Cross-protection Studies In Chickens With H5 Avian Influenza Viruses Or Newcastle Disease Viruses Of Different Genetic Background

1. Construction and characterization of four rFPV expressing HA gene from H5 AIVs of different genetic background respectively.Four strains of H5 avian influenza virus (AIV) were selected for cross-protection study by phylogenetic and antigenic analysis. The HI test with a panel of HA-specific monoclonal antibodies showed different reactivity patterns for the four H5 AIV studied. Phylogenetic analysis revealed genetic diversity among these H5 viruses as well. The analysis also revealed that H5N2 low-pathogenicity WK AIV belongs to the American lineage and is located on distinct branches of the phylogenetic tree; while the other three H5N1 AIV tested belong to the Asia linage (SY and LK viruses from clade 2.3.4, and N36 from clade 0).HA genes were amplified by RT-PCR from the four AIV stains, and then used to construct four recombinant fowlpox viruses (rFPVs). Expression of these HA genes in rFPVs were confirmed by indirect immunofluorescence assay (IFA) in secondary CEF culture using anti-H5 AIV polyclonal antibodies. These rFPVs obtained could be used to study cross-protection.2. Cross-protection studies with H5 influenza viruses on the level of inactivated or rFPV vaccines.The current study was conducted to determine the impact of genetic relatedness among different AIV strains on the ability to protect against clinical sings, death, and infection and shedding of challenge AIV from immunized chickens. These four avian H5 viruses mentioned above SY, LK, N36 and WK, were further characterized in cross protection studies in SPF chickens. The WK, N36 and LK strains had 89.6, 94.2 and 99.5% deduced amino acid sequence similarity to the HA protein of SY challenge strain, respectively. Chickens were immunized either with the four inactivated whole AIV viruses or with the four corresponding rFPVs, and three weeks later challenged with 1×10⁵ EID₅₀ of the SY strain. On the levels of inactivated or recombinant vaccines, the extent of cross protection was evaluated by comparing the mortalities, frequencies and titers of virus isolation from tracheal and cloacal swabs.All the inactivated vaccines provided complete protection from clinical signs and death except WK inactivated vaccine with the mortality of 11/20. The virus titers in tracheal samples collected from WK inactivated group on day 2 post challenge were significantly higher than those from homologous group SY.All the four rFPVs conferred significant protection from the lethal challenge. Both the rFPV-SYHA and rFPV-LKHA induced full protection against clinical sings and death, however, rFPV-WKHA and rFPV-N36HA immunized groups failed to provide full protection, with mortalities of 12/20 and 5/12, respectively. The frequencies of virus shedding from trachea in rFPV-WKHA group on day 2 and 4 post challenge, and rFPV-N36HA on day 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA). The virus recovery rates of swab samples collected at different time points from group rFPV-SYHA and rFPV-LKHA were relatively low, and no significant difference was observed. The cloacal virus titers of rFPV-WKHA on day 2 and 4 post challenge were significantly higher than those from homologous vaccine group (rFPV-SYHA).These data indicated that the homology of the HA between the challenge virus and the virus used as vaccine is important and can significantly influence the levels of protective immunity in terms of clinical protection and reducing virus shedding even in SPF chickens housed under experimental conditions.Therefore, to maintain optimal protection against presently prevailing HPAIV strains by vaccination, it is necessary to select suitable new vaccine strains or HA insert in rFPV to overcome genetic drift at regular intervals. 3. Construction and characterization of four rFPVs expressing HN gene from different NDV strains respectively.Four strains of NDV were selected for cross-protection study by phylogenitic and antigenic analysis from a pool of NDV isolates. The strains GM and H3, isolated from diseased chicken and goose flocks respectively, are phylogenetically clustered to the same genotype VII, but are phylogenetically diverged from the vaccine strain LaSota, a genotype II virus, and the standard virulent strain F48E8, a genotype IX virus isolated originally in 1946.HN genes were amplified by RT-PCR from the four NDV strains, and then used to construct four rFPVs. Expression of these HN genes in rFPVs was confirmed by IFA in secondary CEF culture using anti-NDV polyclonal antibody. These rFPVs obtained could be used to study cross-protection.4. Cross-protection studies with Newcastle disease viruses on the level of inactivated or rFPV vaccines.It is widely recognized that because ND isolates are of one serotype, ND vaccine prepared with any ND lineage, given correctly, can protect poultry from clinical disease and mortality from a virulent NDV challenge. However, those ND vaccines do not protect vaccinates from virulent virus infection and viral shedding from such a challenge. The objective of this study was to compare the protection induced by ND vaccines prepared with viruses of different genotypes and antigenicity. Four NDV strains mentioned above, GM, H3, LaSota and F48E8, were used to prepare inactivated vaccines. These inactivated vaccines were inoculated into SPF chickens, together with four rFPVs, rFPV-GMHN, rFPV-H3HN, rFPV-LASHN and rFPV-F48HN. Twenty-one days after immunization, all the groups were challenged oculonasally with 100μl PBS-diluted allontoic fluid containing 1×10⁵ EID₅₀ of GM. Tracheal and cloacal swabs were collected on day 3, 5 and 7 post challenge. On day 5 post challenge, six birds from each group were sacrificed and six different organ samples were collected. The presences of challenge virus in swab or organ samples were determined by inoculation into 10-day-old embryonated chicken eggs or by Real-time RT-PCR.Significant levels of HI antibodies response were induced in all the vaccinated groups regardless the antigen used. The HI titers in inactivated groups were much higher than those in corresponding rFPV groups. Each vaccine group gave the highest HI titers when the antigen used in the assay was homologous to the vaccine antigen, except for H3 and rFPV-H3HN, in which higher HI titers were determined using heterologous antigen GM rather than homologous H3. All the inactivated vaccines and rFPV immunized chickens were fully protected against morbidity and mortality after challenge, whereas unvaccinated chickens died within 5 days after challenge.The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with inactivated vaccines showed that vaccination with Losota had no effect on tracheal shedding of virus on day 3 post-challenge when compared with controls as measured by frequencies of virus isolation. However, all the other three inactivated vaccines caused a significant reduction in virus shedding when compared with the controls. By day 5 and 7, the number of vaccinated birds shedding virus was reduced, and there was no significant difference among them. The results of virus detection by inoculation into embryonated chicken eggs of samples collected from groups immunized with rFPV showed that none of the rFPV vaccines significantly reduced the frequencies of virus shedding from trachea on day 3 post challenge with the exception of rFPV-GMHN, whereas all the rFPVs decreased the frequencies of virus shedding from cloaca. On day 5 post challenge, the frequencies of virus shedding from cloaca in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.When the swab samples were detected by RRT-PCR, no significant decrease in the frequencies of virus shedding was observed in all the rFPV immunized groups from trachea on day 3 post challenge. On day 5 post challenge, the frequencies of virus shedding from trachea in rFPV-LASHN and rFPV-F48HN groups were significantly higher than those in rFPV-GMHN and rFPV-H3HN.These results indicated that all of the vaccine used in the paper could provide complete protection regardless of the antigenic difference of the challenging virus in terms of preventing clinical disease. However, the antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation do affect viral shedding after a virulent challenge. There was a positive correlation in similarity between challenge virus and vaccines, and the ability to decrease the frequencies of virus recovery from trachea or cloaca.Protection from illness and death alone cannot be a comprehensive measure of effective control against an infectious disease such as ND. Prevention of infection and elimination or reduction of virus shedding, thus reducing the likehood of spread of virus to new flocks, are necessary to achieve optimal control of an outbreak. Therefore NDV vaccines formulated to be phylogenetically and antigenically closer to potential outbreak viruses may provide better ND control.5. Construction and protective efficacies of two recombinant fowlpox viruses expressing the HA Gene of H9 subtype AIV.The maternal antibody interference has been an obstacle in the development of rFPV vaccines. In an earlier report (Sun Lei et al., Acta Microbiologica Sinica.45 (3):359-362, 2005), the expression vector pP12LS was used to solve the problem effectively. In the present study, we used the vector pP12LS to construct rFPV against H9 subtype AIV. The HA gene from subtype H9N2 AIV was directionally inserted into the vector pP12LS, resulting in transfer vector pP12LSH9A. Then the transfer vector pP12LSH9A was used to transfect the chicken embryo fibroblast cells, which were pre-infected with strain 282E4 or LP of FPV respectively. After serial blue plaque screening, we obtained two purified rFPVs: rFPV₂₈₂-12LSH9A and rFPV_LP-12LSH9A. The protective efficacies of the two rFPV were investigated by vaccinating antibody negative SPF and antibody positive commercial chickens. A killed vaccine was included as controls. Results showed that the protective efficacies of rFPV₂₈₂-12LSH9A and rFPV_lp-12LSH9A in SPF chickens were both 100%, while those ones in commercial chickens were 88% and 92%, respectively. We concluded that the rFPV₂₈₂-12LSH9A and rFPV_LP-12LSH9A are two effective vaccines against H9 subtype AIV even in the presence of maternal antibodies.6. Comparison of the immunogenicity of three recombinant fowlpox viruses in the individual and combined vaccinationWhen rFPVs against different pathogens are used in a flock consecutively, the subsequent rFPV vaccination usually can not confer good protection due to the pre-existing vector-specific immunity. Combination of different rFPVs may be a strategy to solve the problem. However, whether the same levels of protection against each pathogen might be conferred by such combined rFPVs as those by individual rFPV alone, is unclear. Our aim in this study is to evaluate the ability of the combined rFPV vaccination to induce protection in chickens.Three rFPVs: rFPV-12LSH9A expressing HA gene of H9N2 AIV, rFPV-12LSH5NA expressing HA and NA genes of H5N1 AIV and rFPV-12LSHN expressing HN gene of Newcastle disease virus (NDV), were constructed using FPV strain LP. These rFPV vaccines were inoculated into SPF or commercial chickens with maternal antibodies to H9, H5 subtypes of AIV and NDV, either individually or in combination. Twenty eight days after vaccination, the combination group was divided into three subgroups, which were challenged with homologous H9N2 AIV, highly pathogenic H5N1 AIV or velogenic NDV, respectively. Individual vaccination groups were also challenged with the corresponding virulent virus. Serum samples were collected at appropriate intervals after immunization. Five days after challenge, tracheal (H9N2 AIV challenged groups) or cloacal (H5N1 AIV and NDV challenged groups) swabs were collected for virus isolation. The birds were monitored for 14 days for mortality. Results showed that in SPF chickens, administration of rFPVs in combination induced significant antibody responses to H9, H5 subtype AIV and NDV, but the HI titers were lower than those of individually vaccinated chickens. No death or virus shedding were seen after respective challenge except for the groups challenged with NDV, in which combined rFPV vaccinated group showed significantly higher virus recovery rate than rFPV-12LSHN immunized group(12/16 Vs 3/16). In commercial chickens, no obvious increase in HI titers after inoculation was detected in all the rFPV vaccinated groups. Among the groups challenged with H5N1 AIV, the combined vaccination group experienced a higher mortality (6/25) than the rFPV-12LSH5NA group (4/25), but the difference was not significant. The mortality in combined vaccination group (13/25) was significantly higher than that of the rFPV-12LSHN group (5/25), suggesting that the protective efficacy of rFPV-12LSHN was influenced by the other two rFPVs when being used in combination. No significant difference in the rate of virus recovery was observed between the combined group (17/25) and rFPV-12LSH9A group (13/25) after being challenged with H9N2 AIV. The results indicated that the protective efficacies of some rFPVs, when vaccinated in combination, might be influenced by other rFPVs. Therefore, it is wise to test each rFPV for efficacies when using multiple rFPVs in combination.7. A vaccination strategy primed with recombinant fowlpox virus and boosted with inactivated vaccine provided significantly better protection in chickens against H5 subtype avian influenza virusTen-day-old chickens with maternal antibodies to H5 subtype AIV were primarily vaccinated with a rFPV, rFPV-12LSH5NA, followed by a boost vaccination with H5 subtype AIV inactivated vaccine 1 week later. Ten-day-old chickens inoculated with rFPV-12LSH5NA and 17-day-old chickens inoculated with inactivated vaccine also served as control groups. One or two week after the boost vaccination, all the groups were challenged intranasally with lethal doses of homologous H5 AIV. All birds in the prime-boost group survived and the virus shedding rate was also significantly reduced, comparing to the groups with a single vaccination with rFPV or inactivated vaccine. These results indicate that the prime-boost strategy is very effective to protect chickens against lethal challenge of HPAI virus, even in the presence of maternal antibodies.