First report of occurrence of Babesia gibsoni in captive Indian wolves.

Blood samples from fifteen captive Indian wolves (Canis lupus pallipes) maintained at Arignar Anna Zoological Park, Vandalur, Chennai were screened for the presence of Babesia spp., Ehrlichia canis and Trypnosoma evansi DNA by PCR. Out of 15 wolf samples, 3 samples were found positive for Babesia spp. The amplified 18S rRNA gene fragments from 3 wolves were sequenced and confirmed as Babesia gibsoni. A maximum likelihood tree was constructed using the three sequences along with other Babesia spp. sequences derived from GenBank adopting HKY nucleotide substitution model based on the Bayesian Information Criterion. The phylogenetic analysis confirmed that the three sequences were of Babesia gibsoni and highly divergent from Babesia canis, B. vogeli and B. vulpes. This might be a possible spill over event of B. gibsoni from community dogs through blood feeding dog ticks. This is the first report and molecular confirmation of B. gibsoni infection in captive Indian wolves.

A cross sectional study on molecular prevalence of Orientia tsutsugamushi in household rat population of South India.

This study aimed to assess the molecular prevalence of mite-borne zoonotic pathogen O. tsutsugamushi in household rats of South India through nested polymerase chain reaction amplification of O. tsutsugamushi 47-kDa htrA gene and to determine the most suitable sample type for screening of O. tsutsugamushi in rats. Out of 85 rats trapped in Tamil Nadu, Karnataka, and Puducherry regions, 47 rats were found positive for the O. tsutsugamushi genome with prevalence of 55.29 %. Among different sample types screened, faecal samples exhibited the highest positivity rate, followed by liver, spleen, kidney, and blood samples. Agreement between faecal and spleen samples of rats for the presence of O. tsutsugamushi was the highest. Principal component analysis revealed a positive correlation between the spleen, liver, and faeces and a negative correlation between blood and faeces for the presence of O. tsutsugamushi genome. These findings underscore the varied distribution of O. tsutsugamushi among different samples and indicate that the faecal and liver samples of rats are an ideal choice of samples for epidemiological studies. This is the first study to report a high level of presence of O. tsutsugamushi in faecal samples of rats.

Genetic evaluation of semen production potential in Indigenous bulls using random regression models in a Bayesian framework.

Genetic potential of indigenous bulls with respect to semen production traits over the age of the bulls at semen collection was analyzed using random regression models (RRMs). Data pertaining to 59,641 ejaculates from 189 bulls of 18 indigenous breeds collected from BAIF (Bharatiya Agro Industries Foundation) was utilized for this study. Six semen production traits, viz., ejaculate volume (EV, ml), sperm concentration (SC, 109/ml), initial sperm motility (ISM, %), post-thaw motility (PTM, %), the total number of spermatozoa per ejaculate (TNS, 109/ejaculate), and the theoretical number of semen doses (TNSD) were studied. Univariate and RRM were used to obtain variance components and genetic parameter estimates. Two hundred thousand Gibbs samples were generated for each trait with a burn-in of 20,000 and thinning interval of 50 in a Bayesian framework. Legendre polynomials with orders of fit up to 5 for additive and permanent environmental effects were used. RRM modeled the heritability and repeatability for all ages between 3 and 10 years (productive lifespan). Heritability estimates ranged from 0.18 to 0.36, 0.18 to 0.45, 0.02 to 0.06, 0 to 0.001, 0.09 to 0.32, and 0.14 to 0.42 while the repeatability estimates ranged from 0.41 to 0.72, 0.36 to 0.79, 0.04 to 0.10, 0 to 0.001, 0.37 to 0.56, and 0.32 to 0.57 for EV, SC, ISM, PTM, TNS, and TNSD, respectively. Variability of estimates over the age of the bulls obtained through RRM could be useful to further refine the breeding program for age at selection, deciding the production period and age at culling.

Microbial diversity and community composition of caecal microbiota in commercial and indigenous Indian chickens determined using 16s rDNA amplicon sequencing.

BACKGROUND: The caecal microbiota plays a key role in chicken health and performance, influencing digestion and absorption of nutrients, and contributing to defence against colonisation by invading pathogens. Measures of productivity and resistance to pathogen colonisation are directly influenced by chicken genotype, but host driven variation in microbiome structure is also likely to exert a considerable indirect influence. METHODS: Here, we define the caecal microbiome of indigenous Indian Aseel and Kadaknath chicken breeds and compare them with the global commercial broiler Cobb400 and Ross 308 lines using 16S rDNA V3-V4 hypervariable amplicon sequencing. RESULTS: Each caecal microbiome was dominated by the genera Bacteroides, unclassified bacteria, unclassified Clostridiales, Clostridium, Alistipes, Faecalibacterium, Eubacterium and Blautia. Geographic location (a measure recognised to include variation in environmental and climatic factors, but also likely to feature varied management practices) and chicken line/breed were both found to exert significant impacts (p < 0.05) on caecal microbiome composition. Linear discriminant analysis effect size (LEfSe) revealed 42 breed-specific biomarkers in the chicken lines reared under controlled conditions at two different locations. CONCLUSION: Chicken breed-specific variation in bacterial occurrence, correlation between genera and clustering of operational taxonomic units indicate scope for quantitative genetic analysis and the possibility of selective breeding of chickens for defined enteric microbiota.

Evaluating the efficacy of LaSota vaccination induced protection in chickens upon challenge with a genotype IV strain of Newcastle disease virus.

Newcastle disease (ND) is a major risk to the poultry industry which results in severe economic loss throughout the world even with vaccination. The vaccine viruses that are used in many countries include the LaSota and other live viruses that were isolated in the early and late 1950s. Reports from several laboratories including ours indicate a greater variance of the circulating strains and recent classification indicates the existence of XVIII different genotypes of NDV strains. The efficiency of the LaSota vaccination in inducing protective immunity to different heterologous strains has been a question and its efficacy upon exposure to a virulent genotype IV strain has not been reported after 1989 world-wide except for India. Serum antibody negative (SAN) chicks of either sex obtained by hatching specific-pathogen-free (SPF) eggs were vaccinated with increasing doses of the vaccine virus from 101 to 107 EID50 per bird delivered through occulo-nasal route and challenged 20 days later with NDV-2K3 (genotype IV) strain of virus isolated in the year 2000 from pigeon in India. The birds were monitored for serum antibody titers and following challenge for morbidity, mortality, viral load in the cloacal swab and different tissues. We could clearly show that a minimum vaccine titre of 104 EID50 could establish protective antibody levels and also prevent viral replication post challenge upon exposure to the virulent genotype IV strain. We conclude based on our results and previous observation that there do exist differences in the levels of the antibody that could limit viral replication and shedding upon exposure to different heterologous genotype of NDV. Developing a strain matched vaccine might less potential to result in better protection by limiting the viral shedding.

Co-culture: A quick approach for isolation of street rabies virus in murine neuroblastoma cells.

BACKGROUND: Laboratory detection of rabies in most cases is based on detection of the antigen by fluorescent antibody test, however, in weak positive cases confirmative laboratory diagnosis depends on widely accepted mouse inoculation test. Cell lines like neuroblastoma have been used to isolate the virus with greater success not only to target for diagnosis, but also for molecular studies that determine the epidemiology of the circulating street rabies strains and in studies that look at the efficiency of the developed monoclonal antibodies to neutralize the different rabies strains. Due to the recent issues in obtaining ethical permission for mouse experimentation, and also the passages required in the cell lines to isolate the virus, we report herewith a co-culture protocol using the murine neuroblastoma (MNA) cells, which enable quicker isolation of street rabies virus with minimum passages. OBJECTIVE: This study is not to have an alternative diagnostic assay, but an approach to produce sufficient amount of rabies virus in minimum passages by a co-culture approach in MNA cells. MATERIALS AND METHODS: The MNA cells are co-cultured by topping the normal cells with infected cells every 48 h and the infectivity was followed up by performing direct fluorescent-antibody test. RESULTS: The co-culture approach results in 100% infectivity and hence the use of live mouse for experimentation could be avoided. CONCLUSION: Co-culture method provides an alternative for the situations with limited sample volume and for the quicker isolation of virus which warrants the wild type strains without much modification.

Comparative analysis of innate immune response following in vitro stimulation of sheep and goat peripheral blood mononuclear cells with bluetongue virus - serotype 23.

Bluetongue is an infectious disease caused by bluetongue virus (BTV), which affects sheep, goat, cattle and certain wild ruminants. However severe clinical signs are usually seen with significant mortality in sheep than cattle and goat. To date, comparative studies on innate immune responses of sheep and goat infected with BTV is lacking. In this study, we compared the innate immune response of sheep and goat by infecting the peripheral blood mononuclear cells (PBMCs) with BTV serotype 23. In our study, we observed that sheep PBMCs supports higher virus replication than goat PBMCs. To delineate the role of innate immune response in differential viral replication observed in this study, we examined TLR3 (Receptor for dsRNA virus) mRNA expression and cytokine profiles (IL-1ß, Il-6, IL-8, Il-10, IL-12p40, TNF-α, IFN-γ and IFN-α) following Poly I:C (TLR3 ligand) stimulation and BTV 23 infection. In our present study, sheep PBMCs had significantly higher TLR3 mRNA levels, TLR3 specific ligand (Poly I:C) stimulation resulted in increased levels of IFN-γ at transcriptional and translational levels along with IL-8 and IL-10 at transcriptional levels. Whereas, the levels of TNF-α was higher in goat PBMCs at transcriptional levels. BTV infected sheep PBMCs expressed significantly higher levels of IFN-γ at transcriptional and translational levels along with IL-6, IL-8 and IL-10 at transcriptional levels. Whereas the expression levels of TNF-α and IFN-α at transcriptional and translational levels were significantly high in goat PBMCs. To examine the potential factor for consistent increase in the expression of TNF-α, we sequenced the promoter region of TNF-α and identified a total of five single nucleotide polymorphisms (SNP) and one indel in goat TNF-α promoter region. Luciferase assay for transcriptional activity of the promoter showed that goat TNF-α has significantly enhanced transcriptional activity in comparison with sheep TNF-α promoter. Altogether, our data suggests that the expression levels of TNF-α and IFN-α and/or IL-10 plays crucial role in replication of BTV 23.

Comparative in vitro toll-like receptor ligand induced cytokine profiles of Toda and Murrah buffaloes-Identification of tumour necrosis factor alpha promoter polymorphism.

The objective of this study was to assess cytokine production upon activation of pattern recognition receptors responsible for sensing bacterial and viral pathogen associated molecular patterns in two genetically diverse buffalo breeds, Toda and Murrah. A very limited molecular-epidemiological analysis showed a higher prevalence of Anaplasma and Theileria in Murrah than Toda buffaloes. Toda buffalo peripheral blood mononuclear cells (PBMC) produced significantly higher levels of IFN γ and/or TNF α mRNAs in response to peptidoglycan, poly I:C, lipopolysaccharide, imiquimod and CpG. Flagellin stimulation did not result in any significant differences in the expression levels of the cytokines tested between these breeds. The levels of ligand induced IFN γ and TNF α mRNA and proteins also correlated except when induced with CpG. The proximal promoter region of TNF α across these two breeds were also sequenced to detect SNPs and promoter assay performed to determine their role in altering the transcriptional activity. Two polymorphisms were identified at -737 (T/A) and -1092 (G/T) positions in Toda buffalo TNF α promoter and promoter assay revealed higher transcription activity in Toda buffalos than in Murrah. This suggests that disease tolerance of these buffalo breeds could be due to the differences in their cytokine transcription levels in response to the respective PAMPs that may be at least in part determined by polymorphisms in the cytokine promoter regions.

Expression profile of toll-like receptor 2 mRNA in selected tissues of shark (Chiloscyllium sp.).

Sharks are a species of delight for immunologists from the evolutionary perspective since it is considered as the first species to have evolved the adaptive immune responses in addition to the innate immune system. One of the components of the highly conserved innate immune system is the toll-like receptors (TLR) which has a conserved overall protein structure throughout deuterostome evolution. There is no report that demonstrates the expression of these receptors in sharks. In this study we successfully amplified a 270 bp amplicon using a degenerate primer design strategy that corresponded to the Toll/IL-1 receptor (TIR) domain of TLR2 (GenBank ID: JF792813). BLAST analysis revealed a maximum nucleotide identity of 87% and 76% with the TLR2 of higher mammals and teleost fishes respectively. Domain prediction revealed a TIR structure between 1 and 87 amino acids that had a maximum identity of 58% and 76% with TLR2 - TIR protein of teleost fishes and higher mammals respectively. Phylogenetic analysis revealed a closer clustering of the shark TIR sequence with those from human, cattle, goat, sheep and chicken than with other fish species. Basal expression levels of the TLR2-TIR mRNA were found to be significantly higher in kidneys followed by fins, spleen and intestinal spiral valve (ISV). In tissues such as spleen and kidney the expression of the TLR2-TIR mRNA could be localized to lymphoid and macrophages like cells and tubular epithelial cells respectively. In-vivo exposure of sharks to peptidoglycan (TLR 2 ligand) resulted in 9 folds higher expression of TLR2-TIR mRNA in gills followed by 5 folds in the fins. However, when inoculated with a TLR ligand pool, the expression levels significantly increased to 12 fold in skin followed by epigonal, kidneys and ISV. These findings not only support the presence of the TLRs in sharks but also their induction upon exposure to specific ligands. Further studies are needed to identify their numbers, their ligand specificity and downstream cytokine responses.

Transcript profiling of pattern recognition receptors in a semi domesticated breed of buffalo, Toda, of India.

The primary objective of this study was to assess the expression profile and levels of toll-like receptor (TLR) mRNAs in the spleen, lung, mediastinal lymph node (MLN), jejunum, rectum, skin and peripheral blood mononuclear cells (PBMC) of Toda and Murrah buffalos. Spleen and PBMC had increased expression of TLR mRNAs 2, 4, 5, 6, 8, 9 and 10; lung had increased expression of TLR mRNAs 2, 4, 5, 6 and 8, MLN TLR mRNA 6, 9, 10 and decrease in TLR 3 and 7 mRNAs in skin. No significant differences were observed in the expression levels of any of the TLR mRNA in jejunum and rectum. Toda buffaloes showed significantly higher expression levels of TLR 9 mRNA in MLN, TLR mRNAs 1, 5, 6, 9 and 10 in skin and TLR mRNAs 2, 4, 7 and 9 in PBMC than Murrah buffaloes living in the vicinity. Toda and Murrah buffaloes were inoculated with TLR5 (flagellin) and TLR9 (CpG ODN) ligands in vivo and expression levels of the respective TLRs analyzed 12h later. Following CpG inoculation, Toda buffaloes had significantly higher levels of TLR 9 mRNA expression but not in Murrah. However, flagellin induction did not increase TLR 5 mRNA expression in both these breeds. Histological sections of the skin were made and infiltrating cell clusters were graded and quantified. Following CpG inoculation, Toda buffaloes showed higher numbers of infiltrating grade 1 and grade 3 cell clusters while Murrah showed lower numbers of infiltrating grade 1 cells as compared to mock-inoculated skin sections. Flagellin treatment revealed no significant differences in infiltrating cell clusters in both the breeds. The results have shown differential expression of TLR mRNAs in various tissues between two divergent buffalo breeds with the highest difference in TLR expression profile seen in the skin, the largest portal of entry of pathogens, of Toda.

Sequence analysis of Toll-like receptor genes 1-10 of goat (Capra hircus).

This study involved cloning and sequencing of the coding regions of all 10 Toll-like receptor (TLR) genes of goat. Goat TLR 1-10 gene sequences revealed a high degree of nucleotide identity with sheep and cattle sequences (>90%) and 75-85% with pig, mouse and human sequences. At the amino acid level, 85-99% similarity was observed with sheep and cattle and 60-85% with pig, mouse and human. TLR9c DNA of goat showed the highest amino acid identity to that of sheep (99%) while TLR8 cDNA showed the lowest identity of 88.7% to that of sheep. Variations were seen in the number of leucine rich repeats (LRRs) of goat TLRs as compared to other ruminant species with maximum differences in the TLR3 gene. Phylogenetic analysis through molecular evolution and genetic analysis (MEGA) software and multi dimensional scaling revealed a high degree of conservation of goat TLRs with those from other species. However when the TIR domain of all the TLRs were compared, goat TLR7 TIR alone showed a high divergence of 19.3 as compared to sheep sequences. This is the first report of the full-length cDNA sequences of all the 10 TLR genes of goats which would be a useful tool for the study of evolutionary lineages and for phylogenetic analysis.

Difference in the level of interferon gamma mRNA transcripts on stimulation of cattle and buffalo mononuclear cells with foot and mouth disease virus-antigen: a possible role of sequence variation in promoter region.

In an attempt to resolve the claim that buffaloes differ from cattle in disease progression, this study was undertaken to compare the mitogen (conA) or antigen (foot and mouth disease virus) induced expression levels of interferon gamma (IFN-γ mRNA in peripheral blood mononuclear cells (PBMCs) by real-time quantitative PCR. In general, the levels of IFN-γ mRNA were lower in buffaloes than in crossbred cattle. Significantly higher levels of IFN-γ mRNA were also observed in crossbred cattle when induced with FMD virus (1 µg). Analysis of the partial promoter sequences of the IFN-γ gene from the respective species revealed a conserved 4 base (GTCT) deletion in all the buffalo promoter sequences. In-silico analysis indicated the binding of glucocorticoid receptor (GR) and erythroid nuclear factor (NF-E) to this region in cattle. GR has been shown to be a transcription factor by itself and also regulates other major transcription factors like NF-κB and AP-1. The differential expression levels of IFN-γ mRNA between these species could be due to this deletion in the promoter region of buffalo. Further studies involving mobility shift and promoter assays would throw more light on the differential expression levels.

Analysis of the Fusion Protein Cleavage Site of Newcastle disease virus Isolates from India Reveals Preliminary Evidence for the Existence of II, VI and VII Genotypes.

Newcastle disease virus (NDV) has been a threat to poultry industry in most of the developing countries with a wide variety of avian species being susceptible, coupled with the presence of mobile wild bird reservoirs contributing not only to the vast genomic diversity of this virus but also to the diagnostic failures. NDV of multiple genotypes (I-XI) is known to be prevalent and reported worldwide. However, there is a paucity of information on the circulating genotypes of NDV in India. This study utilized the fusion protein cleavage site (FPCS) sequence to determine the different genotypes of NDV circulating in India. Our results indicate that majority of NDV isolates from southern states of India namely, Tamil Nadu, Kerala and Karnataka were found to belong to genotype II. However, some of the strains from Tamil Nadu and most from Uttar Pradesh belong to genotype groups VI and VII. Interestingly, three isolates recovered from Tamil Nadu grouped with genotype IV viruses (namely Herts/33) which had not been hitherto reported to GenBank since 1989. This preliminary information points to the existence of multiple genotypes and also the need for efficacy studies with vaccines incorporating multiple genotypes in controlling virulent NDV (vNDV) outbreaks in India.

Differential expression of toll-like receptor mRNA in selected tissues of goat (Capra hircus).

Pattern recognition receptors (PRRs) expressed by various immune cells and tissues have been shown to play a pivotal role in the recognition of pathogens by the host. The present study was carried out to identify toll-like receptors (TLRs) 1-10 mRNA in goat peripheral blood mononuclear cells (PBMCs) and selected tissues including jejunum, lung, lymph node, skin, spleen and uterus using reverse transcriptase polymerase chain reaction (RT-PCR). Our results confirm earlier reports regarding the evolutionarily conserved nature of these receptors as successful amplification of the goat TLR mRNAs could be obtained with bovine TLR mRNA-specific primers. The partial sequences of the purified TLR PCR amplicons had 93.8-99.7% nucleotide identity with sheep TLR cDNA sequences available in the GenBank. Semi-quantification of the expression levels of the TLR mRNAs was done using densitometric analysis of band intensities. All the TLR mRNAs (1-10) were expressed in high amounts in the lymph node while spleen showed lower expression of TLR 6 and 10 mRNAs. PBMC and lung expressed all TLR mRNAs in high amounts except TLR 10 mRNA. In uterus and jejunum, lower expression of TLR 3, 4 and 10 mRNAs was seen. Skin had the lowest repertoire of TLR mRNA expression with lower or no expression of TLR 2, 3, 4, 8, 9 and 10 mRNAs. Another interesting observation was that tissues such as uterus, lung and skin that exhibited lower levels of TLR 2 had higher levels of TLR 6 mRNAs.

Pathotyping of a Newcastle disease virus isolated from peacock (Pavo cristatus).

This report describes Newcastle disease in peacock and the isolation and characterization of the virus. The virus had an intracerbral pathogenicity index of 1.71 and mean death time of 47 h. The isolate had multiple basic amino acids at the fusion protein cleavage site sequence ((110)GGRRQRRFIG(119)) with a phenylalanine at residue 117. Biological and molecular characterization revealed that the virus is velogenic. Phylogenetic analysis placed the isolate in genotype II.

TNF-alpha induced CD38 expression in human airway smooth muscle cells: role of MAP kinases and transcription factors NF-kappaB and AP-1.

In human airway smooth muscle (HASM) cells, the expression of CD38, which synthesizes the calcium-mobilizing molecule cyclic ADP-ribose, is augmented by TNF-alpha, a cytokine implicated in asthma. We determined the role of mitogen-activated protein kinase (MAPK) in the activation of NF-kappaB and AP-1 in the regulation of CD38 expression in HASM cells. In HASM cells exposed to TNF-alpha (40 ng/ml), the inhibitors of extracellular signal-regulated kinase (ERK), p38, or c-Jun NH(2)-terminal kinase (JNK) decreased CD38 expression and ADP-ribosyl cyclase activity. Transfection of HASM cells with a dominant negative MEK decreased while a wild-type ERK increased TNF-alpha-induced CD38 expression. Electrophoretic mobility shift assays (EMSAs) were performed using nuclear proteins and consensus sequences to detect the effect of the MAPKs on NF-kappaB and AP-1 activation. EMSAs confirmed the role of p38 and JNK in mediating NF-kappaB and AP-1 activation. Transfection of a dominant negative c-Jun decreased TNF-alpha-induced CD38 expression indicating involvement of AP-1. Stability of TNF-alpha-induced CD38 transcripts were determined in the presence of MAPK inhibitors after arresting the transcription with actinomycin D. Transcript stability decreased in the presence of ERK and p38 MAPK, but not the JNK, inhibitors. These results indicate that regulation of CD38 expression through p38 and JNK MAPKs involves NF-kappaB and AP-1 activation, and ERK and p38 MAPKs also regulate expression posttranscriptionally through message stability.

Transcriptional regulation of CD38 expression by tumor necrosis factor-alpha in human airway smooth muscle cells: role of NF-kappaB and sensitivity to glucocorticoids.

The transmembrane glycoprotein CD38 catalyzes the synthesis of the calcium mobilizing molecule cyclic ADP-ribose from NAD. In human airway smooth muscle (HASM) cells, the expression and function of CD38 are augmented by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), leading to increased intracellular calcium response to agonists. A glucocorticoid response element in the CD38 gene has been computationally described, providing evidence for transcriptional regulation of its expression. In the present study, we investigated the effects of dexamethasone, a glucocorticoid, on CD38 expression and ADP-ribosyl cyclase activity in HASM cells stimulated with TNF-alpha. In HASM cells, TNF-alpha augmented CD38 expression and ADP-ribosyl cyclase activity, which were attenuated by dexamethasone. TNF-alpha increased NF-kappaB expression and its activation, and dexamethasone partially reversed these effects. TNF-alpha increased the expression of IkappaBalpha, and dexamethasone increased it further. An inhibitor of NF-kappaB activation or transfection of cells with IkappaB mutants decreased TNF-alpha-induced CD38 expression. The results indicate that TNF-alpha-induced CD38 expression involves NF-kappaB expression and its activation and dexamethasone inhibits CD38 expression through NF-kappaB-dependent and -independent mechanisms.

Adenoviral mediated anti-sense CD38 attenuates TNF-alpha-induced changes in calcium homeostasis of human airway smooth muscle cells.

CD38 is a membrane-bound protein involved in the synthesis and degradation of cyclic-ADP-ribose (cADPR). cADPR mobilizes calcium from intracellular stores in airway smooth muscle cells. To determine the role of CD38/cADPR signaling in calcium regulation in human airway smooth muscle (HASM) cells, we downregulated CD38 expression using a recombinant replication-defective adenovirus with anti-sense human CD38 (Ad-asCD38). CD38 expression was determined by RT-PCR and real-time quantitative PCR, and ADP-ribosyl cyclase (cyclase) activity was determined by competitive binding assay. In HASM cells infected with Ad-asCD38, TNF-alpha-induced, augmented-CD38 expression and cyclase activity were significantly lower than in TNF-alpha-treated cells. The net intracellular calcium responses to 10 nmol/L bradykinin were measured in HASM cells by fluorescence imaging. In cells infected with Ad-asCD38 in the presence of TNF-alpha, the net intracellular Ca2+ responses were significantly lower than in cells treated with TNF-alpha in the presence of the control vector (p < 0.001). These results provide evidence for the feasibility of using adenoviral vectors for gene transfer to down regulate gene expression, and confirm the role of CD38 in calcium homeostatis in ASM cells.

Characterization of nucleoprotein gene sequence of an Indian isolate of rabies virus.

Rabies occurs in all parts of Indian sub-continent except Andaman and Nicobar and Lakshadweep group of islands. The full-length nucleoprotein (N) gene sequence of a rabies virus isolate from India is reported for the first time and the same has been compared with available N gene sequences from the database. A central domain of 230 amino acids (aa) from aa 141 to aa 370 exhibited more than 95% similarity. There were 8 amino acid positions (aa 29, 32, 38, 84, 119, 379, 438, and 439) at which substitution was unique for Indian isolates but common for laboratory strains. In antigenic epitopes, except for a single amino acid difference at the antigenic site IV, the amino acids were conserved. The Indian isolate also possessed two Bam HI sites (aa 247 and 278), while the other Asian isolates had only one site at aa 278 or were not digested with Bam HI at all. Phylogenetic analysis also demonstrated that the Indian isolate was closely related to the Sri Lankan isolate and grouped in the cluster that comprised of the isolates from other Asian countries namely China and Pakistan.