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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.

The aim was to clone and sequence hfq gene of Salmonella Typhimurium strain PM-45 and compare its sequence with hfq gene of other serovar of Salmonella. Salmonella Typhimurium strain PM-45 was procured from the G. B. Pant University of Agriculture and Technology, Pantnagar, India. The genomic DNA was isolated from Salmonella Typhimurium. Hfq gene was polymerase chain reaction (PCR) amplified from the DNA using specific primers, which was subsequently cloned into pET32a vector and transformed into Escherichia coli BL21 pLys cells. The recombinant plasmid was isolated and subjected to restriction enzyme digestion as well as PCR. The clone was then sequenced. The sequence was analyzed and submitted in GenBank. PCR produced an amplicon of 309 bp. Restriction digestion of the recombinant plasmid released the desired insert. The hfq sequence shows 100% homology with similar sequences from other Salmonella Typhimurium isolates. Both nucleotide and amino acid sequences are highly conserved. The submitted sequence is having Genbank accession no KM998764. Hfq, the hexameric RNA binding protein is one of the most important post-transcriptional regulator of bacteria. The sequence of hfq gene of Salmonella Typhimurium is highly conserved within and between Salmonella enterica serovars. This gene sequence is probably under heavy selection pressure to maintain the conformational integrity of its product in spite of its being not a survival gene.

Salmonella Typhimurium (STM), a zoonotic pathogen, can adjust its metabolic pathway according to the variations in the partial pressure of atmospheric oxygen and nitrate via fumarate nitrate reductase regulator (Fnr) and NarL, the response regulator for nitrate reductase. Both Fnr and NarL have been individually reported to be the contributors of virulent phenotypes of STM. Hypoxia along with nitrate-rich environment are prevalent in macrophages and the Salmonella -induced inflammatory lumen of the host’s large intestine activates both fnr and narL genes. In this study, the double ( fnr and narL ) knockout STM showed a synergistic reduction in the swimming (62%), swarming (84%) and biofilm density (86%) phenotypes anaerobically in association with its significant aerobic attenuation. The intracellular replication of the double mutant was reduced by 2.3 logs in chicken monocyte-derived macrophages. Furthermore, the competitive index of the double mutant in liver and spleen was found to be 0.3 and 0.44 respectively at 120 h post-infection (PI) in mice. Surprisingly, no double mutant could be recovered from the infected mouse liver 3 days PI. Histopathological findings showed moderate infiltration of mononuclear cells in the large intestine of mice infected with double mutant, but severe infiltration was seen with the wild-type strain.

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.

•Resiquimod (R-848) showed potential adjuvant capacity with Newcastle disease (ND) vaccine in SPF chicken.•R-848 enhanced antigen specific humoral as well as cellular immune responses.•R-848 up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-4, IL-1β, MHC-II and iNOS transcripts in the chicken spleen.•R-848 potentiated the protection capacity of inactivated ND vaccine against virulent ND virus challenge. Resiquimod (R-848), an imidazoquinoline compound, is a potent synthetic Toll-like receptor (TLR) 7 agonist. Although the solitary adjuvant potential of R-848 is well established in mammals, such reports are not available in avian species hitherto. Hence, the adjuvant potential of R-848 was tested in SPF chicken in this study. Two week old chicks were divided into four groups (10birds/group) viz., control (A), inactivated Newcastle disease virus (NDV) vaccine prepared from velogenic strain (B), commercial oil adjuvanted inactivated NDV vaccine prepared from lentogenic strain (C) and inactivated NDV vaccine prepared from velogenic strain with R-848 (D). Booster was given two weeks post primary vaccination. Humoral immune response was assessed by haemagglutination inhibition (HI) test and ELISA while the cellular immune response was quantified by lymphocyte transformation test (LTT) and flow cytometry post-vaccination. Entire experiment was repeated twice to check the reproducibility. Highest HI titre was observed in group D at post booster weeks 1 and 2 that corresponds to mean log2 HI titre of 6.4±0.16 and 6.8±0.13, respectively. The response was significantly higher than that of group B or C (P<0.01). LTT stimulation index (P≤0.01) as well as CD4+ and CD8+ cells in flow cytometry (P<0.05) were significantly high and maximum in group D. Group D conferred complete protection against virulent NDV challenge, while it was only 80% in group B and C. To understand the effects of R-848, the kinetics of immune response genes in spleen were analyzed using quantitative real-time PCR after R-848 administration (50μg/bird, i.m. route). Resiquimod significantly up-regulated the expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-4, iNOS and MHC-II genes (P<0.01). In conclusion, the study demonstrated the adjuvant potential of R-848 when co-administered with inactivated NDV vaccine in SPF chicken which is likely due to the up-regulation of immune response genes.

Canine monocytic ehrlichiosis (CME) is an important hemoprotozoan disease of dogs associated with anemia, leucopenia, and thrombocytopenia. The present study was conducted with the aim of assessing the status of oxidative/nitrosative stress and apoptosis in naturally occurring cases of chronic canine monocytic ehrlichiosis, to investigate potential of darbepoietin alpha in ameliorating oxidative/nitrosative stress and apoptosis associated with CME and to assess the impact of darbepoietin alpha administration on clinical outcome in chronic canine monocytic ehrlichiosis. Thirty naturally occurring cases of chronic CME were allotted randomly in two treatment groups, groups I and II. Animals in group I were treated with doxycycline @10 mg/kg, PO, sid, for 28 days, and other supportive therapy. Whereas, animals in group II received darbapoietin alpha in addition to doxycycline and supportive therapy. Six apparently healthy dogs acted as healthy control. All these animals were monitored closely till 28 days of post treatment. Normocytic normochromic anemia, thrombocytopenia, decreased leukocyte count, increased levels of blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), and alkaline phosphatase (SAP) were observed in dogs suffering from chronic CME. There was significant increase in the levels of lipid peroxidation (LPO), nitric oxide (NO), and apoptosis percentage in dogs suffering from chronic CME. Levels of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase were significantly reduced in dogs suffering from chronic CME. Rapid improvement in the hematological values, oxidative, and nitrosative stress markers and percent apoptosis were observed in group II as compared to group I. Rapid clinical recovery and reduced hospital stay were observed in animals belonging to group II as compared to group I. No adverse effects of darbepoietin alpha administration were observed in the present study.

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.

The use of viruses for treatment of cancer overcomes the bottlenecks of chemotherapy and radiotherapy. Several viruses and their proteins have been evaluated for oncolytic effect. The VP3 protein (apoptin) of chicken anemia virus is one such protein with an inherent ability to lyse cancer and transformed cells while leaving normal cells unharmed. In the present study, the apoptosis inducing potential of VP3 protein of CAV was evaluated in human cervical cancer cell line (HeLa). It was found that in VP3-induced apoptosis, caspase-dependent intrinsic pathway plays an important role with the cleavage of poly (ADP-ribose) polymerase (PARP) and there was no evidence of involvement of death receptor-mediated extrinsic pathway. The results of this study provide intuitive information and strengthen the candidacy of apoptin as a viral oncotherapeutic agent.

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.

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.