Expression of recombinant Apple chlorotic leaf spot virus coat protein in heterologous system: production and use in immunodiagnosis (original) (raw)
Related papers
Molecular characterization and diagnostic development for Apple chlorotic leaf spot virus
2011
Work done in the present thesis entitled "Molecular characterization and diagnostic development for Apple chlorotic leaf spot virus" includes studies on incidence of the viruses infecting pome and stone fruits, virus transmission, host range, purification, molecular characterization, diversity analysis, heterologous over expression of viral coat protein (CP) in E. coli and development of nucleic acid and ELISA based diagnostic systems. Horticultural crops play a unique role in India's economy by supplementing the income of the rural people. These crops form a significant part of total agricultural produce and have become key drivers of economic development in many of the states in the country. In the past one decade, the change in cropping pattern indicates a shift towards the fruit and commercial crops. In Himachal Pradesh (HP) apple is the major fruit accounting for more than 40% of total area under fruits and about 88% of total fruit production from the state. Commercially, apple is the most important of all the fresh fruits grown in HP, Jammu & Kashmir (J&K) and Uttarakhand. Any losses occurring would affect the economy and livelihood of some growers for whom apple is the only cash crop. Some of the economically important major viruses and viroids known to cause diseases in apple and other pome and stone fruits are Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV) and Apple scar skin viroid (ASSVd). During extensive surveys (2007-2009) of pome and stone fruit growing areas in HP and J&K, presence of typical virus like symptoms on these fruits indicated viral infection. Symptoms of leaf mosaic, severe chlorosis, deformation, curling etc were recorded in apple orchards. While plum, cherry, peach/nectarine, almond, cherry and apricots exhibited various symptoms like yellow flecking on leaves, mosaic, mottling, necrotic ring spots and shot holes. The symptoms were not persistent throughout the year, and disappeared late in the season making the plant seem healthy. Most of the orchards surveyed had mixed plantation of pome and stone fruits.
Molecular Detection of Latent Apple chlorotic leaf spot virus in Elite Mother Plants of Apple
Indian Journal of Virology, 2012
Apple chlorotic leaf spot virus (ACLSV; family Betaflexiviridae genus Trichovirus) is one of the economically important latent virus infecting apple (Malus 9 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.
HortScience, 1997
The purpose of this study was to adapt an ELISA test for diagnosing of “Apple Chlorotic Leaf Spot Virus” (CLSV) in apple trees. This work was carried out at Centro de Pesquisa Agropecuária de Clima Temperado–CPACT/EMBRAPA, Pelotas–RS, Brazil, during the 1996 spring season. The application of ADGEN Diagnostic Systems protocol does not give some positive results from diseased apple trees. The procedure modified by FLEGG & CLARK (1979) gives an unsatisfactory result for color reaction in the positive samples. It means it is necessary to adapt this methodology. When the antigen was obtained from leaves grown from the base to the intermediate position in the stem and grounded with extracting buffer—0.02 M, pH 7.4 (1 g tissue: 3 ml extracting buffer) and polyclonal antisera and antibody alkaline phosphatase conjugate was diluted in coating buffer—0.05 M, pH 9.6 (1 μg antisera or antibody: 500 μl coating buffer) the reaction become more intensive and the test was able to diagnosticate the...
Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein
Journal of Biosciences, 2010
The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91–100% and 70–98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.
Indian Phytopathology
Apple stem grooving virus (ASGV) belonging to the genus Capillovirus under family Betaflexiviridae is one of the widely distributed latent viruses mainly on pome fruit trees. It infects apple causing considerable economic losses that is a threat to the apple industry. To study the occurrence and incidence of ASGV disease, sensitive antisera based diagnostic tool has been developed, which would be helpful in large scale indexing, certification and quarantine programmes. Coat protein gene of ASGV was cloned into pET-32a(+) and pHIS-Parallel expression vectors. Expression characteristics of the proteins expressed from both the systems were compared and taken for raising the antisera. Sensitivity and specificity of the antiserum against virus infection was compared by Double Antibody Sandwich (DAS-ELISA). It was observed that the antisera raised from the protein expressed from pHIS-Parallel expression system (smaller ~ 3 kDa His tag) was more sensitive as compared to the antisera raised from the protein expressed from pET-32a(+) expression system (~ 18 kDa His tag) and the commercially available antisera. The antisera could also be successfully used for western blotting of infected samples and in DAS-ELISA. Thus, this study presents the first report on production of polyclonal antiserum against recombinant coat protein of ASGV from India, for reliable detection of the virus.
Molecular variability of Apple chlorotic leaf spot virus in different hosts and geographical regions
2004
A fragment of 500 nt corresponding to 85% of the coat protein (CP) gene of 35 isolates of Apple chlorotic leaf spot virus (ACLSV) from different hosts and geographical areas were sequenced and the results were compared with those already available. Sequence alignments at the amino acid level showed that most of the variability was present in the N-terminal part of the CP cistron (overlapping with the movement protein) whereas the C-terminus was significantly less divergent. Four isolates (APR-EA5, PE 150, PE 154 and PE 297) differed in showing high variability throughout the CP gene. Phylogenetic analysis at the nucleotide and amino acid level clustered the isolates in two groups: A, containing the great majority of the isolates, and B, containing the above four diverging isolates. A few subclusters could be identified in group A: pome fruit isolates clustered together in two different groups, one being close to the two almond isolates and one subcluster containing all the Spanish isolates. One subgroup was composed of three similar sequences each obtained from different hosts (peach, apricot, plum) originating from three different countries, respectively (Italy, Lebanon and Jordan). In Western blot analysis, three different migration rates were found for the CPs of ten representative isolates. No correlation was observed between the electrophoretic mobility of CPs and the phylogenetic groups, indicating that other factors besides the primary structure must account for the different electrophoretic mobilities observed.
HortScience: a publication of the American Society for Horticultural Science
Pear plants (Pyrus pyrifolia var. Hengshen) showing symptoms of chlorotic spots on leaves were observed in orchards in central Taiwan in 2004. The sap of diseased leaves reacted positively to Apple chlorotic leaf spot virus (ACLSV) antiserum. A purified virus isolate (LTS1) from pear was characterized by host range, electron microscopy, phylogenetic analyses, serological property and back-inoculation experiments to pear. Fifteen of 28 species of tested plants were susceptible to this virus after mechanical inoculation. Pathogenicity of ACLSV isolate LTS1 was verified by back-inoculating to pear seedlings. Filamentous virions of ca. 12×750 nm were observed in the preparations of purified virus. Virus particles accumulated in the cytoplasm were observed in the ultrathin sections of LTS1-infected pear leaf tissue. Sequence analyses of the coat protein (CP) gene of LTS1 and the CP gene of LTS2, which originated from a distinct symptomatic-pear sample, shared 81.4-92.6% nucleotide and 87...
Phytoparasitica, 2018
Majority of the apple trees are known to be infected by two latent viruses, Apple stem grooving virus (ASGV) and Apple stem pitting virus (ASPV). The importance of ASGV and ASPV is due to their non expression of symptoms, worldwide occurrence and wide host range on pome and stone fruits. Due to their latent nature in apple, early and rapid diagnostics plays important role for production of virus free quality planting material. The present investigation was conducted to detect and quantify ASPV & ASGV from different plant parts (spatial) in apple trees during different seasons (temporal) for optimisation of tissue and time for their rapid and early detection. Detection and relative quantification using immuno-molecular diagnostic techniques like, Double Antibody Sandwich-ELISA, Reverse Transcription-PCR and Real Time RT-PCR in various plant parts (leaf, whole flower, sepal, petal, anther, stigma with style, bark, fruit, seed and root) during different seasons was done. The DAS-ELISA based detection revealed infection in all plant parts except root and fruit with ASGV and ASPV, showing more expression in leaves followed by bark and whole flower. Similar results were also observed on RT-PCR based detection. Quantitative real time PCR analysis showed variation in expression of ASGV and ASPV in different parts during different seasons. Results confirmed that the ASGV and ASPV expression is higher in leaves followed by bark and whole flower. Periodic detection of these viruses in different plant parts during all the four seasons revealed varied virus titer from one season to another in the same plant. During all the seasons, both ASPV and ASGV were detected in bark in measurable titer using immunomolecular detection tools, however via DAS-ELISA, ASGV remained undetected during dormant season. Hence leaves and bark except leaf during fall, can be directly used as detection material for their early and rapid detection leading to production of virus free planting material.
Phytopathologia Mediterranea, 2006
Apple chlorotic leaf spot virus (ACLSV) isolates from various hosts and geographic locations in Turkey were molecularly characterized by RFLP, nucleotide sequence analysis and the construction of a phylogenetic tree including ACLSV isolates from GenBank. Based on nucleotide sequence alignment and the phylogenetic tree, we proposed a classification of ACLSV isolates in which isolates were divided into three major groups. The first group contained mainly Far-Eastern isolates, the second group the Hungarian (eastern-European) ACLSV isolates, and the third group, which contained isolates of variable characteristics, was again divided into two subgroups, subgroup I containing mixed European isolates, and subgroup II containing central European isolates. Three representative Turkish ACLSV isolates belonged to the third group; of these, one was from the mixed European cluster (subgroup I) and two from the central European cluster (subgroup II). The nucleotide sequence divergence and geogra...