Barkha Ratta

Newcastle disease virus-induced cytopathic effect in infected cells is caused by apoptosis.

The velogenic Newcastle disease virus (NDV) causes highly infectious and economically significant Newcastle disease (ND) in birds of various species. In cell culture NDV induces cytopathic effect (CPE) characterized by rounding, vacuolation, syncytia formation and cell death. Aside from cell to cell fusion caused by the F and HN glycoprotein of the virus molecular events leading to cell death are not known. In the current study, NDV-infected Vero cells, at 48 h p.i., showed nuclear condensation, cytoplasm blebbing, DNA fragmentation, and phosphatidylserine translocation to the cell surface. In addition, virus-infected cells demonstrated decreased DNA content and an increased Bax to Bcl-2 ratio, p53 level and caspase 3, 8, 9 expression compared to mock-infected cells. Based on these results, it was concluded that CPE in NDV-infected cells was caused by to the induction of apoptosis with the involvement of p53 and the Bax, dependent apoptotic pathways.

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.

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.

Time course of Newcastle disease virus-induced apoptotic pathways.

Newcastle disease virus (NDV) causes economically significant Newcastle disease (ND) in almost all birds worldwide. Previous studies have shown that NDV induces caspase dependent apoptotic pathways in infected cells. In the present study, time course induction of apoptotic pathways in Vero cells is described. In NDV-infected cells, caspase-8 activity, percentage of cells showing TRAIL expression was higher at 24h p.i. (post-infection) compared to 48 h p.i. In contrast, caspase-9 activity, efflux of cytochrome c, loss of mitochondrial membrane potential was higher at 48 h compared to 24h p.i. The caspase-3 activity was high both times. Based on these results, it was concluded that at 24h p.i., NDV induces apoptosis through extrinsic apoptotic pathway while at 48 h p.i. predominantly through intrinsic apoptotic pathway.

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.

Isolation of Newcastle disease virus from a non-avian host (sheep) and its implications

Newcastle disease virus (NDV) is an avian virus that has not been isolated from naturally infected non-avian and non-human hosts except for one report in which it was isolated from cattle in 1952. We report here for the first time the isolation and identification of NDV from sheep and suggest that this virus be included in the screening of viruses from non-avian hosts.

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.

Detection of Peste Des Petits Ruminants Virus (PPRV) Genome from Nasal Swabs of Dogs

Peste des petits ruminants virus (PPRV) one of the most important viruses of small ruminants has a restricted host range. We report here the presence of PPRV virus in the nasal swabs of 3 out of 12 dogs in a routine microarray screening. The presence of PPRV sequence was further confirmed by PCR and sequencing. The sequence analysis revealed that the PPRV virus has close similarities with the viruses present in Indian subcontinent but was not identical to the vaccine virus used in India. Results suggest possible crossing of species barrier but requires further serological evidences.

Microarray Chip Based Identification of a Mixed Infection of Bovine Herpesvirus 1 and Bovine Viral Diarrhea 2 From Indian Cattle

Bovine herpesvirus 1 (BHV1) and bovine viral diarrhea virus 2 (BVD2) are endemic in India although no mixed infection with these viruses has been reported from India. We report first mixed infection of these viruses in cattle during routine screening with a microarray chip. 62 of the 69 probes of BHV1 and 42 of the 57 BVD2 probes in the chip gave positive signals for the virus. The virus infections were subsequently confirmed by RT-PCR. We also discuss the implications of these findings.

Viral diagnosis in Indian livestock using customized microarray chips

Viral diagnosis in Indian livestock using customized microarray chips is gaining momentum in recent years. Hence, it is possible to design customized microarray chip for viruses infecting livestock in India. Customized microarray chips identified Bovine herpes virus-1 (BHV-1), Canine Adeno Virus-1 (CAV-1), and Canine Parvo Virus-2 (CPV-2) in clinical samples. Microarray identified specific probes were further confirmed using RT-PCR in all clinical and known samples. Therefore, the application of microarray chips during viral disease outbreaks in Indian livestock is possible where conventional methods are unsuitable. It should be noted that customized application requires a detailed cost efficiency calculation.