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Papers by Ashish Warghane

Journal of Virological Methods, 2017

Tristeza is a devastating disease of citrus and reported to be present in almost all countries wh... more Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20 kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60 min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001 ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India. Citrus tristeza virus (CTV) is economically a very destructive pathogen infecting citrus and has killed more than 70 million citrus plants globally during last six decades (Bar-Joseph et al., 1989; Ahlawat, 2012). It is grouped in the genus Closterovirus with in the family Closteroviridae and is responsible to cause most important and damaging malady of citrus known as Tristeza (Bar-Joseph et al., 1989). The size of this filamentous virus approximately is 2000 nm in length and 11 nm in diameter (Bar-Joseph et al., 1989; Ayllon et al., 2001). The genome of CTV, ss (+) RNA of ∼20 kb size, organized into 12 ORFs which potentially encodes a minimum of 19 proteins (Rubio et al., 2001; Martın et al., 2009). Citrus being vegetatively propagated crop, the virus is disseminated in the nursery by budding, and grafting. However, a number of efficient aphid vectors spread the virus semi-persistently with in a citrus grove and these includes brown citrus aphids (Toxoptera

Phytopathology, Jan 11, 2015

Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting... more Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, non-cultured, Gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat copy number (TRN) into; Class I (TRN ≤5), Class II (TRN >5 ≤10), Class III (TRN >10 ≤15) and Class IV…

Phytoparasitica, 2014

ABSTRACT Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the... more ABSTRACT Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etio- logic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur iso- late formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.

Eurasian Journal of Medicine and Oncology, 2021

In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based ... more In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based on the Spike Protein Gene T he SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2), the causative agent of COVID-19 is found to be similar to Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV). This virus has led to infecting over 223 countries worldwide with over 133 million (133,552,774) confirmed cases and 2 million (2,894,295) confirmed deaths as of World Health Organisation (WHO) reports on 10 th April, 2021. [1] The mortality rate of SARS-CoV-2 lies between 1-35% and is similar to SARS-CoV and MERS-CoV during the year 2003 and 2012 respectively. [2] With a higher infectivity rate than its mortality rate, COVID-19 finds itself easily unfurling across six continents in the form of droplets, sneezing and cough from one individual to another. [3,4] The disease is primarily characterized by fever, sore throat, common cold, fatigue, lack of smell and taste. People having comorbidity such as heart disease, diabetes or chronic lung disease may further develop severe symptoms including pneumonia and acute respiratory distress syndrome. A few people also develop asymptomatic conditions of the disease. [5,6] Objectives: The Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has been the current global pandemic concern. With a high transmission rate, especially through direct contact, this disease spreads from person to person, and this has in turn led to a huge number of infections on a global scale. Methods: In present study, comparative genomic analysis was performed using 151 gene sequences of the viral spike protein retrieved from NCBI and along with its translated nucleotide sequences using MEGAX software. Variation in the nucleotide and amino acid positions were identified.

Molecular and Cellular Probes

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive... more Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The raised polyclonal antibodies detected CTV specifically and gave consistent results for CTV-positive and negative samples discrimination when compared with conventional RT-PCR. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016

A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (C... more A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) belong to Cluster A1 family of solute binding proteins (SBPs). The crystal structures in metal-free, intermediate and metal-bound states, in the previous study, revealed the unusual mechanism of metal binding and release for CLas-ZnuA2. Although CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance seems to be closer to Zn-specific SBPs of Cluster A-I family. The present study reports the binding affinity studies using SPR and CD and crystal structure of CLas-ZnuA2 in Zn(2+)-bound state. Despite a similar overall structure, there are noticeable differences at the metal binding site. The SPR and CD analysis confirmed our previous observation that CLas-ZnuA2 exhibits a low metal-binding affinity. The low metal-binding affinity of CLas-ZnuA2 could be attributed to the presence of a proline in linker helix resulting in relatively higher bending and rigidity of the same. This structural feature fixes the C-domain similar to metal-bound states of related SBPs. Further, the binding of both Mn(2+) and Zn(2+) occurs pentavalently with square pyramidal geometry not preferred by either. The site-specific positive Darwinian selection analysis showed that the proline in linker helix is under purifying selection and might have diverged long ago. Our structural and evolutionary analyses suggest that CLasZnua2 might have evolved, particularly for plant pathogens, to facilitate transport of both Mn(2+) and Zn(2+), with reversible binding to Zn(2+), unlike other Mn-binding SBPs (PsaA).

Journal of Plant Biochemistry and Biotechnology, 2015

Phytopathology, Jan 11, 2015

Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting... more Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, non-cultured, Gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat copy number (TRN) into; Class I (TRN ≤5), Class II (TRN >5 ≤10), Class III (TRN >10 ≤15) and Class IV (TRN>15). The study revealed that the Indian popu...

Available online at www.pacificjournals.com

Journal of Virological Methods, 2017

Tristeza is a devastating disease of citrus and reported to be present in almost all countries wh... more Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20 kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60 min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001 ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India. Citrus tristeza virus (CTV) is economically a very destructive pathogen infecting citrus and has killed more than 70 million citrus plants globally during last six decades (Bar-Joseph et al., 1989; Ahlawat, 2012). It is grouped in the genus Closterovirus with in the family Closteroviridae and is responsible to cause most important and damaging malady of citrus known as Tristeza (Bar-Joseph et al., 1989). The size of this filamentous virus approximately is 2000 nm in length and 11 nm in diameter (Bar-Joseph et al., 1989; Ayllon et al., 2001). The genome of CTV, ss (+) RNA of ∼20 kb size, organized into 12 ORFs which potentially encodes a minimum of 19 proteins (Rubio et al., 2001; Martın et al., 2009). Citrus being vegetatively propagated crop, the virus is disseminated in the nursery by budding, and grafting. However, a number of efficient aphid vectors spread the virus semi-persistently with in a citrus grove and these includes brown citrus aphids (Toxoptera

Phytopathology, Jan 11, 2015

Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting... more Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, non-cultured, Gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat copy number (TRN) into; Class I (TRN ≤5), Class II (TRN >5 ≤10), Class III (TRN >10 ≤15) and Class IV…

Phytoparasitica, 2014

ABSTRACT Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the... more ABSTRACT Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etio- logic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur iso- late formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.

Eurasian Journal of Medicine and Oncology, 2021

In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based ... more In Silico Characterisation of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) based on the Spike Protein Gene T he SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2), the causative agent of COVID-19 is found to be similar to Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV). This virus has led to infecting over 223 countries worldwide with over 133 million (133,552,774) confirmed cases and 2 million (2,894,295) confirmed deaths as of World Health Organisation (WHO) reports on 10 th April, 2021. [1] The mortality rate of SARS-CoV-2 lies between 1-35% and is similar to SARS-CoV and MERS-CoV during the year 2003 and 2012 respectively. [2] With a higher infectivity rate than its mortality rate, COVID-19 finds itself easily unfurling across six continents in the form of droplets, sneezing and cough from one individual to another. [3,4] The disease is primarily characterized by fever, sore throat, common cold, fatigue, lack of smell and taste. People having comorbidity such as heart disease, diabetes or chronic lung disease may further develop severe symptoms including pneumonia and acute respiratory distress syndrome. A few people also develop asymptomatic conditions of the disease. [5,6] Objectives: The Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has been the current global pandemic concern. With a high transmission rate, especially through direct contact, this disease spreads from person to person, and this has in turn led to a huge number of infections on a global scale. Methods: In present study, comparative genomic analysis was performed using 151 gene sequences of the viral spike protein retrieved from NCBI and along with its translated nucleotide sequences using MEGAX software. Variation in the nucleotide and amino acid positions were identified.

Molecular and Cellular Probes

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive... more Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The raised polyclonal antibodies detected CTV specifically and gave consistent results for CTV-positive and negative samples discrimination when compared with conventional RT-PCR. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2016

A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (C... more A periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) belong to Cluster A1 family of solute binding proteins (SBPs). The crystal structures in metal-free, intermediate and metal-bound states, in the previous study, revealed the unusual mechanism of metal binding and release for CLas-ZnuA2. Although CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance seems to be closer to Zn-specific SBPs of Cluster A-I family. The present study reports the binding affinity studies using SPR and CD and crystal structure of CLas-ZnuA2 in Zn(2+)-bound state. Despite a similar overall structure, there are noticeable differences at the metal binding site. The SPR and CD analysis confirmed our previous observation that CLas-ZnuA2 exhibits a low metal-binding affinity. The low metal-binding affinity of CLas-ZnuA2 could be attributed to the presence of a proline in linker helix resulting in relatively higher bending and rigidity of the same. This structural feature fixes the C-domain similar to metal-bound states of related SBPs. Further, the binding of both Mn(2+) and Zn(2+) occurs pentavalently with square pyramidal geometry not preferred by either. The site-specific positive Darwinian selection analysis showed that the proline in linker helix is under purifying selection and might have diverged long ago. Our structural and evolutionary analyses suggest that CLasZnua2 might have evolved, particularly for plant pathogens, to facilitate transport of both Mn(2+) and Zn(2+), with reversible binding to Zn(2+), unlike other Mn-binding SBPs (PsaA).

Journal of Plant Biochemistry and Biotechnology, 2015

Phytopathology, Jan 11, 2015

Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting... more Citrus Huanglongbing (HLB, Citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, non-cultured, Gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat copy number (TRN) into; Class I (TRN ≤5), Class II (TRN >5 ≤10), Class III (TRN >10 ≤15) and Class IV (TRN>15). The study revealed that the Indian popu...

Available online at www.pacificjournals.com