| The available evidence suggests that H9 subtype avian influenza virus (AIV) did not circulate in chicken flocks in china until the early 1990s. H9 viruses were isolated first from chickens in southern China in 1994, and circulated for a few years thereafter in some regions. However, the pandemic of H9 subtype AI, which started in summer of 1998, spread very rapidly to more than 20 provinces within several months. Obviously, the virus responsible for the 1998 pandemic was quite different from the virus isolated in early 1990s. A key feature of the pandemic influenza virus is its ability to spread rapidly and efficiently from infected to noninfected birds by aerosol. In order to investigate the molecular mechanism affecting the route of transmisson for H9N2 AIVs, strains of A/Chicken/Guangdong/SS/94(H9N2)(SS)and A/Chicken/Shanghai /F/98(H9N2)(F) were compared in their pathogenicity and route of transmisson. Our findings suggested that F strain could transmitted in chickens by direct contact and by aerosol, whereas SS only by direct contact, and neither of the two viruses by fecal contact. On the basis of the generation of F by reverse genetics, three reassortants were generated, with one or twosurface genes from SS and six internal genes from F. We tested the route of transmission of these reassortants in chickens and the results indicated that NA gene might play a key role for the ability of in aerosol transmission. An Attenuated reassortant H5N1 subtype AIV vaccine candidate (rgFH5△N1) with all eight genes from avian viruses was also generated and the protective efficacy of the inactivated vaccine made of it was evaluated in SPF and commercial chickens.1. Generation of reassortant AIV with all eight genes from avian virus by reverse geneticsWe had difficulties in the generation of reassortant influenza A virus with all eight genes from avian viruses until working conditions of the eight-plasmid based reverse genetics system were optimized.The parameters, such as the cell type and the proportion and amount of different plasmids used for transfection were adjusted and optimized but they were not the key factor to the success of virus generation. When we tried to cotransfected plasmids from F and A/WSN/33(H1N1) with various combinations, infectious viruses could be rescuedin most cases except the group in which the two polymerase genes of F and the other genes from A/WSN/33. The reason for the failure in rescuing the virus might be the low efficiency of replication of avian virus (F) in COS-1 cell. When we inoculated the culture supernatant harvested 24 h post-cotransfection into embryonated chicken eggs, the F was successfully generated. The rescued virus(RF) was identified by HA and HI tests, virus morphology and sequence analysis. The established system for generation of AIV from cloned cDNAs provided a novel method for studying molecular mechanism that determines the route of transmision for AIV and for developing new vaccine.2. Comparison of the pathogenicity and the route of transmission of F and SS and Sequencing and phylogenetic analysis of the HA and NA genes of SSF and SS were propagated in 10-day-old SPF embryonated chicken eggs. Allantoic fluid containing virus was collected, titrated to determine the ELD50/EID50 and stored at-70 ℃. To better understand the biological feature of transmission of H9 virus infection in chickens, we compared the pathogenicity, replication and route of transmission of the two H9N2 viruses. SS representing the earlier strain isolated in chickens in Guangdong province in 1994, whereas F was isolated in Shanghai during 1998 pandemic. Groups of five 4-week old SPF chickens were inoculated by oral, tracheal and nasal routes. Virus replication was monitored five days after inoculation. The lung lesion of F was more severe than that of SS. Virus load in terms of ELD50/EID50 in trachea after infection was assayed. Transmission experiments were designed. Groups of three chickens were inoculated by oral, intratracheal and intranasal routes respectively. Groups were placed in direct conta... |
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