| Highly pathogenic avian influenza (HPAI) is a severe form of generalized avian influenza among domestic poultry and various other birds which is characterized by a rapid and severe course of disease and a very high mortality, which is caused by highly pathogenic avian influenza virus (HPAIV), avian influenza virus strains of the subtypes H5 or H7. Since 2003, a widespread occurrence of HPAI has been registered in Southeast Asia, and in six countries HPAIV has also caused fatal human infections. During summer 2005, the disease has slowly spread westward. Isolated outbreaks have been reported from Kazakhstan, Russia, Romania, Turkey, Croatia and Ukraine. Avian influenza viruses are continuing to spread in waterfowl in Eurasia, even to Africa. The disease has caused enormous economical loss and threatened the health of humans.HPAIVs belong to the family of Orthomyxoviridae, which are pleomorphic, enveloped RNA viruses. Protruding from the lipid envelope are two distinct glycoproteins, the hemagglutinin (HA) and neuraminidase (NA). HA attaches to cell surface sialic acid receptors, thereby facilitating entry of the virus into host cells. Since it is the most important antigenic determinant to which neutralizing antibodies are directed, HA represents a crucial component of current vaccines.The culling of infected poultry is the time-honored method to control or eradicate the highly pathogenic avian influenza outbreaks and also the best-known way to prevent transmission to humans. However, when the viruses are widely spread over a huge area, culling and physical containment are highly unlikely to be successful. The vaccination program is a reliable strategy in the control of prevalence of these disastrous diseases. The ideal seed virus for vaccine production is a strain of low pathogenicity that is wellmatched antigenically with the prevailing virus and capable of growing well in eggs. All H5N1 viruses isolated in China since 1996 have multiple basic amino acids in the cleavage site of HA and therefore are all either highly pathogenic or potentially highly pathogenic avian influenza viruses unsuitable for vaccine production. It is difficult to construct avian influenza virus H5 subtype vaccine by traditional methods. Plasmid-based reverse genetics is a powerful tool to generate ideal reassortant influenza vaccine candidates.In this study, the six internal genes of the high yield influenza virus A/Goose/Dalian/3/01 (H9N2), the hemagglutinin (HA) gene of A/Goose/HLJ/QFY/ 04 (H5N1) strain and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting site between HA1 and HA2. Eight gene expressing plasmids were constructed and the recombinant virus H5N3 was reassorted by cells transfection. The chickens embryos infection and chickens challenge tests demonstrated the recombinant H5N3 (rH5N3) influenza virus is non-virulent. The rH5N3 oil-emulsified vaccine could induce hemagglutination inhibition (HI) antibodies in chickens in two weeks post-vaccination and maximum geometric mean HI titer were observed on 4～5 weeks post-vaccination and were kept during the eighteen weeks observation. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, both A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04.â… Construction of the high yield avian influenza virus H5 vaccine strainThe six internal genes of the high yield influenza virus A/Goose/Dalian/3/01 (H9N2), the hemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain and the neuraminidase (NA) gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. These amplified gene fragments, PB2, PB1, PA, NP, M, NS, HA and NA, were linked with pHW2000 plasmids respectively, and eight recombinant plasmids, named pML-PB2, pML-PB1, pML-PA, pML-NP, pML-M, pML-NS, pML-HA and pML-NA, were obtained. The virulence associated five continuous basic amino acids (R-R-R-K-K) of the HA gene were removed. The eight recombinant plasmids were transfected into 293T/MDCK mixed cell monolayer. Obvious cellular pathological changes could be observed after 72 hours. The hemagglutination (HA) titer of recombinant virus H5N3 (rH5N3) in the collected supernatant was 1:32. The results indicated that the recombinant vaccine strain rH5N3 whose all gene segments derived from avian influenza viruses was successfully constructed.â…¡The biological characteristics of the recombinant avian influenza vaccine strain rH5N3Analysis of antigenicityAnalyzing the antigenicity by the hemagglutination inhibition(HI) assay and neuraminidase inhibition(NI) assay, it was found that the antibodies from chickens against A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04 could inhibit the hemagglutination activity of rH5N3 and the HI titers were 1:512 and 1:1 024 respectively, while the antibody against the parent avian influenza virus strain, A/Goose/Dalian/01(H9N2), couldn't inhibit the hemagglutination activity. The antibody against the reference avian influenza virus strain, A/Duck/Germany/1215/ 73(H2N3), could acted with rH5N3, whose NI titer was 1:800, but the NI titer of the anti-A/Goose/HLJ/QFY/2004 antibody was less than 1:10. The results demonstrated that the phenotype of the recombinant avian influenza virus strain is H5 and N3 subtypes, as same as that of the experimental design.Analysis of pathogenicitySPF chicken embryonated eggs were inoculated with attenuated (10-1～10-10) rH5N3 virus (0.1 ml/each). The allantoic liguids were collected and analyzed for HA activity. According to Reed-Muench method, in this study, the EID50 (50% embryo infective dose) of rH5N3 is 2×109/mL. The HA titer reached 1:2048 at 48 h post-vaccination (p.v.). Embryonated SPF eggs inoculated with rH5N3 virus of 1×108 EID50 dose were still alive at 96 h. 4-mouth-old SPF chickens were inoculated with attenuated (1: 10) rH5N3 virus (0.1ml/each) by the intravenous route and observed for disease signs and death for 10 days. It was found that the chickens were completely normal and the IVPI is naught. This results indicated that rH5N3 virus is non-pethogenic to embryonated eggs and less pathogenic to chickens.Characteristics of growthMDCK cells and SPF chicken embryonated eggs were inoculated with attenuated allantoic fluids (10-1) of the recombinant virus strain rH5N3 and its parent H5N1 virus strain A/Goose/HLJ/QFY/04 (0.1 ml/each). The HA titers of rH5N3 in the collected supernatants and allantoic fluids were 1:512 and 1:2048 respectively, which are eight times of that parental virus strain A/Goose/HLJ/QFY/04. It was showed that the recombinant virus strain rH5N3 grows well in culture of mammalian cell lines and embryonated eggs, and is a high yield avian influenza vaccine strain.Genetic stability10-day-old SPF chicken embryonated eggs were inoculated with attenuated (10-1) rH5N3 virus (0.1 ml/each), and the allantoic fluid from infected eggs was harvested. After ten sequential passage in chicken embryonated eggs, the nucleic acid sequence recombinant virus rHSN3 didn't change, which indicated that the recombinant virus strain rH5N3 displays high levels of genotypic stability.â…¢The immune efficacy of the recombinant avian Influenza vaccine strain rH5N3Immune efficacySixteen six-week-old SPF chickens were disparted into two groups on average randomly. The chickens in one were injected with the vaccine of different doses (0.1, 0.3, 0.6, 0.9 mL/each) and the chickens in the other were only injected with allantoic fluid from NS-vaccinated chickens' embryos (0.6 mL/each). SPF chicken were vaccinated hypodermically with formalin-inactivated oil-emulsion rH5N3 vaccine, which was prepared by mixing mineral oil and allantoic fluid in the light of 1:2 proportion, and the blood of the chickens was drew at different time and detected for HI antibody titer and calculated the geometric mean of the HI antibody. The HI antibody became detectable but the titers were low in several chickens on day 7. All chickens had obvious antibody response at 2 weeks, even in the group were injected with the vaccine of low dose (0.1mL), with the HI titer exceeding 1:128 (7 log2). The HI antibody titer increased to 1:256 (8 log2) or higher at 4 weeks after vaccination. The geometric mean of the HI antibody titer still exceeded 1:16, which is the critical level in antibody protection.Protective efficacy3-week-old SPF chickens with negative HI antibody before immunizing were hypodermically injected with formalin-inactivated oil-emulsion rH5N3 vaccine. After 3 weeks, the chickens were detected for the titer of HI antibody in sera, and attacked with virulent avian influenza virus H5N1 subtype strains (A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04) using the method of dripping them into their noses and eyes. Oropharyneal and cloacal swabs were collected for virus isolation and titration at 3 and 7 days respectively post-attacking and detected the surviving chickens' HI antibody titer on days 14 post-attacking. It indicated 0.3mL vaccine dose could induce the chickens to produce high titer HI antibody and the geometric mean of the HI antibody titer exceeded 1:512 (9 log2). There was no clinic disease and death appearance and no virus separation among the chickens immunized with rH5N3 vaccine after attacking with 100 CLD50 HPAI virus, but all chickens in the blank comparison group died at 2～5 days after attacking and virus separation were positive. These results indicated that rH5N3 vaccine can not only protect chickens from HPAI virus' fatal attacking but reduce and prevent the transmission of HPAI virus among chickens.By using plasmid-based reverse genetics, the recombinant vaccine strain rH5N3, whose all gene segments derived from avian influenza viruses, was generated. Because of deletion of the virulence associated five continuous basic amino acids of the HA gene, the resulting virus is attenuated for chickens and chicken eggs. N3 neuraminidase subtype, which is infrequent in China, was selected as the molecular marker of the vaccine strain in order to distinguish vaccinated poultry and infected poultry with wild viruses conveniently. The vaccine strain rH5N3 with safety, low virulence and high yield properties improves it's growth property in chickens' embryos. This vaccine strain appears not only good inheritance stability but also favorable immunogenicity, and it can absolutely protect poultry from strong virulent viruses'fatal attack. The development of this vaccine strain provides us with a new tool to control and prevent the infection of avian influenza virus H5N1 subtype.
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