High genetic diversity in the coat protein and 3′ untranslated regions among geographical isolates of<Emphasis Type="Italic">Cardamom mosaic virus</Emphasis> from south India

A survey was conducted to study the biological and genetic diversity ofCardamom mosaic virus (CdMV) that causes the most widespread disease in the cardamom growing area in the Western Ghats of south India. Six distinct subgroups were derived based on their symptomatology and host range from the sixty isolates collected. The serological variability between the virus isolates was analysed by ELISA and Western blotting. The 3′ terminal region consisting of the coat protein (CP) coding sequence and 3′ untranslated region (3′UTR) was cloned and sequenced from seven isolates. Sequence comparisons revealed considerable genetic diversity among the isolates in their CP and 3′UTR, making CdMV one of the highly variable members ofPotyviridae. The possible occurrence of recombination between the isolates and the movement of the virus in the cardamom tract of south India are discussed.     

Expression and immunological characterization of cardamom mosaic virus coat protein displaying HIV gp41 epitopes

The coat protein of cardamom mosaic virus (CdMV), a member of the genus Macluravirus, assembles into virus‐like particles when expressed in an Escherichia coli expression system. The N and C‐termini of the coat protein were engineered with the Kennedy peptide and the 2F5 and 4E10 epitopes of gp41 of HIV. The chimeric proteins reacted with sera from HIV positive persons and also stimulated secretion of cytokines by peripheral blood mononuclear cells from these persons. Thus, a system based on the coat protein of CdMV can be used to display HIV‐1 antigens.     

Occurrence of three distinct begomoviruses in cassava in Madagascar

The presence of East African cassava mosaic virus in association with cassava mosaic disease in Madagascar has previously been reported. We now describe virus isolates from mosaic‐affected Madagascan cassava with epitope profiles typical of African cassava mosaic virus, and an isolate with a nucleotide sequence similar to that of South African cassava mosaic virus. Thus, three distinct begomoviruses occur in cassava in Madagascar.     

The DNA-1 like alphasatellites in association with the bipartite Mungbean yellow mosaic virus in natural infections

Yellow mosaic disease is the major limitation in the production of grain legumes in India. This disease is caused by bipartite begomovirus, Mungbean yellow mosaic virus. In addition to the bipartite genomic components, the yellow mosaic disease affected urdbean plants which contain satellite like DNA-1 component called as alphasatellites. The present study has been attempted to characterise the alphasatellites associated with Mungbean yellow mosaic virus. Nucleotide sequence analysis of alphasatellites showed 98% identity with Vernonia yellow vein Fijian alphasatellite, VYVFA (JF733780). Since the sequence identity is more than 98%, the threshold value for demarcation of alphasatellites species, the alphasatellites of the present study are named as Vernonia yellow vein Fijian alphasatellite. Comparison with other, alphasatellites shared 51–55% identity with alphasatellites associated with monopartite begomovirus and it shared only 41–42% identity with an unusual alphasatellites, DNA-2. This is the first report on characterisation of alphasatellites associated with Mungbean yellow mosaic virus.     

Biological Characterization and Complete Genome Sequence of a Possible Strain of Indian cassava mosaic virus from Jatropha curcas in India

The outbreak of a severe mosaic disease with a significant incidence was noticed on Jatropha curcas plants growing in Lucknow, Northern India. The causal virus was successfully transmitted by whiteflies (Bemisia tabaci) and grafting from naturally infected to healthy J. curcas plants. The association of Begomovirus with the mosaic disease of J. curcas was detected by PCR using primers specific to DNA‐A of Begomoviruses. Further, full‐length DNA‐A genome of ～2.7 kb was amplified by RCA followed by digestion with Bam HI restriction enzyme. Cloning and sequencing of obtained amplicons resulted in 2740 nucleotides of complete DNA‐A consisting of six ORFs and IR region (GenBank Accession HM230683 ). The sequence analysis revealed highest 85% similarities with Jatropha curcas mosaic virus, 77–84% with Indian cassava mosaic virus and 73–76% with Sri Lankan cassava mosaic virus isolates. Phylogenetic analysis of the Begomovirus isolate also showed a clear‐cut distinct relationship with earlier reported Begomoviruses from Jatropha curcas and other Begomoviruses. On the basis of the guidelines of the International Committee on Taxonomy of Viruses (ICTV‐2008), our virus isolate was identified as a possible strain of Indian cassava mosaic virus, and its name Jatropha mosaic India virus (JMIV) is proposed.     

Expression,purification and molecular modeling of the NIa protease of Cardamom mosaic virus

The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8?M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.     

Detection of begomovirus associated with okra leaf curl disease

Leaf curl and yellow vein mosaic viral disease is the major constraint on okra (Abelmoschus esculentus L.) production in India. Amplified fragment sequence of DNA-β showed highest similarity of 91.7% with Bhendi yellow vein mosaic virus-Tamil Nadu (AJ308425, NC_003405) and lowest similarity of 48.5% with OKLCV (NC_004093), whereas coat protein specific amplified sequence showed highest homology with isolate of Madurai, Haryana, Ludhiana and lowest homology of 92% with Mesta yellow vein mosaic Bahraich virus (MYVMBV) (EU360303). The results obtained in the present study confirm that both the viral diseases of okra reported in southern India are caused by a begomovirus associated with DNA-β in which the plants show leaf curl symptoms and never develops yellow vein mosaic and those plants which show yellow vein mosaic, never develops leaf curl symptoms even in the same rows and field. The okra leaf curl is an emerging virus disease in India.     

Molecular diagnosis of apple virus and viroid pathogens from India

Apple is known to be susceptible to various virus and viroid pathogens. Symptomatic apple cultivars and rootstocks were collected and analyzed by ELISA and then through RT-PCR. The study reports the presence of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), the major apple viruses and Prunus necrotic ringspot virus (PNRSV), a minor apple virus, at the molecular level in India. Apple scar skin viroid (ASSVd) infection was also confirmed at the molecular level. Sporadic incidences of Tomato ringspot virus and Arabis mosaic virus infections were also detected by ELISA in nursery plants.     

Mixed infections of begomoviruses in pumpkins with yellow vein mosaic disease in north India

Yellow vein mosaic disease of pumpkin (Cucurbita moschata) is a serious problem for the cultivation of pumpkin throughout India. Symptomatic samples collected from Varanasi region of North India showed mixed infection of the three begomoviruses which could represent up to three species namely Tomato leaf curl New Delhi virus, Squash leaf curl China virus and Tomato leaf curl Palampur virus.     

Genetic diversity and population structure of Sorghum mosaic virus infecting Saccharum spp. hybrids

Sugarcane mosaic disease is widespread in many countries and has been identified to be caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV). Viral surveys of SCMV, SrMV and SCSMV were performed from 104 leaf samples of Saccharum spp. hybrid growing in China and two leaf samples in Myanmar. Sorghum mosaic virus was a major causal agent for sugarcane mosaic disease in China whereby 72.1% (75/104) of samples had SrMV infection alone, 6.7% (7/104) were mixed with SCMV and 17.3% (18/104) were mixed with SCSMV. Sugarcane streak mosaic virus infection alone occurred in 3.8% (4/104) of samples, but no single infections were observed for SCMV. Two viruses (SrMV and SCSMV) were detected in sugarcane mosaic samples in Myanmar. Phylogenetic analysis revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes based on the CP sequences (825 nucleotides) of 113 Chinese and 2 Burmese isolates from this study and 73 isolates reported worldwide. Six clearly distinct SrMV phylogroups (G1–G6) were formed and shared 74.3–94.1% nucleotide identity and 84.7–98.1% amino acid identity of CP sequences. SrMV‐G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces. The unique SrMV‐G6 phylogroup only occurred in Yunnan province. Insertion/deletion mutations, negative selection and frequent gene flow are factors driving the genetic evolution and population structure of SrMV in China.     

An Unusual Feature at the N-terminal end of the Coat Protein of Maize dwarf mosaic virus isolated in Hungary

Maize dwarf mosaic is the most widespread virus disease affecting corn production in Hungary. In attempts to identify the causal virus by test plant reactions, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), only Maize dwarf mosaic virus (MDMV) was detected. To further characterize Hungarian isolates of MDMV, one isolate from each of the sweet corn varieties Dallas, Royalty and GH23‐85 was selected for sequence analysis of its coat protein (CP) gene. The three Hungarian isolates shared CP amino acid sequence similarities of 95–98% not only with one another but also with MDMV isolates from other countries. However, the N‐terminus of the CP of the ‘Dallas’ isolate was unusual in containing a stretch of 13 additional amino acids. This is the first report of variation in the size of the N‐terminus of the MDMV CP.     

Molecular identification of three isolates of Jatropha mosaic India virus associated with mosaic disease of Withania somnifera in India

The association of begomovirus with mosaic disease of Withania somnifera was detected at three locations in India by PCR using begomovirus-specific degenerate primers. The resulting amplicons of ～1.2 kb from three locations (Aligarh, Lucknow and Hindaun City) were sequenced. The begomovirus isolates of three locations shared 96–97% identities among them and 91% identities with Jatropha mosaic India virus (JMIV). Based on highest sequence identities and close phylogenetic relationships with JMIV, these begomovirus isolates associated with mosaic disease of W. somnifera were identified as isolates of JMIV.     

Antisense RNA approach targeting Rep gene of Mungbean yellow mosaic India virus to develop resistance in soybean

The Yellow mosaic disease is caused by Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV) belonging to the genus Begomovirus of the family Geminiviridae. Yellow mosaic disease (YMD) is a major constraint to the production of soybean in South-East Asia. In India, yield losses of 10–88% had been reported due to YMD of soybean. An effort has been made to generate resistant soybean plants, by a construct targeting replication initiation protein (Rep) gene sequences of MYMIV. A construct containing the sequences of Rep gene (566?bp) in antisense orientation was used to transform cotyledonary node explants of three soybean cultivars (JS 335, JS 95-60 and NRC 37). Transformation efficiencies of 0.2, 0.21 and 0.24% were obtained with three soybean cultivars, JS 335, JS 95-60 and NRC 37, respectively. The presence of transgene in T₁ plants was confirmed by polymerase chain reaction (PCR) and sequence analysis. The level of resistance was observed by challenge inoculation with the virus in T₁ lines. The inheritance of transgene showed classical Mendelian pattern in six transgenic lines.     

First Report of a Croton yellow vein mosaic virus (CYVMV) Associated with Tomato Leaf Curl Disease in India

Leaf curl disease symptoms were observed in tomato crop grown in a tomato field at Matera district of Bahraich, India, in March 2013 with an 85% disease incidence. The infected plants exhibited leaf curl symptoms accompanied with puckering, vein swelling and stunting of the whole plant. PCR carried out with begomovirus coat protein gene and DNA beta‐specific primer sets resulted in positive amplification of ~775 bp and 1.35 kbp, respectively, with all symptom‐bearing plant samples. BLASTn and phylogenetic analyses of CP gene sequences showed highest and close relationship with Croton yellow vein mosaic virus (CYVMV) isolates, while the phylogenetic study of betasatellite sequence showed distinct relationships with other begomovirus associated betasatellites reported from India and abroad. This is a first report of a CYVMV associated with tomato leaf curl disease in India.     

Preparation and Characterization of a Monoclonal Antibody to Prunus necrotic ringspot virus

A cell line named PVRSV1D11 secreting monoclonal antibody (McAb) against the prokaryotically expressed coat protein (CP) of Prunus necrotic ringspot virus (PNRSV) was developed using hybridoma technology including animal immunization, cell fusion, cell line culture and enzyme‐linked immunosorbent assay (ELISA)‐based for screening. The specificity, titre and detection sensitivity of the McAb were determined by indirect ELISA to establish optimal conditions. The antibody reacted strongly with PNRSV and showed no cross‐reactions with the proteins of Plum pox virus, Prunus dwarf virus, Apple stem pitting virus, Apple stem grooving virus, Apple mosaic virus or Apple chlorotic leafspot virus. The ascites developed with PNRSV1D11 cell line showed high absorbance until it was diluted to over 6.6 × 10⁷ fold. The McAb belonged to IgG_2a isotype and was diluted by 1.28 × 10⁵ folds as an optimal detection concentration. The detection sensitivity of the monoclonal antibody was 11.7 ng/ml protein of PNRSV. The results indicated that the McAb against the CP of PNRSV is suitable for PNRSV detection in the plants and for monitoring the dynamics of the virus by using indirect ELISA.     

A new chrysanthemum potyvirus: molecular evidence

Abstract

A number of viruses are known to infect chrysanthemum plants, however in the present study a previously unknown potyvirus was detected using techniques such as ELISA, RT-PCR and hybridization. The ELISA-positive samples were amplified using a potyvirus group-specific primer which gave an amplification of ～850 bp. The amplified product was cloned and sequenced, and shows 72 – 73% homology with known potyviruses that infect chrysanthemums such as Potato virus Y potyvirus, Soyabean mosaic virus and Turnip mosaic potyvirus when compared to the sequence available in the database. However, present potyvirus isolates show 93% homology with Chilli veinal mottle virus and Pepper vein banding virus. The results were further confirmed by Northern hybridization. This is the first report of a potyvirus similar to Chilli veinal mottle virus, and Pepper vein banding virus infecting chrysanthemums.     

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.