Satish S. Gaikwad

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.

Complete Genome Sequence of Newcastle Disease Virus Mesogenic Vaccine Strain R2B from India

ABSTRACT Mesogenic vaccine strains of Newcastle disease virus (NDV) are widely used in many countries of Asia and Africa to control the Newcastle disease of poultry. In India, the mesogenic strain R2B was introduced in 1945; it protects adult chickens that have been preimmunized with a lentogenic vaccine virus and provides long-lasting immunity. In this article, we report the complete genome sequence of the hitherto unsequenced Indian vaccine virus strain R2B. The viral genome is 15,186 nucleotides in length and contains the polybasic amino acid motif in the fusion protein cleavage site, indicating that this vaccine strain has evolved from a virulent virus. Phylogenetic analysis of this mesogenic vaccine virus classified it with the viruses belonging to genotype III of the class cluster II of NDV.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry.

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

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.

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.

Rescue of a recombinant Newcastle disease virus strain R2B expressing green fluorescent protein

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.

Complete Genome Sequence of a Newcastle Disease Virus Isolated from Wild Peacock (Pavo cristatus) in India

ABSTRACT We report here the complete genome sequence of a Newcastle disease virus (NDV) isolated from a wild peacock. Phylogenetic analysis showed that it belongs to genotype II, class II of NDV strains. This study helps to understand the ecology of NDV strains circulating in a wild avian host of this geographical region during the outbreak of 2012 in northwest India.

Genetic characterization and evolutionary analysis of Newcastle disease virus isolated from domestic duck in South Korea

Domestic ducks are considered a potential reservoir of Newcastle disease virus. In the study, a Newcastle disease virus (NDV) isolated from a domestic duck during surveillance in South Korea was characterized. The complete genome of the NDV isolate was sequenced, and the phylogenetic relationship to reference strains was studied. Phylogenetic analysis revealed that the strain clustered in genotype I of Class II ND viruses, has highly phylogenetic similarity to NDV strains isolated from waterfowl in China, but was distant from the viruses isolated in chickens and vaccine strains used in South Korea. Pathogenicity experiment in chickens revealed it to be a lentogenic virus. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the avirulent motif (112)GKQGRL(117) at the cleavage site and caused no apparent disease in chickens and ducks. With phylogeographic analysis based on fusion gene, we estimate the origin of an ancestral virus of the isolate and its sister strain located in China around 1998. It highlights the need of continuous surveillance to enhance current understanding of the molecular epidemiology and evolution of the pathogenic strains.

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.