Improving expression of antigen is critical to the immunogenicity of DNA vaccines. To achieve this goal, we modified the NDV F48E9 strain HN gene by optimizing the condon usage and inserting the secretary leader sequence [A/Goose/Guangdong/1/96 (H5N1) HA gene, Accession No. AF144305]. The HN gene modified and knocked the signal peptide off were named SoptiHN and optiHN. The three sequence: SoptiHN, optiHN and the NDV F48E9 strain HN gene were inserted into the vector pVAX1 and vector pVAX1-CpG including CpG-ODN sequence respectively. Then we got six recombinant plasmids: pV-SoptiHN, pVC-SoptiHN, pV-optiHN, pVC-optiHN, pV-HN and pVC-HN. By optimizing condon usage in transiently transfected 293T cells, expression levels of HN gene were higher from the codon-optimized gene than the counterpart. Moreover, both optimization of condon usage and addition of signal peptide could improve expression of HN gene in vitro.
Animals ; Chickens ; Codon ; HN Protein ; genetics ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; Newcastle Disease ; immunology ; prevention & control ; Newcastle disease virus ; classification ; genetics ; Vaccines, DNA ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

In order to determine the challenge dose of pigeon paramyxovirus type 1 (PPMV-1) inactivated vaccine (S-1 strain). The virus titer of PPMV-1 E5 allantoic fluid (Chuansha strain) was determined using SPF chicken embryos in this research. After inoculating 30-day-old and 120-day-old pigeons with low-HI antibody against PPMV-1 (HI antibody < or =2) with different doses of PPMV-1 (Chuansha strain), the clinical symptoms and histopathological lesions of the challenged pigeons were examined. The results showed that the minimal lethal dose (MLD) of PPMV-1 (Chuansha strain) was 102.5 ELD50, so we determined that 10(5.5) ELD50, which was 1000 times the MLD, could be taken as the challenge dose in the vaccine efficacy test for PPMV-1 inactivated vaccine (S-1 strain).
Animals ; Antibodies, Viral ; immunology ; Bird Diseases ; immunology ; mortality ; virology ; Chick Embryo ; Columbidae ; immunology ; virology ; Newcastle Disease ; immunology ; mortality ; virology ; Newcastle disease virus ; immunology ; pathogenicity ; Phylogeny ; Viral Vaccines ; immunology ; Virulence

Despite the intensive vaccination policy that has been put in place to control Newcastle disease virus (NDV), the recent emergence of NDV genotype VII strains in Korea has led to significant economic losses in the poultry industry. We ssessed the ability of inactivated, oil-emulsion vaccines derived from La Sota or Ulster 2C NDV strains to protect chickens from challenge with Kr-005/00, which is a recently isolated Korean epizootic genotype VII strain. Six-week-old SPF chickens were vaccinated once and challenged three weeks later via the eye drop/intranasal route. All vaccinated birds were fully protected from disease, regardless of the vaccine strains used. All vaccinated and challenged groups showed significant sero-conversion 14 days after challenge. However, some vaccinated birds, despite being protected from disease, shed the challenge virus from their oro-pharynx and cloaca, albeit at significantly lower titers than the unvaccinated challenged control birds. The virological, serological, and epidemiological significance of our observations with regard to NDV disease eradication is discussed.
Administration, Intranasal ; Animals ; Chickens ; Cloaca/virology ; Disease Outbreaks/prevention & control/*veterinary ; Korea ; Newcastle Disease/*immunology/prevention & control ; Newcastle disease virus/*immunology ; Ophthalmic Solutions ; Poultry Diseases/*immunology/prevention & control ; *Vaccines, Inactivated/administration & dosage ; Viral Vaccines/*administration & dosage ; Virus Shedding/drug effects

In chicken fibroblast cell (CEF) cultures with antiserum against Newcastle disease virus (NDV) strain TZ060107, the virus was passed serially for 50 passages in 3 independent lineages. HN and F genes were amplified and sequenced every 10 passages. The derived virus A1-50 with most mutations among 3 lineages was further passed for another 50 passages in CEF with or without antiserum against A1-50, each in 3 independent lineages. Sequence comparisons for HN and F genes of 60, 70, 80, 90 and 100 passages indicated that the ratio of nonsynonymous mutations (NS) vs synonymous mutations (S) for HN genes in the lineages passed with antiserum against A1-50 was 5.25, which was obviously higher than 2. 375 of NS/ S in the lineages without the antiserum. The stable NS mutations occurred in the first 50 passages with the antiserum against the original TZ060107 were still maintained and one more new stable NS mutation appeared. For the F gene, 3 new stable NS mutations occurred during the second 50 passages in lineages with antiserum against A1-50 when the original NS mutations obtained in the first 50 passages with antiserum against TZ060107 still existed. Cross hemagglutination inhibition (HI) between original virus and its derivative viruses indicated that the more continuous passages in cell culture with antiserum passed, the bigger difference of antigenicity between the virus and the original virus had.
Amino Acid Sequence ; Animals ; Antibodies, Viral ; immunology ; Base Sequence ; Chickens ; Evolution, Molecular ; HN Protein ; genetics ; immunology ; Hemagglutination Inhibition Tests ; Molecular Sequence Data ; Mutation ; Newcastle Disease ; immunology ; virology ; Newcastle disease virus ; genetics ; immunology ; Poultry Diseases ; Viral Fusion Proteins ; genetics ; immunology

Newcastle disease (ND) caused by virulent Newcastle disease virus (NDV) is a highly contagious viral disease of poultry. Virulent NDVs characteristically have a multibasic amino acid sequence (virulence motif) such as (112)RRQKRF(117) at the cleavage site of the precusor fusion (F0) protein. The antigenic and immunogenic characteristics of the virulence motif (112)RRQKRF(117) in the F0 protein of virulent NDVs were investigated. Epitope mapping analysis revealed that a RRQKRF-specific monoclonal antibody 4G2 recognized the KRF section of the motif. A synthetic peptide bearing the RRQKRF motif reacted strongly with sera from virulent NDV (with RRQKRF motif)-infected chickens. These sera also showed reactivity to peptides bearing other virulence motifs ((112)KRQKRF(117), (112)RRQRRF(117) and (112)RRRKRF(117)) but not an avirulence motif ((112)GRQGRL(117)) by ELISA. The synthetic bearing RRQKRF motif reacted with 60% to 91% of sera taken from surviving chickens on ND outbreak farms but not with sera from vaccinated birds, even though most of the sera had antibody to NDV due to vaccination. This indicates that the virulence motif has the potential to differentiate virulent NDV infected birds from vaccinated birds.
Amino Acid Motifs/*immunology ; Amino Acid Sequence ; Animals ; Chickens ; Enzyme-Linked Immunosorbent Assay/veterinary ; Epitope Mapping/veterinary ; Newcastle Disease/*immunology ; Newcastle disease virus/*genetics/pathogenicity ; Poultry Diseases/*immunology/*virology ; Serologic Tests/veterinary ; Viral Fusion Proteins/*genetics/immunology ; Virulence/genetics

Two hundred thirty specimens of wild birds were collected from some areas in Heilongjiang Province during the period of 2003~2004, including two batches of specimens collected randomly from a same flock of mallards in Zhalong Natural Reserve in August and December, 2004, respectively. Primary virus isolation and identification for avian influenza virus (AIV) and Newcastle disease virus (NDV) were performed. The results showed that only two specimens of young mallards collected from Zhalong Natural Reserve in August, 2004 were positive to AIV (isolation rate 0.9%), and one strain (D57) of these two virus isolates was identified to be H9 subtype by hemagglutination inhibition test. Meanwhile, the two batches of blood serum samples of mallards from Zhalong were also examined for antibodies against AIV and NDV. Among 38 blood serum samples collected in August, antibodies against the hemagglutinin of H1, H3, H5, H6 and H9 subtypes of AIV were found in 1, 0, 2, 0 and 8 samples, respectively; and 11 samples were found with antibody against NDV. Whereas the NDV isolation in both two batches of specimens of mallard was negative, all of the 32 blood serum samples collected in December were negative for antibodies against AIV and NDV.
Animals ; Animals, Wild/*virology ; Antibodies, Viral/isolation&purification ; Birds/virology ; China/epidemiology ; Hemagglutination Tests ; Influenza A virus/*isolation&purification ; Influenza in Birds/epidemiology/immunology/*virology ; Newcastle Disease/epidemiology/immunology/*virology ; Newcastle disease virus/*isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction

The fusion protein (F) gene of Newcastle disease virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-F, and subcloned into eukaryotic expression vector pmcDNA3. 1+. The F gene was identified by sequencing. The recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was designated as SL7207 (pmcDNA3. 1-F). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3. 1-F was apparently higher than that of pcDNA3. 1-F in SL7207. In order to compare the immune response induced by these two re combinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) initiated F-specific serum and mucosal antibodies in immunized mice. Furthermore, 4-day-old SPF chickens were immunized with SL7207(pcDNA3. 1-F) and SL7207(pmcDNA3. 1-F) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dosage. Humoral and intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive controls. The result of protective efficacy showed that the chickens immunized with SL7207(pmcDNA3. 1-F) had the protective rate of 70.0%, higher than that of the SL7207 (pcDNA3. 1-F) with 50.0%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine has been developed and could be useful for controlling the infection and epidemic of Newcastle disease in the poultry.
Animals ; Chickens ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Newcastle disease virus ; immunology ; Plasmids ; Salmonella typhimurium ; genetics ; Vaccines, Attenuated ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.
Animals ; Antibodies, Viral/blood ; Cercopithecus aethiops ; Chickens ; *HN Protein/genetics/immunology ; Immunogenicity, Vaccine/*immunology ; Newcastle Disease/immunology ; Newcastle disease virus/enzymology/*genetics/immunology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/genetics/*immunology ; Vaccines, Inactivated/immunology ; Vero Cells ; *Viral Fusion Proteins/genetics/immunology ; Viral Vaccines/genetics/*immunology/*standards

In order to amplify F gene of NDV HeB02 strain, one pair of primers was designed according to the GenBank sequence, and a 1.66 kb F gene fragment was obtained by RT-PCR. Sequence analysis indicated that the homologies of the nucleotide sequence of HeB02 strain to those of F48 E9, La Sota and Clone30 strains were 88.1%, 84.9% and 83.8% respectively. The expression plasmid pSV-F was constructed by inserting the F gene into the pVAX1 vector, and transfected into the cultured COS 7 cell line via liposomes. The specific 5.9 kD protein was detected by SDS-PAGE and the immunogenicity of the expressed F protein was confirmed by Western blot, ELISA and neutralization test. 3 week-old SPF chickens were subcutaneously immunized twice at week 0 and 3 with 50 microg DNA of plasmid pSV-F by electroporration. 5 weeks later, all chickenss were challenged with 100 x EID50 of NDV HeB02 strain, 1 week post challenge all chickenss were sampled by larynx swabbing to isolate virus and the HI level of NDV was measured. The results indicated that the virus isolation was negtive in all vaccinated chickenss and positive in all control chickens. The HI titres reached to 8.3log2 +/- 1.30 and 7.2log2 +/- 1.23 induced by NDV vaccine and positive cells (pSV-F), respectivily, the HI titres induced by Control cells (pVAX1) was not detected. Furthermore, the HI titres reached to 9.8log2 +/- 1.55 and 8.9log2 +/- 1.77 in vaccinated group with NDV vaccine and positive cells (pSV-F), respectivily, were sinificantly higher than that of the control cells (pVAX1) immunized group( HI titers was 3.0 log2 +/- 1.40, P < 0.01) after challenge. These results showed that the plasmid pSV-F could be as a candidate of DNA vaccine to provide protective immune response against NDV infection.
Animals ; COS Cells ; Cercopithecus aethiops ; Chickens ; Cloning, Molecular ; Hemagglutination Inhibition Tests ; veterinary ; Newcastle Disease ; immunology ; prevention & control ; Newcastle disease virus ; classification ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vaccination ; Vaccines, DNA ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology ; Viral Vaccines ; genetics ; immunology

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera