Anant Rai

Induction of apoptosis in Vero cells by Newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation

Newcastle disease virus causes (NDV) apoptotic death of infected cells. In the present study, the stimulus that provoked the induction of apoptosis in infected cells was examined. Vero cells infected with NDV developed apoptosis as characterized by DNA fragmentation and decreased DNA content. In presence of ammonium chloride, infected cells did not show reduced DNA content indicating the requirement of virus entry for the induction of apoptosis. UV-inactivated NDV did not induce apoptosis in cells suggesting the need of virus replication. Although cycloheximide blocked NDV-induced apoptosis, actinomycin-D did not, suggesting that de-novo viral protein synthesis was critical for the induction of apoptosis. In addition, activation of caspases was also detected by flowcytometry, indirect fluorescent and colorimetric assays. Based on the results, it was concluded that NDV-induced apoptosis in Vero cells required virus replication, de-novo protein synthesis and caspase activation.

Effective protection by high efficiency bicistronic DNA vaccine against infectious bursal disease virus expressing VP2 protein and chicken IL-2.

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). IBDV belongs to genus Avibirnavirus of family Birnaviridae. It is a non-enveloped virus with icosahedral symmetry that contains two segments of double-stranded RNA. The virus affects the lymphoid tissues of chickens, mainly the B cells of bursa of Fabricius, leading to severe and prolonged immunosuppression. VP2, a major structural protein of IBDV, contains antigenic epitopes responsible for induction of neutralizing/protective antibody. In the present study, VP2 gene of IBDV was cloned in a bicistronic vector along with chicken interleukin-2 (chiIL-2) as an adjuvant. An in vivo challenge study of bicistronic DNA vaccine expressing IBDV-VP2 and chicken IL-2 showed effective protection against a lethal IBD infection in chickens. In addition, mortality, gross picture of bursa and histopathological findings demonstrated the efficacy of the vaccine in reducing virulence of the disease.

HN protein of Newcastle disease virus causes apoptosis in chicken embryo fibroblast cells

Newcastle disease virus (NDV), an avian paramyxovirus, induces apoptosis in chicken embryo fibroblast (CEF) cells. In the present investigation, the ability of haemagglutinin-neuraminidase (HN) protein of NDV to cause apoptosis in CEF cells was examined. The results revealed that cells expressing the HN protein demonstrated decreased DNA content, phosphatidylserine exposure and increased cytoplasmic vacuolation. Up-regulation of caspase-1, -9, -8, -3, loss of mitochondrial transmembrane potential and an increase in oxidative stress were also observed in cells expressing the HN protein. Based on the above results it can be concluded that HN protein of NDV causes apoptosis in CEF cells.

Rabies DNA vaccine: no impact of MHC class I and class II targeting sequences on immune response and protection against lethal challenge.

Abstract Rabies is progressive fatal encephalitis. WHO estimates 55,000 rabies deaths and more than 10 million PEP every year world-wide. A variety of cell-culture derived vaccines are available for prophylaxis against rabies. However, their high cost restricts their usage in developing countries, where such cases are most often encountered. This is driving the quest for newer vaccine formulations; DNA vaccines being most promising amongst them. Here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. These strategies include use of signal sequences namely Tissue Plasminogen Activator (TPA), Ubiquitin (UQ) and Lysosomal-Associated Membrane Protein-1 (LAMP-1). TPA, LAMP-1 and their combination were aimed at enhancing the CD4+ T cell and antibody response. In contrast, the UQ tag was utilized for enhancing CD8+ response. The potency of modified DNA vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. Interestingly, the DNA vaccines that had been designed to generate different type of immune responses yielded in effect similar response. In conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies.

Rabies DNA vaccine encoding lysosome-targeted glycoprotein supplemented with Emulsigen-D confers complete protection in preexposure and postexposure studies in BALB/c mice.

The worldwide incidence of rabies and the inability of currently used vaccination strategies to provide highly potent and cost‐effective therapy indicate the need for an improved rabies vaccine. Thus, DNA vaccine based on lysosome‐targeted gly‐coprotein of the rabies virus was evaluated in BALB/c mice. It imparted partial protection (60%) against challenge with 20 LD50 of the challenge virus standard (CVS) strain of rabies virus. To improve the outcome of vaccination, to ultimately enhance the immune response, we investigated different routes for DNA vaccine delivery, varied doses of DNA, and the influence of adjuvant supplementation. The highest immune response pertaining to IgG antibody titer, with a predominantly IgG1/IgG2a subclass distribution, effective cellular immunity, and a high level of rabies virus neutralizing antibodies (RVNAs) was attained by the optimized DNA vaccine formulation comprising intramuscular administration of 100 μg of DNA vaccine supplemented with Emulsi‐gen®‐D. In preexposure prophylaxis, a 3‐dose regimen of this formulation generated a high RVNA titer (32 IU/ml) and conferred complete protection against challenge with 20 LD50 of CVS. For postexposure efficacy analysis, rabies was experimentally induced with 50 LD50 of CVS. Subsequent therapy with 5 doses of the formulation completely prevented rabies in BALB/c mice, which maintained protective RVNA titers of 4 IU/ml. The World Health Organization recommended rabies protective titer threshold is 0.5 IU/ml. Thus, this optimized DNA vaccine formulation provides an avenue for preventing and controlling rabies.—Kaur, M., Saxena, A., Rai, A., and Bhatnagar, R. Rabies DNA vaccine encoding lyso‐some‐targeted glycoprotein supplemented with Emulsi‐gen‐D confers complete protection in preexposure and postexposure studies in BALB/c mice. FASEB J. 24, 173–183 (2010). www.fasebj.org

Detection of canine adenoviral infections in urine and faeces by the polymerase chain reaction

The present study was aimed at developing the polymerase chain reaction (PCR) assay for detection of canine adenoviruses from faecal or urine samples. Urine or faecal samples were treated with chloroform or activated charcoal to eliminate the PCR inhibitory substances and the total DNA was extracted. The PCR was optimized using common set of primers to amplify 508 bp or 1,030 bp DNA sequence within E3 gene of canine adenovirus-1 (CAV-1) and canine adenovirus-2 (CAV-2), respectively. The PCR assay could detect up to 0.016 TCID(50) viruses from CAV-1 infected MDCK cell culture fluid, 1.6 TCID(50) viruses from faeces and 16 TCID(50) viruses from urine. In addition, the PCR assay was validated using clinical samples. Based on the results, it is concluded that, the present PCR assay can be used for rapid detection of canine adenoviral infections.

Immune responses induced by DNA vaccines encoding Newcastle virus haemagglutinin and/or fusion proteins in maternal antibody-positive commercial broiler chicken.

1. The immune responses induced by recombinant plasmids containing Newcastle disease virus (NDV) F (pVAX.nd.f) or HN (pcDNA.nd.hn) genes separately or in combination in bi-cistronic (pIRES.nd.hn.f) constructs were evaluated in maternal antibody-positive commercial chicks. 2. Immunofluorescence and immunoperoxidase tests demonstrated the expression of both F and HN proteins in Vero cells. Real-time PCR analysis revealed the expression of HN and/or F genes in muscle, peripheral blood mononuclear cells (PBMC), spleen and liver after immunisation. 3. Chicks inoculated intramuscularly thrice (two booster doses) with pVAX.nd.f and pcDNA.nd.hn did not develop detectable haemagglutination inhibiting (HI) antibodies. In contrast, an increase in a NDV-specific cell-mediated immune response was demonstrated. 4. After challenge with virulent NDV, chicks immunised with the recombinant plasmids as well as those in control groups succumbed to Newcastle disease. 5. Based on these results, it is concluded that DNA vaccines containing HN and/or F genes fail to protect commercial chicks, possibly due to interference from maternal antibodies.

Role of immunostimulatory molecules in poultry vaccines.

Immunization by vaccination is the most suitable and safest method for preventing infectious diseases in the poultry worldwide. Vaccines alone cannot effectively protect birds from variety of pathogens under field conditions. The combined use of potent immunostimulants in vaccines is an alternative to increase the efficacy of vaccines that can be achieved by the development of better adjuvant. One such adjuvant is cytokine; cytokines have been used extensively as adjuvant in vaccines and are responsible for the type and extent of an immune response following vaccination. Although the innate immune system in birds is not fully characterized but their immune system is very much similar to that of mammals, and moreover with the recent discovery of a number of avian cytokine genes it is now possible to study their effectiveness in enhancing the immune response during vaccination. This review focuses on the recent studies and developments involving the role of immunomodulating agents especially cytokines of avian origin in poultry vaccines.

Immunogenicity of Infectious Bursal Disease Virus Strains Isolated in India

Abstract Pahar, B. and Rai, A. 1997. Immunogenicity of infectious bursal disease strains isolated in India. J. Appl. Anim. Res., 12: 137–144. Out of the three isolates of infectious bursal disease virus (IBDV), only IBDV S394 gave 100% protection in birds without any immunosuppressive effect and mortality in day old chicks, while IBDV S494 and IBDVS194 gave 88% and 76% protection, respectively. Thus IBDV S394 may serve as prophylactic agent against infectious bursal disease in poultry.

Restriction Map of Cloned 3.8 kb PstI Fragment of Bovine Herpes Virus-1 DNA

Abstract Gupta, P.K. and Rai, A. 1994. Restriction map of cloned 3.8 kb PstI fragment of bovine herpes virus-1 DNA. J. Appl. Anim. Res., 6: 143–149. A 3.8 kb Pstl fragment of bovine herpesvirus-1 DNA was selectively cloned into pUC9 plasmid vector. The recombinant plasmid with the insert was analyzed using restriction endonucleases. On the basis of restriction digested fragment pattern, partial restriction site map was prepared for a few restriction endonucleases. Shot gun cloning of PstI digested DNA fragments yielded two clones having double inserts.