| Highly pathogenic avian influenza (HPAI), is an economically important disease of poultry with high morbility and mortality, which is caused by avian influenza virus of the H5N1 subtype. Vaccination is frequently used for the prevention and control of HPAIV in some countries when the diseases spread widely. Conventional HPAIV vaccines in poultry are mainly inactivated whole-virus vaccines that have been proved to confer adequate protection against the disease. However, there are several disadvantages for these vaccines, such as interfering with the serological surveillance, time-consuming preparation and inconvenient administration, high risks of disseminating HPAIV. Development of more efficacious vaccines that can be administered by mass application routes, such as oculonasal administration, is urgently needed. Reverse genetics can allow for the rapid and abundant production of live candidate influenza virus vaccines, overcoming time and quantity limitation of conventional inactivated vaccines.In this paper, a full-length cDNA clone of the genome of attenuated NDV strain ZJ1 was used for insertion of the ORF encoding the hemagglutinin (HA) of the highly pathogenic AIV isolate A/duck/shangdong/ 093/2004 in the intergenic region between the NDV phospoprotein (P) and matrix (M) gene. Infectious recombinant NDV expressing avian influenza HA was generated by reverse genetics. In addition, we also evaluate its genetic stability and protective efficacy in commercial chickens with low NDV- and HPAIV-specific maternal antibodies. The purpose of this study is to develop bivalent live attenuated vaccine against both velogenic NDV and HPAI H5N1 virus infection in poultry.1. Construction and generation of recombinant Newcastle disease virus expressing H5N1 HPAIV HA geneThe fragment spanning nucleotides 2864-5226 of the ZJ1 genome was amplified using specific primers, cloning into the pCR2.1 T vector and subcloned into the plasmid pUC18 vector using Hind III and Xba I, resulting in plasmid pUCR11. The AIV H5N1 ORF, amplified by PCR from plasmid pHA with Apa I-tagged primers, was inserted into pUCR11, generating pUCR11/HA. Finally, the Hind III-Xba I fragment of pNDV/ZJ1, or pNDV/ZJ1FM was substituted by a corresponding fragment of pUCR11/HA to creat full-length clones NDV/ZJ1 or NDV/ZJ1FM, respectively.H5N1 HA sequence that resembles a transcription termination-like sequence of NDV was modified by silent mutations by using primers. The resulting PCR fragment was cloned into pCR2.1. The Age I-Sac II fragment of pCRmHA was excised to replace the corresponding fragment in the pNDV/ZJ1FMHA, resulting in the formation of pNDV/ZJ1FM-mHA.The full-length clones were transfected together three helper plasmids, pCIneoNP, pCIneoP, and pCIneoL, into BSR-T7/5 cells. Supernatants of transfections were inoculated into 9-day-old embryonated SPF eggs to generate the infectious recombinant NDV, rNDV/ZJ1HA, rNDV/ZJ1FMHA, and rNDV/ZJ1FM-mHA, respectively.The presence of the modified HA gene and F gene in the virus from each passage was examined by RT-PCR and sequencing. Sequence analysis showed that the sequence identity of the HA gene was preserved. The expression of HPAIV H5N1 HA protein by rNDV/ZJ1FM-mHA was examined by IFA, indirect ELISA, respectively.2. Biological characterization and genetic stability of recombinant Newcastle disease virus expressing H5N1 HPAIV HA geneThe recovered virus was passaged 15 times in 9-day-old embryonated SPF chicken eggs, and the presence of the modified HA gene and F gene in the virus from each passage was examined by RT-PCR and sequencing. Sequencing analysis showed that the sequence identity of the HA gene was preserved and stably maintained even after 15 serial passages in developing chicken embryos. The expression of HPAIV H5N1 HA protein by rNDV/ZJ1FM-mHA was examined by IFA, indirect ELISA. These results showed that the recombinant rNDV/ZJ1FM-mHA stably expressed the modified HA protein and that modified HA protein was incoporated into the virions.rNDV/ZJ1FM and rNDV/ZJ1FM-mHA at an M.O.I of 0.01, were inoculated into 10-day-old emryonated SPF chicken eggs. Allantoic fluids were harvested to determine virus titers at 24 h intervals. TCID50 of each virus present in the allantoic fluid was determined by IFA. The results showed that the kinetics and magnitude of replication for both rNDV/ZJ1FM and rNDV/ZJ1FM-mHA were very similar, though the replication of rNDV/ZJ1FM-mHA was delayed slightly.The pathogenicity of the parental rNDV/ZJ1FM and that of the recombinant rNDV/ZJ1FM-mHA were determined by using two in vivo tests of mean death time(MDT) for the minimum lethal dose to kill the embryos and intracerebral pathogenicity index(ICPI) in 1-day-old chickens.The values of MDT for rNDV/ZJ1FM and rNDV/ZJ1FM-mHA were more than 120 h. ICPI scores of rNDV/ZJ1FM and rNDV/ZJ1FM-mHA were 0.16 and 0, respectively. These results indicated that the pathogenicity of rNDV/ZJ1FM-mHA was not increased after insertion of the HPAIV H5N1 HA gene.3. Inmmunization and challenge of commercial chickens with low NDV- and HPAIV-specific maternal antibodiesGroups of ten 15-day-old commercial chickens with low NDV- and HPAIV-specific maternal antibodies were inoculated by oculonasal administration with 106EID50 of rNDV/ZJ1FM-mHA, rNDV/ZJ1FM, and wt La Sota in a 0.2 mL volume. Blood samples were collected at 0, 7, 14, and 21 day post-immunization (p.i) and 14 day post-challenge (p.c). Sera were subjected to HI antibody detection using the OIE standard method. Three weeks p.i, the chickens were challenged oculonasally with 106.5ELD50 of velogenic NDV I4 or with 106.64ELD50 of highly pathogenic H5N1, respectively.After challenge infections, all chickens were observed daily for a period of 14 days for clinical signs and mortality. Oropharyngeal and cloacal swabs of the challenged chickens were collected to analyze AIV shedding at 2, 4, 8, and 14 d p.c.100% of the rNDV/ZJ1FM-mHA-vaccinated chickens were protected against the lethal NDV challenge, whereas all sham-infected animals died within 4 days, exhibiting typical signs of Newcastle disease. Challenge infection of with HPAI H5N1 virus caused severe disease and death in all control chickens inoculated with rNDV/ZJ1FM, and wt La Sota or PBS (sham) within 36 h, with a mortality rate of 100%. In contrast, 92.3% the rNDV/ZJ1FM-mHA-immunized chickens survived the lethal challenge. Thirteen of 14 chickens remained completely healthy, whereas only one chicken died at 4 d p.c. Furthermore, vaccination of rNDV/ZJ1FM-mHA could remarkably reduce virus shedding and virus transmission.In summary, we can draw the following conclusions from this study:1. Both of wild-type and a mutated HA ORF from HPAI mallard strain, A/Mallard/Huadong/S/2005 (H5N1) were inserted into the intergenic region between P and M gene of attenuated NDV, rNDV/ZJ1FM. Two infectious recombinant NDVs expressing avian influenza HA were generated by reverse genetics.2. The recombinant rNDV/ZJ1FM-mHA grew efficiently in embryonated chicken eggs and stably expressed the HA protein even after 15 serial passages.3. The pathogenicity of rNDV/ZJ1FM-mHA was not increased after insertion of the HPAIV H5N1 HA gene.4. Immunization of chickens with rNDV/ZJ1FMmHA induced NDV- and AIV-specific antibodies and protected commercial chickens with low NDV- and HPAIV-specific maternal antibodies against clinical disease after challenge with a lethal dose of velogenic NDV or highly pathogenic AIV, respectively.5. Shedding of NDV and HPAIV was remarkably reduced.6. rNDV/ZJ1FM-mHA is suitable as a bivalent vaccine against velogenic NDV and HPAIV and may be used as marker vaccine for the control of highly pathogenic avian influenza. |
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