western blot assay. The pCAGGS-OptiE expressed more E proteins than pCAGGS-E in transfected293T cells. Both pCAGGS-OptiE and pCAGGS-E could stimulate mouse immuno-system to produce specific antibodies against duck Tembusu virus, although pCAGGS-OptiE was more efficiency than pCAGGS-E. This experiment provided primary dates for DNA vaccine against the disease caused by duck Tembusu virus.In conclusion, this study we described partial characteristics of H9N2AIVs and duck Tembusu virus, and developed primary vaccines against the diseases caused by H9N2AIVs and duck Tembusu virus. The results would be helpful in the prevention the two diseases.