The Protection Efficacity of DNA Vaccine Encoding Hemagglutinin of H5 Subtype Avian Influenza Virus

The DNA vaccine pCIHA5 encoding hemagglutinin can protect SPF chicken against lethal H5N1 avian influenza virus challenge. The more characters about its protection efficacity were studied. The protective rates in 10, 40, 70, 100 and 150 μg groups immunized with pCIHA5 were 12.5 (1/8), 58.3 (7/12), 72.7 (8/11), 50.0 (6/12) and 66.7% (8/12), respectively. The protective rates in 5, 20, 35 and 50 μg groups were 145.5 (5/11), 58.3 (7/12), 58.3 (7/12) and 91.7% (11/12), respectively. The 70, 100 and 5 μg groups have virus shedding of 1/8, 2/6 and 1/5. Though the inactived oil-emulsion vaccine has high HI antibody titers and 100% protective rate, the AGP antibody could be detected after vaccination. Results show that the pCIHA5 is fit to boost by intramuscular injection. This would be useful to the study on gene engineering vaccine of avian influenza virus.

Generation of Recombinant Equine Influenza Vaccine Candidate RgH3N1 Virus by Reverse Genetics

The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/ 8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/Jl/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.

Immune Efficacy of a Recombinant Fowlpox Virus Co-Expressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.

Immune Responses in Mice Injected with gD Plasmid DNA of Infectious Bovine Rhinotracheitis Virus

The gene encoding gD of isolate Luojing of infectious bovine rhinotracheitis virus(IBRV)was amplified, sequenced, and cloned into plasmid pcDNA 3.1, resulting in a recombinant pcDNA-gD. Groups of BALB/c mice were injected with 100 μg of plasmid only or together with liposome. After immunization, serum samples were collected from mice every 2 weeks for a 10-week period and tested for protein-specific antibody with enzyme-linked immunosorbent assay(ELISA). It was showed that the plasmid encoding IBRV glycopretein D developed gene-specific antibody. This report indicates the potential of DNA injection as a method of vaccination.

Cloning and Sequence Analysis of Glycoprotein D Gene of Bovine Herpesvirus-1 Strain Luojing

By means of PCR,the gene encoding gD of bovine herpesvirus-1 (BHV-1) strain Luojing was amplified,cloned and sequenced.The nucleotide sequence of this gD gene was (1 251 bp,)encoding 417 amino acids.Comparied with the published P8-2 strain,the homology of the necleotide sequence is 99.92%,and that of the deduced amino acid sequence is 100%.The results indicated that gD of BHV-1 was highly conservative.

Molecular Cloning and Prokaryotic Expression of Non-Structural Protein NS1 Gene of Porcine Parvovirus

Porcine parvovirus (PPV) is one of the major agents causing swine reproductive failure. NS1 protein is a non-structural protein of PPV and can be used as a reagent for differentiation of vaccinated animals and infected ones. In present study, a recombinant plasmid pET28a/NS1 was constructed by cloning the coding sequence for NS1 of PPV into pET28a, a bacterial expression vector. The NS1 protein was expressed in E. coli BL21(DE3) after induced by IPTG and the recombinant fusion protein was purified with affinity chromatography. Expression amount of NS1 protein was improved by optimizing the inducing parameters. The recombinant NS1 protein is reactive to PPV positive sera in Western blot and ELISA test and therefore can be applicable in differential diagnosis of PPV infections.

Combined immunity of DNA vector and recombinant vaccinia virus expressing Gag proteins of equine infectious anemia virus

In order to develop a new vaccine candidate for equine infectious anemia virus (EIAV), gag gene of Chinese donkey leukocyte attenuated strain (EIAV DLV) and its pa- rental virulent strain (EIAV LN) were inserted respectively into the TK region of the Tiantan strain (VV) of vaccinia virus by homologous recombination and the positive clone was confirmed by blue plaque assay. Protein expression was examined by Western blot. Prime and prime-boost proce- dures were used to immunize mice with two DNA vectors and two recombinant vaccinia viruses expressing EIAV Gag pro- teins. The results showed that the specific lysis of CTL re- sponses in the DNA+rVV groups was stronger than those in the DNA groups, amounting to 31%. Although the levels of specific antibodies were not significantly different, we could conclude that the recombinant vaccinia virus could boost the cellular responses following DNA vector priming. There was no detectable difference between the immune responses in- duced by DLV and LN Gag proteins. This data demonstrates that the combined immunity of DNA vector and recombinant vaccinia virus expressing EIAV gag proteins, utilizing the prime-boost procedure, can drive immunized mice to pro- duce powerful cellular responses. These results lay an im- portant foundation for the development of a new EIAV ge- netic engineering vaccine.