Mixed infection of two mandariviruses identified by high-throughput sequencing in Kinnow mandarin and development of their specific detection using duplex RT-PCR.

In the current study, high-throughput sequencing (HTS) was used to identify viruses associated with the Kinnow mandarin (Citrus reticulata) plants exhibiting yellow vein clearing, mottling, and chlorosis symptoms at experimental farm of ICAR-Indian Agricultural Research Institute, New Delhi, India. During November 2022, leaf samples of symptomatic and asymptomatic Kinnow mandarin trees were collected, subjected to HTS and one of the representative symptomatic samples was subjected to leaf-dip electron microscopy (EM). In the EM results, flexuous virus particles typical of mandarivirus were observed. Ribosomal RNA was depleted from total RNA of pooled samples and RNA sequencing was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs, which were annotated through BLASTn using database of plant viruses/viroids reference genomes (NCBI). Results of assembled contigs revealed near-complete genomes of two mandariviruses, i.e., citrus yellow vein clearing virus (CYVCV) and citrus yellow mottle-associated virus (CiYMaV). The values of fragments per kilo base transcript length per million fragments mapped estimation indicated the dominance of CYVCV in HTS data and it was also confirmed through krona plot distribution of viruses in the pooled samples. A rapid and reliable duplex RT-PCR assay was also developed and standardized for the simultaneous detection of both CYVCV and CiYMaV in a pooled Kinnow mandarin sample. The developed duplex RT-PCR was then validated for the presence of these viruses in individual Kinnow mandarin samples. The specificity and sensitivity results confirmed that primers were highly specific to their targets and able to detect viruses up to 10-2 dilutions of RNA in standard and duplex RT-PCR. Therefore, the developed rapid duplex RT-PCR can be used for virus indexing and production of virus-free Kinnow mandarin plants for certification programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04011-9.

Transient expression analysis of promoters of okra enation leaf curl virus in Nicotiana benthamiana, cotton and okra plants.

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.

Progress Toward Poliomyelitis Eradication - Pakistan, January 2022-June 2023.

Since the establishment of the Global Polio Eradication Initiative in 1988, Pakistan remains one of only two countries (along with Afghanistan) with continued endemic transmission of wild poliovirus (WPV). This report describes Pakistan's progress toward polio eradication during January 2022-June 2023. During 2022, Pakistan reported 20 WPV type 1 (WPV1) cases, all of which occurred within a small geographic area encompassing three districts in south Khyber Pakhtunkhwa. As of June 23, only a single WPV1 case from Bannu district in Khyber Pakhtunkhwa province has been reported in 2023, compared with 13 cases during the same period in 2022. In addition, 11 WPV1 isolates have been reported from various environmental surveillance (ES) sewage sampling sites to date in 2023, including in Karachi, the capital of the southern province of Sindh. Substantial gaps remain in the quality of supplementary immunization activities (SIAs), especially in poliovirus reservoir areas. Despite the attenuation and apparently limited geographic scope of poliovirus circulation in Pakistan, the isolation of WPV1 from an ES site in Karachi is cause for concern about the actual geographic limits of transmission. Interrupting WPV1 transmission will require meticulous tracking and sustained innovative efforts to vaccinate children who are regularly missed during SIAs and rapidly responding to any new WPV1 isolations.

Progress Toward Poliomyelitis Eradication - Pakistan, January 2021-July 2022.

After reporting a single wild poliovirus (WPV) type 1 (WPV1) case in 2021, Pakistan reported 14 cases during April 1-July 31, 2022. Pakistan and Afghanistan are the only countries where endemic WPV transmission has never been interrupted (1). In its current 5-year strategic plan, the Global Polio Eradication Initiative (GPEI) has set a goal of interrupting all WPV1 transmission by the end of 2023 (1-3). The reemergence of WPV cases in Pakistan after 14 months with no case detection has uncovered transmission in southern Khyber Pakhtunkhwa province, the most historically challenging area. This report describes Pakistan's progress toward polio eradication during January 2021-July 2022 and updates previous reports (4,5). As of August 20, 2022, all but one of the 14 WPV1 cases in Pakistan during 2022 have been reported from North Waziristan district in Khyber Pakhtunkhwa. In underimmunized populations, excretion of vaccine virus can, during a period of 12-18 months, lead to reversion to neurovirulence, resulting in circulating vaccine-derived polioviruses (cVDPVs), which can cause paralysis and outbreaks. An outbreak of cVDPV type 2 (cVDPV2), which began in Pakistan in 2019, has been successfully contained; the last case occurred in April 2021 (1,6). Despite program improvements, 400,000-500,000 children continue to be missed during nationwide polio supplementary immunization activities (SIAs),* and recent isolation of poliovirus from sewage samples collected in other provinces suggests wider WPV1 circulation during the ongoing high transmission season. Although vaccination efforts have been recently complicated by months of flooding during the summer of 2022, to successfully interrupt WPV1 transmission in the core reservoirs in southern Khyber Pakhtunkhwa and reach the GPEI goal, emphasis should be placed on further improving microplanning and supervision of SIAs and on systematic tracking and vaccination of persistently missed children in these reservoir areas of Pakistan.

CRISPR/Cas-Mediated Resistance against Viruses in Plants.

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.

Agrobacterium-mediated co-inoculation of okra plants with cloned okra enation leaf curl virus DNA and bhendi yellow vein mosaic beta-satellite DNA furthers Koch's postulates for enation leaf curl disease.

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.

Progress Toward Poliomyelitis Eradication - Pakistan, January 2020-July 2021.

When the Global Polio Eradication Initiative began in 1988, wild poliovirus (WPV) transmission was occurring in 125 countries; currently, only WPV type 1 (WPV1) transmission continues, and as of August 2021, WPV1 transmission persists in only two countries (1,2). This report describes Pakistan's progress toward polio eradication during January 2020-July 2021 and updates previous reports (3,4). In 2020, Pakistan reported 84 WPV1 cases, a 43% reduction from 2019; as of August 25, 2021, Pakistan has reported one WPV1 case in 2021. Circulating vaccine-derived poliovirus (cVDPV) emerges as a result of attenuated oral poliovirus vaccine (OPV) virus regaining neurovirulence after prolonged circulation in underimmunized populations and can lead to paralysis. In 2019, 22 cases of cVDPV type 2 (cVDPV2) were reported in Pakistan, 135 cases were reported in 2020, and eight cases have been reported as of August 25, 2021. Because of the COVID-19 pandemic, planned supplementary immunization activities (SIAs)* were suspended during mid-March-June 2020 (3,5). Seven SIAs were implemented during July 2020-July 2021 without substantial decreases in SIA quality. Improving the quality of polio SIAs, vaccinating immigrants from Afghanistan, and implementing changes to enhance program accountability and performance would help the Pakistan polio program achieve its goal of interrupting WPV1 transmission by the end of 2022.

Comparative genome analysis of novel coronavirus (SARS-CoV-2) from different geographical locations and the effect of mutations on major target proteins: An in silico insight.

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.

Diversity and recombination analysis of Cotton leaf curl Multan virus: a highly emerging begomovirus in northern India.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Occurrence and variability of begomoviruses associated with bhendi yellow vein mosaic and okra enation leaf curl diseases in south-western India.

Bhendi yellow vein mosaic disease (BYVMD) and Okra enation leaf curl disease (OELCuD) are common diseases of okra/bhendi [Abelmoschus esculentus (L.) Moench] affecting both pod yield and quality in the Indian subcontinent. BYVMD is caused by the infection of a begomovirus and associated betasatellite. In this study, we have made an attempt to investigate the diversity of begomoviral and the satellite sequences in okra samples showing BYVMD and OELCuD, by using a rapid PCR-based approach on 46 samples collected from 23 locations of Southern and Western India. We have also analyzed nine RCA-generated full-length begomoviral clones, some generated from the above samples displaying BYVMD and some OELCuD. By the PCR approach, we find the presence of begomovirus okra enation leaf curl virus (OELCuV) in most samples, irrespective of the disease being displayed (BYVMD or OELCuD). The nine apparently full-length sequences also show high identities with OELCuV and show instances of both intra-specific as well as intra-strainal recombination. We have also analyzed the begomoviral sequences associated with BYVMD and OELCuD from publicly available nucleotide sequence databases and show much higher sequence diversity amongst BYVMV, as compared to OELCuV. This is the first study which comprehensively demonstrates the presence of OELCuV in okra samples showing BYVMD and those showing OELCuD.

Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus.

Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L-1 (BAP) and 0.5 mg L-1 (NAA) while 80% shoot percentage obtained with 4.0 mg L-1 (BAP) and 0.05 mg L-1 (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.

Characterization of a new begomovirus and betasatellite associated with chilli leaf curl disease in India.

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.

Functional characterization of a strong bi-directional constitutive plant promoter isolated from cotton leaf curl Burewala virus.

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 ß-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.

Update on polio eradication in the World Health Organization South-East Asia Region, 2013.

There has been a tremendous amount of progress toward polio eradication in the World Health Organization South-East Asia Region particularly over the past 4 years. In 1988, there were >25,000 reported cases of wild poliovirus infection in the South-East Asia Region, and because of substantial underreporting the estimated polio burden was probably 10-fold higher. Following the initiation of mass polio immunization campaigns in the mid-1990s and years of intense effort, the 11 countries of the South-East Asia Region reported no cases of wild poliovirus infection in 2012. With India reporting the last wild poliovirus case in the region, on 13 January 2011, and its subsequent removal from the list of polio-endemic countries, in February 2012, the South-East Asia Region is firmly on track for polio-free certification in early 2014.