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.

Development of Simplified Recombinase Polymerase Amplification Assay for Rapid and Robust Detection of Citrus Yellow Vein Clearing Virus.

Citrus is an economically important fruit crop, belongs to family Rutaceae, cultivated commercially in over 130 countries, which holds a leading profitable position in the international market. The most important citrus varieties are mandarins, oranges, lemons, sweet limes, grapefruits and pomelos. Citrus yellow vein clearing virus (CYVCV) is an important graft transmissible plant pathogen known to reduce productivity of citrus fruits due to its predominant association and widespread occurrence. Requirement of fast, reliable, efficient & economical CYVCV indexing assay is a prerequisite for production of healthy planting material. Currently, nucleic acid isolation and thermal cycler-based assay available for CYVCV indexing is a cumbersome lab intensive method. The present study was undertaken to develop and validate reverse transcription-recombinase polymerase amplification (RT-RPA) assay requiring no tedious RNA isolation, separate cDNA synthesis and costlier instrument like thermo-cycler. Optimized RT-RPA assay was able to amplify CYVCV up to 10-7 dilution (equivalent to 0.1 pg/µl) with the prepared templates of both RNA and crude saps and showed higher sensitivity in detection of CYVCV infection in field samples as compared to the conventional RT-PCR. Developed RT-RPA assay showed high specificity without any cross-reaction with other citrus pathogens (Indian citrus ringspot virus, citrus yellow mosaic virus, citrus tristeza virus, citrus exocortis viroid and huanglongbing). RT-RPA using crude leaf sap as template is quite simple, robust, highly sensitive, time and cost effective; therefore, it can be used in resource constrained laboratories as screening tool, for field surveys and on-site testing programs in farms, nurseries and biosecurity. Present study, first time reports the development, optimization and validation of crude sap-based RT-RPA assay for the detection of CYVCV infection in citrus plants namely; Kinnow mandarin, Mosambi and Grape fruit.

Development of Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807 bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31 kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

Development and application of crude sap-based recombinase polymerase amplification assay for the detection and occurrence of grapevine geminivirus A in Indian grapevine cultivars.

Geminiviruses are known to infect several fields and horticultural crops around the globe. Grapevine geminivirus A (GGVA) was reported in the United States in 2017, and since then, it has been reported in several countries. The complete genome recovered through high-throughput sequencing (HTS)-based virome analysis in Indian grapevine cultivars had all of the six open reading frames (ORFs) and a conserved nonanucleotide sequence 5'-TAATATTAC-3' similar to all other geminiviruses. Recombinase polymerase amplification (RPA), an isothermal amplification technique, was developed for the detection of GGVA in grapevine samples employing crude sap lysed in 0.5 M NaOH solution and compared with purified DNA/cDNA as a template. One of the key advantages of this assay is that it does not require any purification or isolation of the viral DNA and can be performed in a wide range of temperatures (18°C-46°C) and periods (10-40 min), which makes it a rapid and cost-effective method for the detection of GGVA in grapevine. The developed assay has a sensitivity up to 0.1 fg µl-1 using crude plant sap as a template and detected GGVA in several grapevine cultivars of a major grapevine-growing area. Because of its simplicity and rapidity, it can be replicated for other DNA viruses infecting grapevine and will be a very useful technique for certification and surveillance in different grapevine-growing regions of the country.

Fabrication and Evaluation of Basil Essential Oil-Loaded Halloysite Nanotubes in Chitosan Nanocomposite Film and Its Application in Food Packaging.

Increasing health concerns regarding the use of plasticware have led to the development of ecofriendly biodegradable packaging film from natural polymer and food additives. In the present study, basil essential oil (BEO) loaded halloysite nanotubes (HNTs) composite films were synthesized using a solution casting method. The effects of BEO and nanotube concentration on the mechanical, physical, structural, barrier, and antioxidant properties of films were evaluated. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) demonstrated well-dispersed HNTs and BEO in tailored composite films. The addition of BEO in Chitosan (Ch) film caused darkening of the film color; furthermore, the incorporation of HNTs in varied concentrations increased opaqueness in Ch/BEO film. The Ch/BEO film, upon adding HNTs 5-30 wt%, exhibited a corresponding increase in the film thickness (0.108-0.135 mm) when compared with the Ch/BEO film alone (0.081 mm). The BEO-loaded HNTs composite films displayed reduced moisture content and characteristic barrier and UV properties. The Ch/BEO film with 15 wt% HNTs was found to have enhanced antioxidant activity. The Ch/BEO/HNTs composite also managed to prevent broccoli florets from losing weight and firmness during storage. The enhanced barrier and antioxidant qualities of the nanocomposite film suggest its potential application in the food processing and packaging sector. This is the first ever report on the fabrication of nanocomposite film using BEO and HNTs for food packaging. The low production cost and ecofriendly approach make the film acceptable for further research and commercialization thereafter.

Chasing COVID-19 through SARS-CoV-2 spike glycoprotein.

An ongoing pandemic Coronavirus disease (COVID-19), caused by a newly emerged Coronavirus, SARS-CoV-2 has affected millions of people globally. One of the most crucial structural proteins of SARS-CoV-2 is the Spike glycoprotein (S-glycoprotein), for which the first de novo modelling was envisaged by our group in early 2020, and was superimposed to its predecessor SARS-CoV S-glycoprotein, to determine structural divergence, glycosylation and antigenic variation between SARS-CoV-2 and SARS-CoV. S-glycoprotein is involved in binding with the cellular receptor, membrane fusion, internalization via angiotensin-converting enzyme 2 (ACE2) receptor, and tissue tropism. Upon internalization into the target host cells, the viral genome encodes two precursor polypeptides which get processed into 16 mature nonstructural proteins that play a crucial role in replication and transcription of SARS-CoV-2. Currently S-glycoprotein is one of the most vital targets for vaccine and therapeutics development for COVID-19.

Recombinant vaccines for COVID-19.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation. Considering the crucial role of SARS-CoV-2 spike (S) glycoprotein in virus attachment, entry, and induction of neutralizing antibodies, S protein is being widely used as a target for vaccine development. Based on advances in techniques for vaccine design, inactivated, live-vectored, nucleic acid, and recombinant COVID-19 vaccines are being developed and tested for their efficacy. Phase3 clinical trials are underway or will soon begin for several of these vaccines. Assuming that clinical efficacy is shown for one or more vaccines, safety is a major aspect to be considered before deploying such vaccines to the public. The current review focuses on the recent advances in recombinant COVID-19 vaccine research and development and associated issues.

"Rapid diagnosis of Cucumber mosaic virus in banana plants using a fluorescence-based real-time isothermal reverse transcription-recombinase polymerase amplification assay".

Cucumber mosaic virus (CMV) is a widespread plant virus infecting important vegetables, plantation and flower crops. Currently, CMV is detected by enzyme-linked immunosorbent assays (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. ELISA requires polyclonal antibodies and is time-consuming. PCR requires skilled manpower and complex procedures of RNA isolation as well as a thermal cycler. To overcome these difficulties, a portable rapid, simple and visual fluorescence-based reverse transcription-recombinase polymerase amplification (portable RT-exo-RPA) assay for the detection of CMV was developed. A specific primer pair of 30-33 bp targeting a conserved region of the coat protein (CP) gene of CMV and a probe to function in the RT-exo-RPA assays were designed and synthesized. A total of 62 symptomatic as well as 58 asymptomatic banana plant samples, collected from banana orchards located in Jalgaon, Maharashtra, India, were evaluated for CMV infections using crude leaf extracts as templates by a reverse transcription-recombinase polymerase amplification (RT-RPA) assay as well as a real-time RT-exo-RPA assay and the results were compared with those of a reverse transcription-polymerase chain reaction (RT-PCR) assay using purified total plant RNAs as templates. CMV was as efficiently detected using the crude leaf extract template in the RT-RPA and real-time RT-exo-RPA assays as using the purified RNA template in the RT-PCR assay. To dispense with the use of real-time PCR, a portable RT-exo-RPA assay was developed and the alternative methods for the visualization of CMV detection using either a fluorometer or direct viewing with a UV transilluminator were evaluated. To our knowledge, this is the first report of the rapid and reliable diagnosis of CMV infections by a real-time RT-exo-RPA assay using a crude leaf extract as template.

Development of a recombinase polymerase amplification assay for the diagnosis of banana bunchy top virus in different banana cultivars.

Recombinase polymerase amplification (RPA) is a rapid, isothermal amplification method with high specificity and sensitivity. In this study, an assay was developed and evaluated for the detection of banana bunchy top virus (BBTV) in infected banana plants. Three oligonucleotide primer pairs were designed from the replicase initiator protein gene sequences of BBTV to function both in RPA as well as in polymerase chain reaction (PCR). A total of 133 symptomatic as well as asymptomatic banana leaf samples from various cultivars were collected from the different regions of India and evaluated for BBTV infection using the RPA assay. BBTV was efficiently detected using crude leaf sap in RPA and the results obtained were consistent with PCR-based detection using purified DNA as template. To our knowledge, this is the first report of reliable diagnosis of BBTV infection by RPA using crude leaf sap as a template.

Green synthesis and structural characterization of selenium nanoparticles and assessment of their antimicrobial property.

In the present study, selenium nanoparticles were biologically synthesized by non-pathogenic, economic and easy to handle bacterium Ralstonia eutropha. The selenium oxo anion was reduced to selenium nanoparticles in the presence of the bacterium. The bacterium was grown aerobically in the reaction mixture. An extracellular, stable, uniform, spherical selenium nanoparticle was biosynthesized. The TEM analysis revealed that the biosynthesized selenium nanoparticles were spherical in shape with size range of 40-120 nm. XRD and SAED analysis showed that nanocrystalline selenium of pure hexagonal phase was synthesized. The formation of actinomorphic trigonal selenium nanorods was also observed. A mechanism of biosynthesis of selenium nanoparticles by R. eutropha was proposed. The biosynthesized selenium nanoparticles were investigated for their antimicrobial activity against potential pathogens. Selenium nanoparticles showed excellent antimicrobial activity. The 100, 100, 250 and 100 µg/ml selenium nanoparticles were found to inhibit 99 % growth of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Streptococcus pyogenes, respectively. Similarly, the 500 µg/ml of selenium nanoparticles was found to inhibit the growth of pathogenic fungi Aspergillus clavatus. The antimicrobial efficacy of selenium nanoparticle was comparable with commercially available antibiotic drug Ampicillin.

Detection, eye-hand coordination and virtual mobility performance in simulated vision for a cortical visual prosthesis device.

In order to assess visual performance using a future cortical prosthesis device, the ability of normally sighted and low vision subjects to adapt to a dotted 'phosphene' image was studied. Similar studies have been conduced in the past and adaptation to phosphene maps has been shown but the phosphene maps used have been square or hexagonal in pattern. The phosphene map implemented for this testing is what is expected from a cortical implantation of the arrays of intracortical electrodes, generating multiple phosphenes. The dotted image created depends upon the surgical location of electrodes decided for implantation and the expected cortical response. The subjects under tests were required to perform tasks requiring visual inspection, eye-hand coordination and way finding. The subjects did not have any tactile feedback and the visual information provided was live dotted images captured by a camera on a head-mounted low vision enhancing system and processed through a filter generating images similar to the images we expect the blind persons to perceive. The images were locked to the subject's gaze by means of video-based pupil tracking. In the detection and visual inspection task, the subject scanned a modified checkerboard and counted the number of square white fields on a square checkerboard, in the eye-hand coordination task, the subject placed black checkers on the white fields of the checkerboard, and in the way-finding task, the subjects maneuvered themselves through a virtual maze using a game controller. The accuracy and the time to complete the task were used as the measured outcome. As per the surgical studies by this research group, it might be possible to implant up to 650 electrodes; hence, 650 dots were used to create images and performance studied under 0% dropout (650 dots), 25% dropout (488 dots) and 50% dropout (325 dots) conditions. It was observed that all the subjects under test were able to learn the given tasks and showed improvement in performance with practice even with a dropout condition of 50% (325 dots). Hence, if a cortical prosthesis is implanted in human subjects, they might be able to perform similar tasks and with practice should be able to adapt to dotted images even with a low resolution of 325 dots of phosphene.

Environmental impact of coal washery and Damodar River water on the morphometry and biochemical changes of maize (Zea mays L.) and soil health.

Enormous quantity of water is used for coal beneficiation and accordingly huge amount of effluents are being generated. In this study an attempt was made to evaluate the potential of this effluent water for irrigation. Water samples were collected from three different points (a) feeding point, (b) thickening point, and (c) outlet point of coal washery, and from Damodar River for monitoring the water quality. The samples were analyzed for various parameters and compared with prescribed standard, which revealed that the total suspended solids of thickening point and Damodar River were higher. A pot experiment with maize was conducted to study the suitability of this coal washery water for irrigation. Pots were irrigated with water from the three points of washery and Damodar River in two concentrations (100% and 50% dilution with distilled water); pure distilled water was used for control. There was 100% germination in all the treatments. The plant growth, chlorophyll content and soil quality parameters were significantly better in washery and Damodar River water treated pots. The Damodar River water and washery water from feeding and outlet point could be successfully used for irrigation. In general mixing with good quality water has shown better results.

Management of lignite fly ash for improving soil fertility and crop productivity.

Lignite fly ash (LFA), being alkaline and endowed with excellent pozzolanic properties, a silt loam texture, and plant nutrients, has the potential to improve soil quality and productivity. Long-term field trials with groundnut, maize, and sun hemp were carried out to study the effect of LFA on growth and yield. Before crop I was sown, LFA was applied at various doses with and without press mud (an organic waste from the sugar industry, used as an amendment and source of nutrients). LFA with and without press mud was also applied before crops III and V were cultivated. Chemical fertilizer, along with gypsum, humic acid, and biofertilizer, was applied in all treatments, including the control. With one-time and repeat applications of LFA (with and without press mud), yield increased significantly (7.0-89.0%) in relation to the control crop. The press mud enhanced the yield (3.0-15.0%) with different LFA applications. The highest yield LFA dose was 200 t/ha for one-time and repeat applications, the maximum yield being with crop III (combination treatment). One-time and repeat application of LFA (alone and in combination with press mud) improved soil quality and the nutrient content of the produce. The highest dose of LFA (200 t/ha) with and without press mud showed the best residual effects (eco-friendly increases in the yield of succeeding crops). Some increase in trace- and heavy-metal contents and in the level of gamma-emitters in soil and crop produce, but well within permissible limits, was observed. Thus, LFA can be used on a large scale to boost soil fertility and productivity with no adverse effects on the soil or crops, which may solve the problem of bulk disposal of fly ash in an eco-friendly manner.