Indian Journal of Virology最新文献

Large cardamom chirke virus (LCCV), genus Macluravirus, family Potyviridae is an important constrain in large cardamom production in India. Purification of LCCV from large cardamom tissues is difficult and therefore immunodiagnostic reagents are not available. In the present study, we have successfully expressed coat protein (CP) gene of LCCV in Escherichia coli. The purification of expressed protein by Ni-NTA affinity chromatography was inefficient due to precipitation of protein during renaturation. We have optimized a simple, inexpensive and efficient method for purification of the expressed CP through gel extraction with 5 % SDS followed by renaturation in Milli-Q water, which resulted in high yield (4.7 mg/ml) and good quality of the protein. A higher titer (1:256,000) polyclonal antibody (PAb) to the recombinant CP was produced, which strongly recognized LCCV in crude leaf extract and showed minimal background reaction with the healthy leaf extract in enzyme linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA). The sensitivities of the ELISA and DIBA were 5 and 0.1 ng of expressed protein, respectively. Both the ELISA and DIBA were validated with 100 % accuracy in detecting LCCV in field samples. The PAb differentiated Cardamom mosaic virus, another close relative of LCCV. Our study is first to report highly efficient immunodiagnosis with PAb to E. coli expressed recombinant CP of a virus under the genus Macluravirus. The antigen expression construct and PAb developed in the present study will be useful in production of virus free planting materials of large cardamom.

Highly pathogenic avian Influenza (HPAI) is an important zoonotic disease and is becoming a great threat to poultry industry. India has experienced continual outbreaks of H5N1 HPAI virus since February, 2006 especially in Eastern India. Survivability in poultry faeces is an important determinant in evaluating the persistence of the virus in the poultry sheds and their vicinity. In this paper, survivability of Indian H5N1 HPAI virus in dry and wet poultry faeces at 42, 37, 24 and 4 °C, respectively is reported. The effect of different temperatures was determined by linear regression model and defined in terms of linear equation. The virus survived up to 18 h at 42 °C, 24 h at 37 °C, 5 days at 24 °C and 8 weeks at 4 °C in dry and wet faeces, respectively. The coefficients of determination (R(2)) values for dry and wet faeces revealed that the difference in viral persistence in dry and wet faeces at all temperatures was not very marked. Results of the present study indicated that H5N1 HPAI virus may remain viable for extended periods of time in faeces at low temperatures and may act as a long term source of influenza virus in the environment.

Five Potato leafroll virus (PLRV) isolates were collected from five states representing different potato growing parts of India. The ssRNA genome sequences of these isolates were determined. The genome comprised of 5,883 nucleotides and deduced genome organization resembled other PLRV isolates. About 97.6-98.7 % similarities was observed within the Indian isolates and were more close to European, Canadian, African, American and Czech isolates (95.8-98.6 %) than to an Australian isolate (92.9-93.4 %). These isolates were 43.7-53.1 % similar to other poleroviruses and 29.1-29.3 % to Barley yellow dwarf virus, a luteovirus. Out of five isolates, the isolate PBI-6 was recombinant one as detected by RDP3 software. Multiple sequence alignment of nucleotide and amino acid sequences of different ORFs indicated that the ORF 3 and ORF 4, corresponding to coat protein and movement proteins are more conserved than other ORFs. Amino acid changes specific to Indian isolates were observed and it was more in ORF 2 than in ORF 0, ORF 3 and ORF 4. This is the first report of complete genome sequence of PLRV isolates from India, which reveals low level genetic diversity.

The seasonal outbreaks of human rotavirus (RV) infection occur every winter. Most patients are diagnosed clinically by a rapid latex agglutination detection kit or polymerase chain reaction assays for RV from stool samples, but some problems have been reported on the specificity and sensitivity of such rapid detection assays. To ratify these issues, a sensitive, specific, simple, and rapid nucleic acid based diagnostic method is expected to be introduced and the reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect the RV in human stool samples by incubation at 60 °C for 1 h and amplification was confirmed by electrophoretic laddering, restriction enzyme digestion, and hydroxynapthol blue discoloration. The assay established in this study was found to detect only the RVs and no cross-reaction with other viruses, demonstrating its high specificity. By using serial samples dilution as template, the detection limit of LAMP was 10 times more than that of PCR. The results showed the potential clinical feasibility of RT-LAMP as a useful diagnostic tool for the detection of RV with high sensitivity in comparison to conventional RT-PCR.

Present era of molecular biology is witnessing revolutionary developments in sequencing technology. This advancement has considerably influenced plant virology in the field of diagnostics and host virus interaction. Next generation high-throughput sequencing technology has made it possible to directly detect, identify and discover novel viruses in several plants in an unbiased manner without antibodies or prior knowledge of the virus sequences. Entire viral genome could be sequenced from symptomatic or asymptomatic plants through next generation sequencing of total nucleic acids including small RNAs. It provides census of viral population in a particular ecosystem or cropping system. Viral genome variability, evolution within the host and virus defence mechanism in plants can also be easily understood by massive parallel sequencing. In this article, we provide an overview of the applications of next generation sequencing technology in characterization, discovery and molecular interaction of plant viruses.

During an investigation in the year 2010, on the weed reservoir of begomovirus, Abutilon pictum showing bright yellow mosaic symptoms was observed in Udhagamandalam, Tamil Nadu, India. The complete bipartite genome of a begomovirus was cloned and sequenced which revealed association of Abutilon mosaic virus (AbMV). Nicotiana benthamiana plants inoculated biolistically with the concatemers generated through rolling circle amplification of the cloned DNAs were asymptomatic; however three out of nine plants showed presence of viral DNA A. A recombination event in the ORF BC1 with ToLCNDV DNA B (HM989846) was detected. This is the first molecular evidence of AbMV in India.

Newcastle disease virus (NDV), the causative agent of Newcastle disease (ND) in chicken causes significant economic loss for the poultry industry worldwide. The mechanism involved in host response to NDV infection is not well understood. For better understanding of the virus-host interaction; transcriptional profile of some genes of chicken embryo (CE) cells infected with NDV vaccine strain D58 was established using quantitative RT-PCR SYBR Green method. The relative standard curve method was used to measure the level of transcripts of the cellular genes against an endogenous control (β actin) gene. Among the genes studied, IFN α, IFN γ, MHC I and DDX 1 were up-regulated while IL 6 was down regulated. The expression of viral genes (M and F) in the infected CE cells was also confirmed by relative quantification. The host cellular genes involved in pro-inflammatory response, interferon-regulated proteins and the cellular immune response were affected by NDV infection, indicating involvement of complex signaling pathways of host cell responses to the infection. Thus, this study contributes to the understanding of the pathogenesis of ND and provides an insight into the virus-host interaction.

Studies show that hepatitis B virus (HBV) DNA isolation methods vary in their efficiency to extract DNA from serum samples. The purpose of the present study was to develop an improved method for isolation of HBV DNA and compare it with commonly used HBV DNA isolation protocols. In order to develop HBV DNA isolation protocol, serum samples were collected from patients and screened for the presence of hepatitis B surface antigen, hepatitis B e antigen and HBV DNA. Highly viremic samples were pooled and used to compare commonly used HBV DNA isolation methods; namely alkaline lysis, microwave treatment, organic, inorganic with modified inorganic method. DNA isolated by these methods was detected qualitatively by polymerase chain reaction and quantitatively with competitive polymerase chain reaction (cPCR). The modified inorganic method gave maximum yield of HBV DNA followed by inorganic, organic, microwave treatment and alkaline lysis method. Our data also demonstrated a critical role of proteinase K in HBV DNA isolation. DNA isolation method described here, in combination with a reproducible and sensitive quantitative technique would further help in accurate classification of HBV infected patients, designing suitable drug regimen for treatment and monitoring antiviral treatment as well as emergence of drug resistant mutants.

Rotavirus (RV) infections are worldwide in distribution causing high morbidity and mortality in human and animal neonates. Human settlements in close proximity of animals aids for genetic re-assortment of the virus by interspecies transmission and consequent emergence of new viral antigenic strain. Therefore, the present study was designed to explore RV incidence in a single approach from human and animal neonates sharing similar environment. Altogether, 200 diarrheal samples from children (50), piglets (80) and calves (70) were collected during the year of 2010-2012 from various locality, farms and hospitals, initially screened through monoclonal antibody based enzyme immunoassay followed by RNA-PAGE and VP7 gene amplification by Reverse transcription PCR. The overall prevalence of rotavirus was found to be 41.5 % (83/200) where maximum numbers of positive cases were found in piglets (46.3 %) followed by human (40 %) and cow (37.1 %). Majority of samples demonstrated characteristic group A rotavirus (RVA) electropherotype of 4:2:3:2 pattern. Moreover, RNA profiles of seven samples from piglets and calves revealed variation in the migration pattern of class II, III and class IV segments. The study, for the first time from the valley, detected 43.7 % of neonatal RVA positive cases from human and animal sharing similar setting. The variation in RNA migration pattern in seven cases signifies tentative cases of gene re-assortment that warrant further evaluation.

A begomovirus isolate (OY136A) collected from okra plants showing upward leaf curling, vein clearing, vein thickening and yellowing symptoms from Bangalore rural district, Karnataka, India was characterized. The sequence comparisons revealed that, this virus isolate share highest nucleotide identity with isolates of Cotton leaf curl Bangalore virus (CLCuBV) (AY705380) (92.8 %) and Okra enation leaf curl virus (81.1-86.2 %). This is well supported by phylogentic analysis showing, close clustering of the virus isolate with CLCuBV. With this data, based on the current taxonomic criteria for the genus Begomovirus, the present virus isolate is classified as a new strain of CLCuBV, for which CLCuBV-[India: Bangalore: okra: 2006] additional descriptor is proposed. The betasatellite (KC608158) associated with the virus is having more than 95 % sequence similarity with the cotton leaf curl betasatellites (CLCuB) available in the GenBank.The recombination analysis suggested, emergence of this new strain of okra infecting begomovirus might have been from the exchange of genetic material between BYVMV and CLCuMuV. The virus was successfully transmitted by whitefly and grafting. The host range of the virus was shown to be very narrow and limited to two species in the family Malvaceae, okra (Abelmoschus esculentus) and hollyhock (Althaea rosea), and four in the family Solanaceae.