Evolutionary analysis of rabies virus using the partial Nucleoprotein and Glycoprotein gene in Mumbai region of India.

Nearly 1.7 million cases of dog bites are reported every year in India and many cases of animal rabies are left unattended and undiagnosed. Therefore, a mere diagnosis of rabies is not sufficient to understand the epidemiology and the spread of the rabies virus (RV) in animals. There is a paucity of information about the evolutionary dynamics of RV in dogs and its biodiversity patterns in India. In total, 50 dog-brain samples suspected of rabies were screened by the nucleoprotein- (N) and glycoprotein- (G) gene PCR. The N and G genes were subsequently sequenced to understand the molecular evolution in these genes. The phylogenetic analysis of the N gene revealed that six isolates in the Mumbai region belonged to a single Arctic lineage. Time-scaled phylogeny by Bayesian coalescent analysis of the partial N gene revealed that the time to the most recent common ancestor (TMRCA) for the sequences belonged to the cluster from 2006.68 with a highest posterior density of 95 % betweeen 2005-2008, which is assigned to Indian lineage I. Migration pattern revealed a strong Bayes factor between Mumbai to Delhi, Panji to Hyderabad, Delhi to Chennai, and Chennai to Chandigarh. Phylogenetic analysis of the G gene revealed that the RVs circulating in the Mumbai region are divided into three lineages. Time-scaled phylogeny by the Bayesian coalescent analysis method estimated that the TMRCA for sequences under study was from 1993 and Indian clusters was from 1962. In conclusion, the phylogenetic analysis of the N gene revealed that six isolates belonged to single Arctic lineages along with other Indian isolates and they were clustered into a single lineage but divided into three clades based on the G-gene sequences. The present study highlights and enhances the current molecular epidemiology and evolution of RV and revealed strong location bias and geographical clustering within Indian isolates on the basis of N and G genes.

Molecular epidemiology of Canine parvovirus shows CPV-2a genotype circulating in dogs from western India.

Canine parvovirus (CPV) has emerged as an acute pathogen of young canine causing haemorrhagic enteritis and myocarditis. It is widely distributed and underreported in India. Therefore the study was conducted to type the CPV circulating in western Maharashtra. The faecal samples (n = 150) from clinically ill dogs showing diarrhoea and vomition were collected and subjected to haemagglutination (HA) with porcine RBC's. The DNA was extracted from the samples showing HA titres above 64 and subjected for amplification of VP2 gene fragment by PCR. The amplicons were subjected for restriction fragment length polymorphism (RFLP), sequencing and BEAST phylogenetic analysis. The results revealed 6% positivity by PCR. The RFLP results indicated single cleavage site for ApaLI and HinfI with an exception of two sites for HinfI. The nucleotide sequences showed nonfunctional nucleotide changes at different locations. The sequence analysis indicated that the nucleotide divergence within isolates under study was 0.00-0.42%, while the nucleotide homology was 99.58-100%. The most recent common ancestor was determined by molecular clock analysis using Bayesian methods. The sequence and phylogenetic analysis suggested the isolates as CPV-2a and KATN1 (KU866391, 2014) isolate from Tamilnadu, India as time to most recent common ancestor (TMRCA). The results revealed the circulating CPV in canines from western India as CPV2a genotype.

Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein.

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.

Evolutionary and bioinformatic analysis of the spike glycoprotein gene of H120 vaccine strain protectotype of infectious bronchitis virus from India.

The infectious bronchitis virus is a causative agent of avian infectious bronchitis (AIB), and is is an important disease that produces severe economic losses to the poultry industry worldwide. Recent AIB outbreaks in India have been associated with poor growth in broilers, drop in egg production, and thin egg shells in layers. The complete spike gene of Indian AIB vaccine strain was amplified and sequenced using a conventional reverse transcription polymerase chain reaction and is submitted to the GenBank (accession no KF188436). Phylogenetic analysis revealed that the vaccine strain currently used belongs to H120 genotype, an attenuated strain of Massachusetts (Mass) serotype. Nucleotide and amino acid sequence comparisons have shown that the reported spike gene from Indian isolates have 71.8%-99% and 71.4%-96.9% genetic similarity with the sequenced H120 strain. The study identifies live attenuated IBV vaccine strain, which is routinely used for vaccination, for the first time. Based on nucleotide and amino acid relatedness studies of the vaccine strain with reported IBV sequences from India, it is shown that the current vaccine strain is efficient in controlling the IBV infection. Continuous monitoring of IBV outbreaks by sequencing for genotyping and in vivo cross protection studies for serotyping is not only important for epidemiological investigation but also for evaluation of efficacy of the current vaccine.

Protective effects of recombinant glycoprotein D based prime boost approach against duck enteritis virus in mice model.

Duck virus enteritis, also known as duck plague, is an acute herpes viral infection of ducks caused by duck enteritis virus (DEV). The method of repeated immunization with a live attenuated vaccine has been used for the prevention and control of duck enteritis virus (DEV). However, the incidence of the disease in vaccinated flocks and latency reactivation are the major constraints in the present vaccination programme. The immunogenicity and protective efficacy afforded by intramuscular inoculation of plasmid DNA encoding DEV glycoprotein D (pCDNA-gD) followed by DEV gD expressed in Saccharomyces cerevisia (rgD) was assessed in a murine model. Compared with mice inoculated with DNA (pCDNA-gD) or protein (rgD) only, mice inoculated with the combination of gD DNA and protein had enhanced ELISA antibody titers to DEV and had accelerated clearance of virus following challenge infection. Furthermore, the highest levels of lymphocyte proliferation response, IL-4, IL-12 and IFN-γ production were induced following priming with the DNA vaccine and boosting with the rgD protein. For instance, the specially designed recombinant DEV vector vaccine would be the best choice to use in ducks. It offers an excellent solution to the low vaccination coverage rate in ducks. We expect that the application of this novel vaccine in the near future will greatly decrease the virus load in the environment and reduce outbreaks of DEV in ducks.

Adaptation and growth kinetics study of an Indian isolate of virulent duck enteritis virus in Vero cells.

Duck virus enteritis, also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with chicken embryo adapted live virus that is known to be poorly immunogenic and elicits only partial protection. Further, the embryo propagated vaccine virus pose a threat of harboring other infectious agents. Seeing these limitations, the present study reports for the first time regarding propagation and adaptation of a virulent Indian isolate of duck enteritis virus in Vero cell line. In this study isolation of an outbreak virus from Kerala state was done in chicken embryo fibroblast cell culture (CEF). Then adapted the DEV isolate in the Vero cell line. The characteristic cytopathic effects (CPE) of clumping and fusion of Vero cells were observed starting from the 7th passage onwards. The presence of the virus and its multiplication in Vero cells was confirmed by detection of viral specific DNA and antigen by using polymerase chain reaction (PCR) and indirect immuno fluorescent assay (IIFA), respectively. PCR detection of DEV using self designed primers for US4 (gD) and UL30 (DNA Polymerase) gene has been reported for the in the present study. The kinetics of DEV in Vero cells revealed a maximum infectivity titer of 10(5.6) TCID 50/ml after 48hr of viral infection. Compared to chicken embryo adapted DVE vaccine virus, the Vero cell culture system is free from other infectious agents. So it will be a good candidate for cultivation and propagation of duck enteritis virus vaccine strain. Further research studies are suggested to explore the feasibility of utilizing this Vero cell culture adapted DEV isolate for developing an attenuated vaccine virus against duck virus enteritis.

Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock.

The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 3'-N-P-M-F-HN-L-5', which was limited by 55-nts leader region at the 3' end and a 114-nts trailer sequence at 5' end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site (112)R-R-Q-K-R-F(117), a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry.

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Complete Genome Sequence of a Newcastle Disease Virus Isolated from Wild Peacock (Pavo cristatus) in India.

We report here the complete genome sequence of a Newcastle disease virus (NDV) isolated from a wild peacock. Phylogenetic analysis showed that it belongs to genotype II, class II of NDV strains. This study helps to understand the ecology of NDV strains circulating in a wild avian host of this geographical region during the outbreak of 2012 in northwest India.