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The low potency of genetic immunization has to date impeded development of commercial vaccines against major infectious diseases. The aim of this study was to develop and evaluate a fusion gene-based DNA prime-protein boost vaccination strategy to improve the efficacy of both DNA and subunit vaccines against Newcastle disease virus (NDV). The fusion (F) protein, a viral surface glycoprotein, is responsible for the cell membrane fusion and spread, also is one of the major targets for immune response. In this study, groups of chickens were vaccinated twice intramuscularly at 14-day interval either with plasmid DNA encoding F protein gene of NDV or with recombinant F protein alone or with plasmid DNA and boosted with the recombinant F protein and compared with birds that were vaccinated with live NDV vaccine. The immune response was evaluated by indirect ELISA, lymphocyte transformation test, virus neutralization test, cytokine analysis, immunophenotyping of peripheral blood mononuclear cells, and protective efficacy study against virulent NDV challenge virus infection. Chickens in prime-boost group developed a higher level of humoral and cellular immune responses as compared with those immunized with plasmid or protein alone. The DNA prime-protein boost using F protein of NDV yielded 91.6% protection against virulent NDV challenge infection better than immunization with DNA vaccine (66.6%) or rF protein (83.3%) alone. These findings suggest that the "DNA prime-protein boost" approach using full-length F gene could enhance the immune response against NDV in the chickens.

Newcastle disease (ND) is a highly contagious and fatal disease of chickens. Newcastle disease virus (NDV) strain R2B is an Indian mesogenic strain used for secondary vaccination in chickens. Mesogenic strains have increased virulence and immunogenicity but may cause disease in vaccinated birds, thus rendering them ineffective for use. In this study, we generated a recombinant NDV by changing the fusion protein cleavage site of mesogenic rNDV-R2B from a polybasic amino acid motif RRQKRF to a dibasic amino acid motif GRQGRL leading to generation of an attenuated virus, rNDV-R2B-FPCS. The modified recombinant virus had similar growth characteristics as rNDV-R2B, but was less virulent in susceptible chickens. Immunization of the recombinant attenuated virus to one week of age SPF chickens generated a protective immune response with a substantial reduction in virus shed after challenge with virulent NDV. The results of the study indicate that the modified rNDV-R2B-FPCS virus can be used for primary immunization in birds without any adverse reactions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Newcastle disease virus (NDV), strain R2B is a mesogenic vaccine strain used for booster vaccination in chickens against Newcastle disease in India and many south East Asian countries. A full-length cDNA clone of the virus was generated by ligating eight overlapping fragments generated by reverse transcription polymerase chain reaction having unique restriction enzyme sites within them. This full-length cDNA clone was flanked by hammerhead ribozyme and hepatitis delta virus ribozyme sequences. Defined genetic markers were introduced into the NDV genome to differentiate the rescued virus from the parent virus. A gene cassette containing the reporter gene, green fluorescent protein flanked by NDV gene-start and gene-end signals was generated by PCR and introduced into the full-length clone of NDV between the P and M genes. Recombinant NDV encoding the GFP gene was rescued having precise termini when transfected into permissive Vero cells along with support plasmids harbouring the nucleoprotein, phosphoprotein and polymerase genes. The recombinant virus had similar growth kinetics as that of the parent virus with a moderate reduction in the virulence. The generation of reverse genetics system for NDV strain R2B will help in the development of multivalent vaccines against viral diseases of livestock and poultry.

The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 3'-N-P-M-F-HN-L-5', which was limited by 55-nts leader region at the 3' end and a 114-nts trailer sequence at 5' end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site (112)R-R-Q-K-R-F(117), a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.

Newcastle disease is an avian pathogen causing severe economic losses to the Indian poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. India being an endemic country, advocates vaccination against the virus using lentogenic and mesogenic strains. Two virus strains which are commonly used for vaccination are strain F (a lentogenic virus) and strain R2B (a mesogenic virus). Strain F is given to 0-7 days old chicks and R2B is given to older birds which are around 6-8 weeks old. To understand the genetic makeup of these two strains, a complete genome study and phylogenetic analysis of the F, HN genes of these vaccine strains were carried out. Both the viral strains had a genome length of 15,186 nucleotides and consisted of six genes with conserved complimentary 3' leader and 5' trailer regions. The fusion protein cleavage site of strain F is GGRQGRL and strain R2B is RRQKRF. Although both the viral strains had different virulence attributes, the length of the HN protein was similar with 577 amino acids. Phylogenetic analysis of F, HN and complete genome sequences grouped these two strains in genotype II category which are considered as early genotypes and corroborated with their years of isolation.

A velogenic Newcastle disease virus isolate was passaged 50 times in Vero cell culture and the virus was assessed for the molecular changes associated with the passaging. At every 10th passage, the virus was characterized conventionally by mean death time (MDT) analysis, intracerebral pathogenicity index (ICPI) and virus titration. At increasing passage levels, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription-polymerase chain reaction. Sequence analysis revealed a total of 135 nucleotide substitutions which resulted in the change of 42 amino acids between the velogenic virus and the 50th passage virus. The predicted amino acid motif present at the cleavage site of the virulent virus was (109)SRRRRQRRFVG(119) and the corresponding region of the adapted adapted virus was (109)SGGRRQKRFIG(119). Pathogenicity studies conducted in 20-week-old seronegative birds revealed gross lesions such as petechial haemorrhages in the trachea, proventricular junction and intestines, and histopathological changes such as depletion and necrosis of the lymphocytes in thymus, spleen, bursa and caecal tonsils in the birds injected with the velogenic virus and absence of the lesions in birds injected with the adapted virus. The 50th-passage cell culture virus was back-passaged five times in susceptible chickens and subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor difference found between them.

A recombinant haemagglutinin neuraminidase (HN) antigen-based single serum dilution enzyme linked immuno-sorbent assay (ELISA) was developed to measure the specific antibody in sera of chickens against Newcastle disease virus. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed the demonstration of this correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the standard haemagglutination inhibition (HI) test.

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4+ and CD8+ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds. [ABSTRACT FROM AUTHOR]