[Molecular mechanism affecting route of transmission for H9N2 subtype AIV].

The available evidence suggests that H9 subtype avian influenza virus (AIV) did not circulate in Chicken flocks in China until the early 1990s. 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. In order to investigate the molecular mechanism affecting the route of transmission for H9N2 AIVs, strains of A/Chicken/Guangdong/SS/94 (H9N2) (SS) and A/Chicken/ Shanghai/F/98 (H9N2) (F) were compared in their route of transmission. SS strain representing the earlier strain was isolated in chickens in Guangdong province in 1994, whereas F strain was isolated in Shanghai during 1998 pandemic. The findings suggested that F strain could transmitted in chickens by direct contact and by aerosol route. Whereas SS strain only by direct contact, and neither of two viruses by fecal contact. The cDNAs derived from the HA and NA genes of SS strain were cloned into vector pHW2000 to construct two transcription/expression plasmids respectively, and the cDNAs derived from 8 genes of F strain was done in the same way. Three recombinants were generated by reverse genetics: RF7/SSHA with the HA gene from SS strain and the remaining seven genes from F strain, RF7/SSNA with the NA gene from SS strain and the remaining seven genes from F strain, and RF7/SSHA/SSNA with the HA and NA genes of SS strain and the remaining six genes from F strain. In order to identify three recombinants, a total of seven genes from them were amplified by using PCR with universal primer pairs of H9N2 influenza virus and sequenced. In addition, three recombinants were characterized by HA and HI tests and sequence analysis. The results indicated that three recombinants were successfully rescued by reverse genetics. To determine the genes associated with the ability to transmit by aerosol route in chickens, a set of transmission experiments were designed. Groups of three chickens were inoculated with equal dose of virus by oral, intratracheal and intranasal routes. Each group was placed in direct, aerosol or fecal contact with three uninoculated chickens. Virus isolation and identification showed that only the RF7/SSHA recombinant was transmitted from inoculated to uninoculated chickens by aerosol route, whereas three recombinants were transmitted by direct contact, but not by fecal contact. The results were further confirmed by HI test of serum samples from uninoculated chickens. The data suggest that the NA gene might be the major determinant of the ability of aerosol transmission for H9N2 subtype AIVs in chickens.

[Influence of nonessential region on protective efficacy of recombinant fowlpox viruses].

Because of the interference of maternal antibodies, the recombinant fowlpox virus (rFPV) vaccine has not been used widely. The selection of a well-defined FPV nonessential region might be a desirable way to solve this problem. Two pairs of primers were designed according to the genome of a pathogenic FPV to amplify two flanking regions (FPV1 and FPV2) of the supposed nonessential region by PCR, and then a series of plasmid vectors were constructed to generate the expression vector pP12LS, which containing FPV1, FPV2, the expression cassette of P11-LacZ reporter gene and the promoter Ps. To abtain the vector pP12LSF, the F gene of ZJ1 strain Newcastle Disease Virus (NDV) was inserted into pP12LS, in which the F gene was located downstream of the promoter Ps. pP12LSF was transfected into chicken embryo fibroblast (CEF) pre-nfected with 282E4 strain FPV. The recombinant FPV, rFPV-FSC, was purified by blue plaque selection. The LacZ and F genes could be expressed by rFPV-FSC after 20 successive passages in CEF. The FPV nonessential region was the only difference between rFPV-FSC and rFPV-FSB. These two rFPVs could induce completely protection in SPF chickens against lethal challenge with F48E8 strain NDV. However, the protective efficacy showed a significant difference in commercial chickens with maternal antibodies. The protective efficacy of rFPV-FSC was 100% and rFPV-FSB was 61.54%. The results indicate that the selection of a well-defined FPV nonessencial region is an effective way to increase the protective efficacy of rFPVs, especially in chickens with maternal antibodies.

[Generation of A/Chicken/Shanghai/F/98 (H9N2) avian influenza virus from eight plasmids].

Eight-plasmid system was used for the generation of Avian influenza virus (AIV) strain A/Chicken/Shanghai/F/98 (H9N2) which was isolated in China in 1998. In this plasmid-based expression system, viral cDNA was inserted beteen the RNA polymerase I (pol I) promoter and terminator sequences. The entire pol I transcription unit was flanked by an RNA polymerase II (pol II) promoter and a poly (A) site. Twenty-four hours after the transfection of eight expression plasmid into cos1 cells, the supernatant and cos1 cells transfected were inoculated into the allantoic cavity of 10-day-old specific-pothgen-free (SPF) chicken eggs. The HA titer was determined after passage of the rescued virus in chicken eggs, and as high as that of the parental wild-type virus.

[Generation of newcastle disease virus strain ZJI isolated from an outbreak in the goose using reverse genetics technique].

The full-length cDNA clone, NDV3GM122, and the three helperplasmids pCI-NP, pCI-P and pCI-L of Newcastle disease virus strain ZJI isolated from an outbreak in the goose were cotransfected into BSR-T7/5 cell expressing T7 RNA polymerase. Meanwhile, the full-length cDNA clone NDV3GM122 and the three helperplasmids, pCIneoNP, pCIneoP and pCIneoL which were derived from NDV strain La Sota, were also cotransfected into the cell, respectively. Indiect immunofluorescence assay (IFA) was performed 48 to 96 hours post-transfection using NDV HN-specific monoclonal anbtibody (McAb) 6B1 and bright stainings were found in the transfectants, indicating that the full-length clone was functional and the HN protein was expressed. The transfected cell and the supernatant were mixed well and thereafter the mixture was inoculated into specific pathogen free (SPF) chicken eggs. The allanotoic fluid of the injected eggs gave a positive hemagglutinin( HA) titer ranging from 16 to 32 in the secondary passage and increased to 128 in the third passage, which was same to the level of parent wild-type virus. The allantoic fluid containing the recovered NDV was analyzed in hemagglutination inhibition( HI) test by using McAb 6B1 and the specific inhibition was found. The typical morphology of the produced NDV was detected in the electronic microscope. The results mentioned above demonstrated that infectious NDV of strain ZJI was successfully generated, which laid good foundation for the further related research.