Evolutionary and bioinformatic analysis of the spike glycoprotein gene of H120 vaccine strain protectotype of infectious bronchitis virus from India.

The infectious bronchitis virus is a causative agent of avian infectious bronchitis (AIB), and is is an important disease that produces severe economic losses to the poultry industry worldwide. Recent AIB outbreaks in India have been associated with poor growth in broilers, drop in egg production, and thin egg shells in layers. The complete spike gene of Indian AIB vaccine strain was amplified and sequenced using a conventional reverse transcription polymerase chain reaction and is submitted to the GenBank (accession no KF188436). Phylogenetic analysis revealed that the vaccine strain currently used belongs to H120 genotype, an attenuated strain of Massachusetts (Mass) serotype. Nucleotide and amino acid sequence comparisons have shown that the reported spike gene from Indian isolates have 71.8%-99% and 71.4%-96.9% genetic similarity with the sequenced H120 strain. The study identifies live attenuated IBV vaccine strain, which is routinely used for vaccination, for the first time. Based on nucleotide and amino acid relatedness studies of the vaccine strain with reported IBV sequences from India, it is shown that the current vaccine strain is efficient in controlling the IBV infection. Continuous monitoring of IBV outbreaks by sequencing for genotyping and in vivo cross protection studies for serotyping is not only important for epidemiological investigation but also for evaluation of efficacy of the current vaccine.

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Protective efficacy of a DNA vaccine construct encoding the VP2 gene of infectious bursal disease and a truncated HSP70 of Mycobacterium tuberculosis in chickens.

Infectious bursal disease (IBD) is an acute, infectious, immunosuppressive disease affecting young chicken worldwide. The etiological agent IBD virus (IBDV) is a double stranded RNA virus with outer capsid protein VP2 of IBDV is the major antigenic determinant capable of inducing neutralizing antibody. DNA vaccines encoding VP2 has been extensively studied achieving only partial protection. However, the efficacy of DNA vaccines against IBDV can be augmented by choosing a potential molecular adjuvant. The goal of the present study is to evaluate the immune response and protective efficacy of a DNA vaccine encoding the C-terminal domain of the heat shock protein 70 (cHSP70) of Mycobacterium tuberculosis gene genetically fused with the full length VP2 gene of IBDV (pCIVP2-cHSP70) in comparison to a 'DNA prime-protein boost' approach and a DNA vaccine encoding the VP2 gene (pCIVP2) alone. The results indicate that both pCIVP2-cHSP70 and 'DNA prime-protein boost' elicited humoral as well as cellular immune responses. Chickens in the pCIVP2-cHSP70 and 'DNA prime-protein boost' groups developed significantly higher levels of ELISA titer to IBDV antigen compared to the group immunized with pCIVP2 alone (p<0.01). However, significantly higher levels of lymphocyte proliferative response, IL-12 and IFN-γ production were found in the pCIVP2-cHSP70 group compared to 'DNA prime-protein boost' group. Additionally, chickens immunized with pCIVP2-cHSP70 and 'DNA prime-protein boost' vaccines were completely protected against the vvIBDV whereas pCIVP2 DNA vaccine alone was able to protect only 70%. These findings suggest that the truncated C-terminal HSP70 mediated DNA vaccine genetically fused with the VP2 gene construct stimulated both humoral and cell mediated immune responses and conferred complete protection against IBDV. This novel strategy is perhaps a seminal concept in utilizing HSP70 as an adjuvant molecule to elicit an immune response against IBD affecting chickens.

Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry.

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Recombinant flagellin and its cross-talk with lipopolysaccharide--effect on pooled chicken peripheral blood mononuclear cells.

Toll-like receptors (TLRs) are one of the types of pattern recognition receptors (PRRs) that recognize conserved pathogen molecules. TLRs link innate and adaptive arms of immune system and are implicated in the development of defense against invading pathogens. Lipopolysaccharide (LPS) and flagellin are recognized by TLR4 and TLR5, respectively. In this study, the effect of flagellin and lipopolysaccharide alone and in combination on chicken peripheral blood mononuclear cells (PBMCs) was investigated. The FliC gene of Salmonella typhimurium was expressed in a prokaryotic expression system and the recombinant flagellin was used to stimulate the chicken PBMCs. A combination of recombinant flagellin and LPS synergistically upregulated nitric oxide production, IL-12 and IL-6 expression but antagonistically down regulated IL-4 expression in comparison to recombinant flagellin alone. The results indicate that these agonists synergistically interact and enhance macrophage function and promote Th1 immune response in chicken PBMCs.

Recombinant UL30 antigen-based single serum dilution ELISA for detection of duck viral enteritis.

A recombinant UL30 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed to measure specific antibody in the sera of ducks against duck enteritis virus (DEV). The partial UL30 gene of DEV was cloned, expressed, purified and tested for its diagnostic use by designing a single serum dilution enzyme linked immuno-sorbent assay (ELISA). A total of 226 duck sera samples were tested using the assay. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to determine a standard curve from which an equation was derived which demonstrated 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 specific, sensitive and accurate as compared to the virus neutralization test with a specificity, sensitivity and accuracy being 96%, 95% and 95% respectively.

Diagnosis of leptospirosis by recombinant antigen based single serum dilution ELISA.

BACKGROUND & OBJECTIVES: Leptospirosis, a zoonosis with a worldwide distribution is an acute febrile illness caused by spirochaetes of the pathogenic Leptospira interrogans. Microscopic agglutination test (MAT), the reference method for diagnosis was successively done to evaluate the modified ELISA which was developed with the recombinant LipL32 antigen for the detection of anti-leptospiral antibodies in human serum samples. METHODS: The recombinant LipL32 antigen was developed from the serovar Pomona strain Pomona of the pathogenic L. interrogans species. The predicted titre at a single working dilution was plotted against the observed antiserum titre. Subsequently, predicted antibody activity titres were determined directly from the standard curve by solving the regression line equation. The relative sensitivity, specificity and accuracy of the single dilution ELISA for the detection of anti-leptospiral antibodies were determined in comparison to the MAT. RESULTS: A linear relationship was found between the predicted antibody titres at a single working dilution of 1:250 and the corresponding observed serum titres by the standard serial-dilution method. Regression analysis was used to determine a standard curve from which an equation was derived that allowed demonstration of the mentioned correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. A high level of sensitivity of 96 per cent and specificity of 91 per cent between ELISA and MAT titres was found. The kappa value was almost 1.0 indicating perfect agreement. INTERPRETATION & CONCLUSIONS: The r LipL32 ELISA was proved to be sensitive, specific and accurate as compared to the standard MAT and the test could be efficiently utilized as a screening test for a large number of human serum samples for the detection of leptospiral antibodies.

Recombinant antigen-based dipstick ELISA for the diagnosis of leptospirosis in dogs.

A recombinant LipL 32 antigen-based dipstick ELISA was developed as a screening test for the detection of leptospiral antibodies in serum samples from dogs. The antibodies were detected by a change in the colour of the substrate solution when the recombinant antigen-coated dipsticks were dipped into it. The relative sensitivity, specificity and accuracy of the test, compared with the standard microscopic agglutination test, were 95.9 per cent, 93.8 per cent and 94.8 per cent, respectively.

Adaptation of a velogenic Newcastle disease virus to vero cells: assessing the molecular changes before and after adaptation.

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.

Recombinant haemagglutinin neuraminidase antigen-based single serum dilution ELISA for rapid serological profiling of Newcastle disease virus.

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.

Restriction enzyme analysis of tissue culture-adapted velogenic Newcastle disease virus.

A velogenic Newcastle disease virus isolate typed to belong to group C1 by monoclonal antibody typing was adapted 50 times in chicken embryo fibroblast cell culture and 60 times in Vero cells. At every 10th passage the virus was characterized on the basis of mean death time, intracerebral pathogenicity indices and viral titration studies. A gradual reduction in the virulence of the virus was noted as the passage number increased. RT-PCR of a 254 bp region of the fusion gene encompassing the fusion protein cleavage site was carried out for the virulent as well as cell culture-adapted viruses at every 10th passage level. The amplicons were subsequently digested with three restriction enzymes, viz. AluI, HaeIII and PstI. It was found out that there was difference in banding patterns between the virulent and adapted viruses, indicating nucleotide substitutions in the virulent virus when it was sequentially passaged onto cell culture systems.

Molecular changes of the fusion protein gene of chicken embryo fibroblast-adapted velogenic Newcastle disease virus: effect on its pathogenicity.

Molecular changes of cell culture-adapted Newcastle disease virus (NDV) were studied by adapting a velogenic NDV isolated from commercial layer chicken-to-chicken embryo fibroblast (CEF) cells. The isolate was passaged 50 times in CEF cells. At every 10th passage the virus was characterized conventionally by mean death time analysis, intracerebral pathogenicity index, and virus titration. As the passage level increased, 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 134 nucleotide substitutions, which resulted in the change of 41 amino acids between the parent and the 50th passage virus. Pathogenicity studies conducted in 20-wk-old seronegative chickens revealed gross and histopathologic changes in the chickens injected with the parent virus and absence of the lesions in chickens injected with the adapted virus. The 50th passage cell culture virus was back-passaged five times in susceptible chickens and was subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor differences between them.

Recombinant LipL32 antigen-based single serum dilution ELISA for detection of canine leptospirosis.

A recombinant antigen-based single serum dilution enzyme-linked immunosorbent assay (ELISA) was developed to measure the specific antibody activity in sera of dogs with leptospirosis. The recombinant antigen developed and used in the assay was specific for the pathogenic serovars of Leptospira. A linear relationship was found to exist 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 can be derived that allows demonstration of the mentioned 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 was proved to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT).