Indian Journal of Virology最新文献

The control of an infectious viral disease as rabies is made easier by rapid and accurate diagnosis. Successful rabies prophylaxis is dependent upon the active immunization with vaccine along with passive administration of rabies virus neutralizing antibodies which together clear the virus before widespread infection of central nervous system occurs. The present study aimed at the development of monoclonal antibodies (MAbs) suitable for rabies virus antibody and antigen detection. For the production of rabies specific MAbs, immunization of Swiss albino mice with a commercially available vaccine was done and Polyethylene glycol mediated fusion of spleenocytes with myeloma cells was performed. The positive clones were selected on the basis of distinct reactivity by cell Enzyme linked immunosorbent assay and fluorescence in Indirect Fluorescent antibody test. The positive clones obtained were subjected to single cell cloning by limiting dilution method. The reactive clones were further titrated and employed for virus titration and virus neutralization. The neutralizing activity was evaluated using Fluorescence Activated Cell Sorter technique. Three MAb clones showed a distinct percent inhibition in the presence of positive serum. One of the MAb clone No. 5C3 was relatively more specific in detecting rabies antibodies and also found suitable for competitive ELISA to assess the antibody level in vaccinated subjects.

Bovine herpesvirus -1 (BHV-1) is the etiological agent of many clinical syndromes in cattle which causes huge economic losses to the animal husbandry sector annually. Since the first report of its presence in India in 1976, the disease is considered to be endemic in the country. In the present study, a case of keratoconjunctivitis in a cow was investigated to find out the underlying cause of the condition. The clinical material (ocular swab) was tested by BHV-1 glycoprotein D gene specific PCR using in house designed primers and found to be positive by the presence of a 212 bp DNA product in agarose gel electrophoresis. The virus was isolated in MDBK cell line in the third passage and the serum from the animal, was positive for antibodies against BHV-1 by ELISA. A 575 bp segment of the glycoprotein C gene of the isolate was amplified by PCR, cloned and sequenced. On phylogenetic analysis, it was seen that the sequence matched with published BHV-1.1 sequences from USA and Uruguay whereas it was divergent from Brazilian BHV-1.1 isolates. This study highlights the isolation, rapid and sensitive detection of BHV-1 virus from clinical cases and its subtyping by nucleotide sequencing and subsequent phylogenetic analysis which gives invaluable information about the molecular epidemiology of BHV-1 subtypes prevalent in the country.

Ophiusa disjungens nucleopolyhedrovirus (OpdiNPV) was newly found in Guangdong Province, China. Using BamHI, EcoRI, HindIII, PstI, XhoI, XbaI digestion, the size of the OpdiNPV genome was estimated to be 92,000 base pair. The pstI-G genomic fragment of OpdiNPV was cloned and sequenced. Searches of databases identified at least four open reading frames (ORFs) within this fragment. These ORFs encode odv-e66 (EU 623602), p87/vp80 (EU 732665), odv-ec43 (EU617337) and ac108 gene (EU 732666) respectively. The phylogenetic tree of NPVs based on the combined sequences of odv-ec43 and ac108 indicated that OpdiNPV was most closely related to Mamestra configurata NPV-A and Mamestra configurata NPV-B. The characterization of OpdiNPV in this paper would provide better understanding molecular properties of this virus and be helpful in the development of the newly isolated virus as a biopesticide or an engineered pesticide to control more species of insect pests.

Picobirnaviruses (PBVs) are small, non-enveloped, 35-41 nm virion with bisegmented double-stranded RNA genome. PBVs are widespread and were detected in feces of humans and a wide variety of animals. Domestic pig, one of the ubiquitous farm animal reported incessant association with a variety of viral zoonoses. The objective of our study is to find out the incidence of PBV infection in healthy domestic pigs. The study was conducted by collecting feces of healthy/asymptomatic pigs from a piggery located in an urban slum at Kolkata, India to detect PBV infections. All the 11 fecal samples were tested by polyacrylamide gel electrophoresis and reverse transcription-polymerase chain reaction assay. In this study, we report the first incidence of detection and molecular characterization of porcine PBV (BG-Por-2/2010 and BG-Por-7/2010) in feces of domestic pigs from India using the human PBV genogroup I specific primer pair: PicoB25(+) and PicoB43(-). Sequence comparison and phylogenetic analysis of partial RNA-dependent RNA polymerase gene of genome segment 2 revealed genetic relatedness to hitherto reported porcine, murine and human genogroup I PBVs from different geographical regions. This warrants a stringent global surveillance to study the potential zoonotic and emerging PBV infections.

The 50 % tissue culture infectious dose (TCID50) is still one of the most commonly used techniques for estimating virus titers. However, the traditional TCID50 assay is time consuming, susceptible to subjective errors and generates only quantal data. Here, we describe a colorimetric-based approach for the titration of Enterovirus 71 (EV71) using a modified method for making virus dilutions. In summary, the titration of EV71 using MTT or MTS staining with a modified virus dilution method decreased the time of the assay and eliminated the subjectivity of observational results, improving accuracy, reproducibility and reliability of virus titration, in comparison with the conventional TCID50 approach (p < 0.01). In addition, the results provided evidence that there was better correlation between a plaquing assay and our approach when compared to the traditional TCID50 approach. This increased accuracy also improved the ability to predict the number of virus plaque forming units present in a solution. These improvements could be of use for any virological experimentation, where a quick accurate titration of a virus capable of causing cell destruction is required or a sensible estimation of the number of viral plaques based on TCID50 of a virus is desired.

Apple chlorotic leaf spot virus (ACLSV; family Betaflexiviridae genus Trichovirus) is one of the economically important latent virus infecting apple (Malus × domestica Borkh.). Reverse transcriptase polymerase chain reaction (RT-PCR) procedures were used to amplify coat protein gene of ACLSV. Among 5 primer sets used, two primer sets (1F1R and 1F2R) amplified fragments of expected size (432 bp). Products visible on agarose gel were produced using templates extracted from apple leaves. The results were further validated by sequencing fragment of 432 bp which was amplified from leaf of apple by using primer set 1F 1R. Comparisons with published sequences indicated that the isolate have very high 91 % identity values to the corresponding region of ACLSV isolate from apple. Selected primer pair (1F1R) was further used for screening 42 elite mother plants collected from apple growing areas of Himachal Pradesh, India, where in 17 were found free from ACLSV. Use of NAD5 gene in mitochondrial mRNA of the apple as an internal control, reduced the risk of false negative results that may occur with routine RT-PCR assays.

Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 μg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 μg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.

Transmissible gastroenteritis virus (TGEV) is the etiological agent of TGE, and dogs are potential carriers of TGEV. In this study, genomic RNA were extracted from TGEV designated HYM-09 isolated from dog naturally infected with TGEV. The nucleocapsid (N) gene of HYM-09 was amplified by RT-PCR and cloned into pMD18-T vector. The N gene cDNA was sequenced and encompassed an open reading frame of 1,149 nucleotides, encoding a 382-amino acids protein. Sequence analyses of the N genes were performed, including homologous comparison, phylogenetic tree analysis and residue substitution analysis. The results showed that there existed some unique mutations in the HYM-09 isolate N gene, but HYM-09 N gene shared over 96 % homologous identities compared with 12 TGEV reference strains derived from other regions or countries respectively. The phylogenetic tree analysis revealed that the HYM-09 branched into the most strains group. This study shows that the nucleotide sequence analysis can form a base or further study on the mutation trend of non-porcine TGEV.

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

We studied the prevalence and effects of host genetic polymorphisms for the three AIDS restriction genes (ARGs) namely CCR2-64I, SDF1-3'A and MCP1-2518 G/A for HIV infection and progression to AIDS using PCR-RFLP analysis on a total of 568 HIV seronegative serum samples collected from a specific high risk and young population hailing from the seven Northeastern states of India (n = 346), West Bengal (n = 96) and Gorkha population (n = 101). In addition, 181 HIV seropositive cases of which 92 inpatient cases in a large tertiary care hospital located at Kolkata were included in the study. HIV prevalence in our study group was 0.52 %. Four cases seroconverted, 25 cases progressed to AIDS and 05 died during the follow up period of 41 months. The genotype percentage of CCR2-64I, SDF1-3'A and MCP1-2518 G/A in the Northeastern states were 18.5, 40.3 and 54 % respectively in the seronegative population. Allele frequencies for SDF1-3'A in Northeastern states were significantly higher as compared to the Gorkha (21 %) and the North Indian population (24 %). Relative Hazard values were more than 0.9 for progression to AIDS and death. Kaplan-Meier survival analysis using Cox proportional regression model did not reveal any significant survival benefit (p value <0.05) for any of the 3 ARGs individually or in combination either to seroconversion or disease progression. This is the first study on host genetic polymorphism amongst the Eastern, Northeastern and Gorkha regions in India. We are also the first to report the MCP1-2518 G/A polymorphism in India that is known to increase the rate of neuropsychological impairment (NPI) in AIDS patients.