Three newly identified Immediate Early Genes of Bovine herpesvirus 1 lack the characteristic Octamer binding motif- 1.

Only three immediate early genes (IE) BICP0, BICP4 and BICP22 of Bovine herpesvirus 1 (BoHV-1) are known. These genes are expressed coordinately and their promoters are well characterized. We provide evidence for expression of three additional IE genes of BoHV-1 i.e. UL21, UL33 and UL34. These genes are expressed in the presence of cycloheximide (CH) at the same time as known IE genes. Surprisingly, the promoters of newly identified IE genes (UL21, UL33, UL34) lack the OCT-1 binding site, a considered site of transactivation of the BoHV-1 IE genes. The other difference in the promoters of the newly identified IE genes is the presence of TATA box at near optimal site. However, all the IE genes have similar spatial placements of C/EBPα, DPE and INR elements.

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.

Bovine Herpes Virus 1 Major Immediate Early Transcription Unit 1 (IETU-1) Uses Alternative Promoters to Transcribe BICP0 and BICP4 Transcripts.

Immediate early (IE) genes are transcribed immediately after infection in BHV1 from two different immediate early transcription units. It is reported that the immediate early transcription unit I (IE TU1) of Bovine herpesvirus 1 (BHV1) transcribes two proteins BICP0 and BICP4 from a single promoter by alternative splicing but with identical 5'UTR. We found that the transcripts of BICP0 and BICP4 have different 5'UTRs. The bioinformatics analysis shows two similar spatially arranged TATA less promoter for the two transcripts. The bioinformatics analysis also showed a similar promoter for the IE TU2 which transcribes BICP22. The data strongly suggest that BICP0 and BICP4 are transcribed from two different promoters. The transcript produced by each promoter is spliced specifically as opposed to what has been reported earlier. The BICP0 and BICP4 also show different levels of expression. The expression level of BICP4 continuously declines after attaining a peak level at 1 h, while BICP0 shows biphasic expression supporting the earlier observation that it is expressed from two different promoters.

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.

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.

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.

Development and in vitro characterization of a bivalent DNA containing HN and F genes of velogenic Newcastle disease virus.

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.

Characterization and expression of e2 glycoprotein of classical Swine Fever virus in a eukaryotic expression system.

Classical swine fever (CSF) is an economically important Office International des Epizooties (OIE) list A disease of swine characterized by high fever and multiple haemmorhages. The E2 glycoprotein of CSFV is immunogenic and induces neutralizing antibodies against CSFV. In the present study, complete coding region of the E2 gene from Indian virulent field isolate (Mathura) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and subsequently cloned into a mammalian expression vector; pcDNA3.1(+) at BamHI and XbaI site. The recombinant plasmid; pcDNA.E2.CSFV. was confirmed by restriction enzyme digestion. The pcDNA.E2.CSFV. transfected Vero cell expressed E2 protein which was confirmed by western blotting, immunoperoxidase and indirect immunofluorescent tests. Additionally, flow cytometry analysis also confirmed that 15% of transfected Vero cells expressed the E2 glycoprotein compared to mock or vector alone transfected cells. Further study is under way to evaluate recombinant pcDNA.E2.CSFV. Mathura clone as DNA vaccine against CSFV.

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.

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.

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.

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.