Isolation And Identification Of Newcastle Disease Virus From Shandong Province And Development Of Inactived Vaccine (â…¡+â…¦ Gene Types)

The current epidemic of Newcastle disease (ND) in our country is very complicated, and there is a lot of vaccination failure in some chicken farms, which has caused economic losses to poultry husbandry, especially the chicken industry. In order to prevent and control this disease, we firstly isolated and identified the pathogenicity of 12 ND viruses (NDV) from ill chickens after epidemiological survey of the diseases in Shandong province, and we analyzed the molecular characteristics of one virulent isolate LY1 by sequencing F gene and HN gene. Then, we developed inactive vaccine with LY1 isolated strain and LaSota strain. Meanwhile, we constructed and detected a new DNA vaccine of F gene of NDV fused with C3d P29 as molecular adjuvant. It laid important foundation to prevention treatment technology against NDV. This research consists of four parts as follows:1. Isolation, identification and biological characteristic of NDV in Shandong provinceAfter epidemiological survey of the diseases in Shandong province,12 NDV isolates were isolated from 82 chicken lung samples with clinical signs which were collected from 22 farms in Shandong province, and the virulent of the isolates were detected with MDT. ICPI and IVPI. The results showed that MDT of 6 isolates (WF10、WF13、LY1、LY7、ZB10 and JN2) were less than 60h. ICPI of all isolates was more than 1.6. IVPI of all isolates was above 2.00. It indicated that those 6 isolated, WF10、WF13、LY1、LY7、ZB10 and JN2, were velogen strains. Meanwhile, DY2. LC3 and BZ7 had MDT of 63.6h.69.6h and 62.4h, ICPI of 1.4、1.44 and 1.35; and IVPI of 1.74、1.85 and 1.49, respectively. It showed that DY2. LC3 and BZ7 were intermediate strains. And other 3 isolates, WF3. LY14 and TA6, were lentogen strains. In addition, the results of animal experiments indicated that the chickens infected the isolates had typically clinical symptoms gross lesion. But mortality or morbidity of chickens were different because of the inoculation of different NDV isolates. 2. Cloning and molecule characteristics of F gene and HN gene of NDV LY1 isolateThe HN gene and the main fragment of F gene were cloned and analyzed from LY isolate by reverse transcription PCR (RT-PCR). The results showed that F gene is consisted of 1662 nucleotides encoding 489 amino acids. Compared with the sequences of NDV which were published in foreign literature, it had 84.1%～88.7%identity in deduced nucleotides sequences, and 88.1%～93.3%in deduced amino acid sequences. The amino acid composition (112～117) in cleavage site region of F gene was the same as the composition of velogen strain’s. It proved that the LY1 isolate was velogen strain. Moreover, the deduced amino acid at 101st locus and at 121st were K (Lysine) and I (Valine), which being the typical characteristic of NDVⅦstrain. Compared with the sequences of velogen NDV strain F48E9 and lentogen strain C30, the homology were only 86.3 % and 84.2%, which were lower than those compared with other strains in our country. It indicated that LY1 isolate had changed greatly. Meanwhile, HN gene had about 1716 nucleotides in length encoding 571 amino acids. Compared with the sequences of NDV which were published in foreign literature, the identity were 82.1%～87.4%in nucleotides sequences and 87.6%～90.9% in deduced amino acid sequences. And it also had the molecular characteristic of NDV group C. It was consist with results of biological assay.3. Development of new Newcastle disease oil emulsion vaccine (typeⅦ+Ⅱ)In order to develop a bivalent inactive vaccine, LY1 (typeⅦ) and NDV LaSota strain (typeⅡ) was mixed with oil emulsion together at the rate of 1:3 and the protective efficacy was detected in chickens. The results showed that the vaccine had high safety after doubling injection to the chicken. The anti NDV-HI antibodies in bivalent inactive vaccine group were similar to those in commercial LaSota inactive vaccine group at 14,21 and 28 days post immunization (DPI) (P>0.05). The titers were reached 7.2Log2 at 35DPI. But antibody titer in LY1 oil emulsion vaccine group was lower than those in the bivalent inactive vaccine at 14,21 and 8 days post immunization (DPI) (P<0.05). After challenge with LY1 at 28 DPI and observed for 4 weeks, the immune protective efficiency of LY1+ LaSota bivalent oil emulsion was 100%, but the protective efficiency of LY1 oil emulsion vaccine and commercialization inactived vaccine(lasota strain) was 90% and 70%, respectively. It indicated that the LY1+ LaSota bivalent oil emulsion vaccine had higher protective efficiency than the commercialization inactived vaccine (Lasota strain).4. Construction and immuno-protection of DNA vaccines containing HN gene of NDV fused with C3d as molecular adjuvant HN gene of LY1 strain was amplified by reverse transcription polymerase chain reaction (RT-PCR) and directly inserted into eukaryotic expression vector pCDNA-P29n that contains Cd3-P29n gene, which resulting in 2 DNA Vaccines, pCDNA-HN-P29.4 and pCDNA-HN-P29.6. After vaccination with 3-weeks old SPF chicken, the results showed that the chicken in pCDNA-HN-P29.4. pCDNA-HN-P29.6 and pCDNA-HN groups all could produce HI antibody against NDV. But the HI antibody levels in pCDNA-HN-P29.4 and pCDNA-HN-P29.6 groups were obviously higher than that in pCDNA-HN group (P<0.01), but lower than that in NDV inactivated vaccine group. After challenge test, the results showed that pCDNA-HN-P29.6 and inactivated vaccine could provide 90% protective efficacy against NDV at lethal dose, which being apparently higher than pCDNA-HN group. It indicated that C3d of chicken had a synergistic effect with NDV-HN gene.