A novel severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) causing COVID-19 pandemic in humans, recently emerged and has exported in more than 200 countries as a result of rapid ...spread. In this study, we have made an attempt to investigate the SARS-CoV-2 genome reported from 13 different countries, identification of mutations in major coronavirus proteins of these different SARS-CoV-2 genomes and compared with SARS-CoV. These thirteen complete genome sequences of SARS-CoV-2 showed high identity (>99%) to each other, while they shared 82% identity with SARS-CoV. Here, we performed a very systematic mutational analysis of SARS-CoV-2 genomes from different geographical locations, which enabled us to identify numerous unique features of this viral genome. This includes several important country-specific unique mutations in the major proteins of SARS-CoV-2 namely, replicase polyprotein, spike glycoprotein, envelope protein and nucleocapsid protein. Indian strain showed mutation in spike glycoprotein at R408I and in replicase polyprotein at I671T, P2144S and A2798V,. While the spike protein of Spain & South Korea carried F797C and S221W mutation, respectively. Likewise, several important country specific mutations were analyzed. The effect of mutations of these major proteins were also investigated using various in silico approaches. Main protease (Mpro), the therapeutic target protein of SARS with maximum reported inhibitors, was thoroughly investigated and the effect of mutation on the binding affinity and structural dynamics of Mpro was studied. It was found that the R60C mutation in Mpro affects the protein dynamics, thereby, affecting the binding of inhibitor within its active site. The implications of mutation on structural characteristics were determined. The information provided in this manuscript holds great potential in further scientific research towards the design of potential vaccine candidates/small molecular inhibitor against COVID19.
CRISPR/Cas9 provides a robust and widely adaptable system with enormous potential for genome editing directed towards generating useful products. It has been used extensively to generate resistance ...against viruses infecting plants with more effective and prolonged efficiency as compared with previous antiviral approaches, thus holding promise to alleviate crop losses. In this review, we have discussed the reports of CRISPR/Cas-based virus resistance strategies against plant viruses. These strategies include approaches targeting single or multiple genes (or non-coding region) in the viral genome and targeting host factors essential for virus propagation. In addition, the utilization of base editing has been discussed to generate transgene-free plants resistant to viruses. This review also compares the efficiencies of these approaches. Finally, we discuss combinatorial approaches, including multiplexing, to increase editing efficiency and bypass the generation of escape mutants.
Viral promoters can be used to drive heterologous gene expression in transgenic plants. As part of our quest to look for new promoters, we have explored, for the first time, the promoters of okra ...enation leaf curl virus (OELCuV), a begomovirus infecting okra (
Abelmoschus esculentus
). The Rep and CP promoters of OELCuV fused with the
gfp
reporter gene, were expressed transiently in the natural host okra and the laboratory host cotton and
Nicotiana benthamiana
. The expression levels of the promoters were quantified through confocal laser scanning microscopy and GFP assay in
N
.
benthamiana
and okra. The results indicated that the Rep promoter was more active than the CP promoter, whose activity was similar to that of CaMV 35S promoter. Additionally, the Rep and CP promoters showed increase of expression, probably due to transactivation, when assayed following inoculation of OELCuV and betasatellite DNAs in cotton plants. A moderate increase in promoter activity in
N
.
benthamiana
was also seen, when assayed following the inoculation of the heterologous begomovirus Sri Lankan cassava mosaic virus.
Cotton leaf curl disease (CLCuD), caused by begomoviruses in association with satellite molecules, is a major threat to cotton production causing enormous losses to cotton crop in most of the cotton ...growing countries including Indian subcontinent. In this study, isolates of begomovirus and satellite molecules associated with CLCuD were collected from North India (Haryana, New Delhi). They were amplified employing rolling circle replication mechanism, cloned, sequenced and, their phylogenetic and recombination analysis was performed.
The five Cotton leaf curl Multan virus (CLCuMuV) isolates investigated in this study showed monopartite organization of the genome typical of Old World begomoviruses. Nucleotide sequence analyses assigned them as the strains of CLCuMuV and were designated as CLCuMuV-SR13, CLCuMuV-SR14, CLCuMuV-ND14, CLCuMuV-ND15 and CLCuMuV-SR15. The genome of CLCuMuV-SR13 shared a highest level of nucleotide sequence identity (98%) with CLCuMuV (JN678804), CLCuMuV-SR14 and CLCuMuV-SR15 exhibited 96% with CLCuMuV (KM096471), while isolates CLCuMuV-ND15 and CLCuMuV-SR15 revealed 96% sequence identity with CLCuMuV (AY765253). The four betasatellite molecules investigated in this study shared 95-99% nucleotide sequence identity with Cotton leaf curl Multan betasatellite (CLCuMB) from India. The betasatellite molecules were designated as CLCuMB-SR13, CLCuMB-SR14, CLCuMB-ND14 and CLCuMB-ND15. Alphasatellite molecules in this study, designated as GLCuA-SR14, GLCuA-ND14 and GLCuA-SR15, revealed 98% identity with Guar leaf curl alphasatellite (GLCuA) reported from Pakistan.
The phylogenetic and recombination studies concluded that the isolates of CLCuMuV genomes undertaken in this study have a potential recombinant origin. Remarkably, significant recombination was detected in almost all the genes with contribution of Cotton leaf curl Kokhran Virus (CLCuKoV) in IR, V1, V2, C1, C4 and C5 regions and of CLCuMuV in C2 region of CLCuMuV-SR14. CLCuKoV also donated in C2, C3 regions of CLCuMuV-ND14; V1, V2, C2 and C3 regions of CLCuMuV-ND15 and C1 of CLCuMuV-SR15. Altogether, these observations signify the uniqueness in Indian CLCuMuV isolates showing contribution of CLCuKoV in all the genes. An interesting observation was frequent identification of GLCuA in CLCuD leaf samples.
Genistein is an isoflavanoid abundantly found in soy. It has been found to play an important role in the prevention of various chronic diseases including cancer. In this study, we evaluated potential ...therapeutic properties of Genistein against d-Galactosamine (d-GalN) induced inflammation and hepatotoxicity in male Wistar rats. Fulminant hepatic failure (FHF) was induced in rats by intraperitoneal injection of d-GalN (700mg/kgBW). Genistein (5mg/kgBW/day) was given as pre-treatment for 30days via intra-gastric route followed by d-GalN (700mg/kgBW) injection. The hepatoprotective and curative effects of Genistein were evident from a significant decrease in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels as well as prevention of histological damage by pre-treatment of Genistein. Genistein pre-treatment significantly inhibited the increased protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing nitric oxide (NO) and prostaglandin-E2 (PGE) levels, respectively. In addition Genistein significantly suppressed the production of d-GalN-induced proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. These inhibitory effects were associated with the suppression of nuclear factor-kappa B (NF-ĸB) activation, IKKα/β and Mitogen activated protein kinase (MAPK) phosphorylation by Genistein in d-GalN-treated animals. In conclusion, our results suggest that Genistein may serve as a potential supplement in the prevention of hepatic and inflammatory diseases. Furthermore Genistein is able to maintain the redox potential and strengthens the antioxidant defense system of a cell.
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•First study to evaluate hepatoprotective effect of Genistein against d-GalN•Genistein prevents oxidative damage induced by d-GalN.•Genistein blunts iNOS, COX-2, NF-ĸB, IKKα/β and MAPK expression.•Genistein prevents d-GalN induced apoptosis and necrosis.
The DNAs of the okra enation leaf curl virus (OELCuV) and bhendi yellow vein mosaic betasatellite (BYVMB) were cloned from okra plants displaying symptoms of yellow vein mosaic disease. The cloned ...DNAs were re-introduced onto okra and Nicotiana benthamiana plants, following which the okra plants developed symptoms of okra enation leaf curl disease, whereas the N. benthamiana plants displayed stunting and leaf curling.
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The enation leaf curl disease (ELCuD) is one of the several viral diseases affecting the cultivation of okra (Abelmoschus esculentus L.) in the Indian subcontinent. Several begomoviruses and satellites are associated with ELCuD. However, to date, there are no reports of the re-introduction of any cloned ELCuD-associated viral DNA back into okra to cause ELCuD-like symptoms. Okra enation leaf curl virus (OELCuV) and various satellites, which includes bhendi yellow vein mosaic beta-satellite (BYVMB) have earlier been reported to be associated with ELCuD and with other okra diseases such as bhendi yellow vein mosaic disease. In this report, it is shown that agrobacterium-mediated inoculation of a cloned DNA of OELCuV and BYVMB to the shoot apex of virus-free okra plants led to symptoms resembling ELCuD. The OELCuV and the BYVMB DNAs could be PCR- amplified from the symptomatic leaves of the agro-inoculated plants. Full-length OELCuV DNA could also be amplified from the same symptomatic leaves, part of whose DNA sequence matched with that of the DNA which was inoculated. Hence, this work is an important step towards the fulfilment of Koch’s postulates for ELCuD.
Chilli leaf curl disease (ChiLCD) is a serious problem and a major limitation to chilli (
Capsicum
spp.) cultivation in India. Leaves of a chilli plant showing leaf curl symptoms were collected from ...the Gonda district in Uttar Pradesh, India, in April, 2013. Full-length genomes of a begomovirus and an associated betasatellite were amplified, cloned and sequenced. The size of the begomovirus genome and the betasatellite were 2760 bp and 1374 bp, respectively. The nucleotide sequence of the begomovirus genome shared maximum identity (89 %) with pepper leaf curl Bangladesh virus-India isolate Chhapra (PepLCBV, JN663853), below the threshold for species demarcation. Sequence analysis showed that the begomovirus is a potential recombinant between viruses related to PepLCBV and chilli leaf curl virus (ChiLCV). The name chilli leaf curl Gonda virus (ChiLCGV) is being proposed. The betasatellite associated with ChiLCGV was identified as tomato leaf curl Bangladesh betasatellite (ToLCBDB). Agroinoculation of the viral genome along with betasatellite induced severe leaf curl symptoms in
Nicotiana benthamiana
. ChiLCGV and ToLCBDB characterized in this study represent a new begomovirus-betasatellite complex infecting
Capsicum
.
Cotton leaf curl Burewala virus (CLCuBuV), belonging to the genus Begomovirus, possesses single-stranded monopartite DNA genome. The bidirectional promoters representing Rep and coat protein (CP) ...genes of CLCuBuV were characterized and their efficacy was assayed. Rep and CP promoters of CLCuBuV and 35S promoter of Cauliflower mosaic virus (CaMV) were fused with β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS activity in individual plant cells driven by Rep, CP and 35S promoters was estimated using real-time PCR and fluorometric GUS assay. Histochemical staining of GUS in transformed tobacco (Nicotiana tabacum cv. Xanthi) leaves showed highest expression driven by Rep promoter followed by 35S promoter and CP promoter. The expression level of GUS driven by Rep promoter in transformed tobacco plants was shown to be two to four-fold higher than that of 35S promoter, while the expression by CP promoter was slightly lower. Further, the expression of GFP was monitored in agroinfiltrated leaves of N. benthamiana, N. tabacum and cotton (Gossypium hirsutum) plants using confocal laser scanning microscopy. Rep promoter showed strong consistent transient expression in tobacco and cotton leaves as compared to 35S promoter. The strong constitutive CLCuBuV Rep promoter developed in this study could be very useful for high level expression of transgenes in a wide variety of plant cells.
•First comprehensive report on virome analysis of Indian grapevine rootstocks.•The Study revealed the presence of nine important grapevine viruses and five viroids.•HTS analysis indicated the ...possible role of rootstocks in viruses and viroids dissemination.•Grapevine virus B was the highly prevalent virus in rootstocks and scions.•Recombinase polymerase amplification assay was adapted for GVB diagnosis.
Grapevine stocks are generally vegetatively propagated for the commercial cultivation. The propagation is predominantly done on rootstocks with >90 % of plant stocks propagated on Dogridge rootstock alone in India. Three additional rootstocks i.e. Paulsen, SO4 and Ramsey are also used for grapevine propagation on a limited scale. However, no information is available on the health status of these rootstocks being used by nurserymen and growers. Therefore, these rootstocks were collected from nurseries of five different locations and subjected to high-throughput sequencing (HTS) to determine the spectrum of viruses and viroids. The de novo assembly of unaligned reads revealed the presence of genome sequences of nine viruses viz. grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 (GLRaV-4), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus F (GVF), grapevine virus L (GVL), grapevine fleck virus (GFkV) and five viroids viz. hopstunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and GYSVd-2), Australian grapevine viroid (AGVd) and grapevine latent viroid (GLVd). The identified viruses and viroids were further confirmed through RT-PCR and Sanger sequencing. Indexing for the presence of these graft-transmissible pathogens in 43 rootstocks and 23 scion samples collected through field surveys indicated that infected rootstocks are the probable source for dissemination of these viruses and viroids in grapevine scions. As GVB was the most prevalent virus in all the rootstocks and scions as observed in RT-PCR testing, a simplified crude sap-based recombinase polymerase amplification (RPA) assay was optimized for its rapid detection. The present study highlights that grapevine rootstocks are reservoir of viruses and viroids and source for their dissemination in grapevine vineyards. Findings of the study including the optimized simplified cost-effective isothermal nucleic acid-based detection technique for GVB will be highly useful for production of certified clean rootstocks of grapevine in India.
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