Establishment Of Reverse Genetics Systems For H7N3 Avian Influenza Viruses

The outbreak of the H7 avain influenza has caused the enormous economic loss to the poultry industry, and caused a serious threat to the public health. In 2013, in Shanghai and Anhui, the spread of H7N9 AIV caused a large number of human infections and deaths. The HA gene of H7N9 AIV, which was the pathogenic determinant of H7N9, was provided by H7N3.Although really many extensive researches about H7N9 were carried out after the report of the H7N9, the study of the H7N3 that provided the critical HA gene for H7N9 was rare and seemed much more necessary.In order to establish the reverse genetics system of A/duck/Zhejiang/690/2009 (H7N3) (ZJ690) and A/duck/Zhejiang/766/2009/(H7N3) (ZJ766), the viral RNA were extracted, and reverse transcribed to cDNA. Then the whole genomes were amplified using RT-PCR,. The available PCR products were cloned into pBD bidirectional expression plasmids. Then the 8 purified plasmids of each genome of the virus were transfected into 293T cells and two rescued viruses were abtained. The rescued viruses were identified by the HA test,PCR and gene sequencing.The results indicated that two strains of viruses were rescued successfully and they had the same sequences with the wild viruses. The rescued viruses were named R-ZJ690, R-ZJ766 respectively.To explore the differece of pathogenicity between rescued and wild viruses, mice were inoculated with 106 EID50 of the indicated virus intranasally. Body weight were measured daily for 14 days, and the viruses titers of organs were determined by EID50. The results showed that although none of the mice died during the observation period, but body weight of infected mice was lose slightly. The four viruses were only detected in the nasal turbinates and lungs but not in other tissues at 4th and 6th day of postinfection.. The consistent distribution and virus titers of ZJ690, ZJ766 and R-ZJ690, R-ZJ766 indicating that those four virus had same pathogencity to mice.The study would provide a promising technique support for further study on the pathogenic and transspecies transmission mechanisms of the H7 avian influenza.