Probing of plant transcriptomes reveals the hidden genetic diversity of the family Secoviridae.

Secoviruses are single-stranded RNA viruses that infect plants. In the present study, we identified 61 putative novel secoviral genomes in various plant species by mining publicly available plant transcriptome data. These viral sequences represent the genomes of 13 monopartite and 48 bipartite secovirids. The genome sequences of 52 secovirids were coding-complete, and nine were partial. Except for small open reading frames (ORFs) determined in waikaviral genomes and RNA2 of torradoviruses, all of the recovered genomes/genome segments contained a large ORF encoding a polyprotein. Based on genome organization and phylogeny, all but three of the novel secoviruses were assigned to different genera. The genome organization of two identified waika-like viruses resembled that of the recently identified waika-like virus Triticum aestivum secovirus. Phylogenetic analysis revealed a pattern of host-virus co-evolution in a few waika- and waika-like viruses and increased phylogenetic diversity of nepoviruses. The study provides a basis for further investigation of the biological properties of these novel secoviruses.

Identification of a novel monopartite begomovirus associated with leaf curl disease of Citharexylum spinosum in India.

The present study reports the complete genome of a novel monopartite begomovirus, named tentatively as "Citharexylum leaf curl virus" (CitLCuV), associated with leaf curl disease of Citharexylum spinosum in India. CitLCuV genome (2767 nucleotide) contained the typical genome organization of Old World begomoviruses and shared the maximum nucleotide sequence identity of 89.7% with a papaya leaf crumple virus (PaLCrV) isolate. In addition, two small non-canonical open reading frames (C5 and C6) were determined in the complementary strand of CitLCuV genome. Phylogenetic analysis revealed the relatedness of CitLCuV to PaLCrV and rose leaf curl virus. Recombination analysis detected a possible recombination event in CitLCuV genome. Based on begomovirus species demarcation criteria, CitLCuV can be regarded as a novel begomoviral species.

Identification of a putative novel cholivirus in the transcriptome of Gymnema sylvestre R. Br.

Gymnema sylvestre is a tropical climber species that is widely used in traditional medicine since ages. In the present study, the transcriptome datasets of G. sylvestre available in public domain were screened for the presence of novel plant viral sequences and a putative novel virus tentatively named as Gymnema sylvestre virus 1 (GysV1) was identified. Coding-complete genome segments of GysV1 that are 6.35 kb (RNA1) and 3.98 kb (RNA2) long possessed a single large open reading frame coding for a polyprotein. BLASTp, sequence identity and phylogenetic analyses revealed the relatedness of GysV1 to the members of the subgenus Cholivirus (genus Sadwavirus; family Secoviridae; order Picornavirales). Based on the species demarcation criteria of the family Secoviridae, GysV1 can be regarded as a new cholivirus member.

Identification of two putative novel deltapartitiviruses and an enamovirus in coriander transcriptomes.

Coriander is a herbaceous spice and condiment crop also known for its medicinal properties. The present study identified two putative novel deltapartitiviruses and an enamovirus tentatively named as Coriandrum sativum deltapartitivirus 1, 2 (CsDPV1, 2) and Coriandrum sativum enamovirus (CsEV) in the publicly available transcriptome-assembled contigs derived from coriander grown in India. CsDPV1 and 2 contained tripartite and bipartite genomes, respectively, with each genome segment encoding a single open reading frame (ORF). CsEV contained five ORFs encoding proteins P0, P1, P2, P3 and P5. Phylogenetic analysis revealed three distinct subgroups of deltapartitiviruses wherein CsDPV1 and 2 grouped in subgroup 3 and 1, respectively, whilst CsEV formed a distinct sub-clade within enamoviruses. Further, the presence of CsDPV2 in fruit samples of one of the cultivars from where the virus was identified was confirmed through RT-PCR assay and Sanger sequencing. The study highlights the need for further studies on understanding the importance and the biological properties of identified novel viruses.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Exploration of plant transcriptomes reveals five putative novel poleroviruses and an enamovirus.

Transcriptome datasets available in public domain serve as valuable resource for identification and characterization of novel viral genomes. Poleroviruses are economically important plant-infecting RNA viruses belonging to the family Solemoviridae. In the present study, we explored the plant transcriptomes available in public domain and identified five putative novel poleroviruses tentatively named as Foeniculum vulgare polerovirus (FvPV), Kalanchoe marnieriana polerovirus (KmPV), Paspalum notatum polerovirus (PnPV), Piper methysticum polerovirus (PmPV), Trachyspermum ammi polerovirus (TaPV) and a novel enamovirus named as Celmisia lyallii enamovirus (ClEV) in Foeniculum vulgare, Kalanchoe marnieriana, Paspalum notatum, Piper methysticum, Trachyspermum ammi and Celmisia lyallii, respectively. Coding-complete genomes (5.56-5.74 kb) of CIEV, KmPV, PnPV, PmPV and TaPV were recovered while only the partial genome of FvPV could be recovered. The genome organization of identified viruses except ClEV is 5'-ORF0-ORF1-ORF2-ORF3a-ORF3-ORF4-ORF5-3' while that of ClEV is 5'-ORF0-ORF1-ORF2-ORF3-ORF5-3'. Phylogenetic analysis revealed that poleroviruses of apiaceous plants formed a monophyletic clade within the genus Polerovirus.

Analysis of public domain plant transcriptomes expands the phylogenetic diversity of the family Secoviridae.

Secoviruses are mono-/bipartite plant-infecting, icosahedral RNA viruses that incite economically important diseases in plants. In the present study, nine secoviruses tentatively named as Ananas comosus secovirus (AcSV), Artocarpus altilis secovirus (AaSV), Boehmeria nivea secovirus (BnSV), Gynostemma pentaphyllum secovirus (GpSV), Orobanche cernua secovirus (OcSV), Paris polyphylla secovirus 1 (PpSV1), Paris polyphylla secovirus 2 (PpSV2), Rhododendron delavayi secovirus (RdSV), and Yucca gloriosa secovirus (YgSV) were identified by probing publicly available transcriptomes of eight plant species. Coding-complete genome/genome segments of all the identified viruses encoding a polyprotein were recovered. Two of the nine identified viruses-AcSV and GpSV were discovered in few of the small RNA libraries of respective plant species. Putative cleavage sites were predicted in polyproteins encoded by AcSV, GpSV, PpSV2 and YgSV genome segments. Phylogenetic and sequence identity analyses revealed that AcSV, GpSV and YgSV, PpSV1 and RdSV putatively belong to the genera- Sadwavirus (sub genus: Cholivirus), Fabavirus, Nepovirus and Waikavirus, respectively, while AaSV, BnSV, and PpSV2 may represent a distinct group of viruses within the family Secoviridae as they could not conclusively be assigned to a single genus.

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica).

The major viruses infecting apple cultivars throughout the world including India are apple mosaic virus (ApMV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), apple chlorotic leaf spot virus (ACLSV), and recently, a new virus, apple necrotic mosaic virus (ApNMV), was reported from mosaic-infected apple cultivars in India. The aim of this study was to detect the ApNMV virus along with the other three viruses (ApMV, ASPV and ASGV) simultaneously by multiplex RT-PCR. Four primer-pair-produced amplicons of 670, 550, 350 and 210 bp corresponding to ApNMV, ApMV, ASPV and ASGV, respectively, were found to be specific for these viruses when tested individually. The annealing temperature (55°C), primer concentration (0·8 µl) and other components of the master mix were standardized for the development of one-step m-RT-PCR assay. The m-RT-PCR protocol developed was further validated with 30 samples from seven symptomatic or asymptomatic apple cultivars, which revealed the presence of more than one virus in these cultivars. Most of the viruses were found to be present either alone or in mixed infection; however, ASPV was more common in tested cultivars. An easy, cost-effective and rapid multiplex RT-RCR protocol was developed to detect the four viruses, which infect apple plants either in individually or together in the field. This assay will help in the surveying and indexing of apple germplasm and the distribution of all four viruses in the apple growing regions of India.

Mining of the water hyssop (Bacopa monnieri) transcriptome reveals genome sequences of two putative novel rhabdoviruses and a solendovirus.

The genomes of three putative novel viruses, tentatively named "Bacopa monnieri virus 1" (BmV1), "Bacopa monnieri virus 2" (BmV2), and "Bacopa monnieri virus 3" (BmV3) were identified in the transcriptome dataset of a medicinally important herb - water hyssop (Bacopa monnieri (L.) Wettst.). The BmV1 and BmV2 genomes resemble those of plant rhabdoviruses. The 13.3-kb-long BmV1 genome contains eight antisense ORFs in the order 3' l-N-P2'-P-P3-M-G-P6-L-t 5', with P2' ORF overlapping with P, while the 13.2-kb BmV2 genome contains six interspersed ORFs in the antisense orientation (3' l-N-P-P3-M-G-L-t 5'). The 8-kb BmV3 genome possesses five overlapping ORFs, with ORFs 2 to 5 being similar to those of solendoviruses. Based on genome organization, sequence similarity, and phylogeny, BmV1, BmV2, and BmV3 can be regarded as new members of the genera Cytorhabdovirus, Betanucleorhabdovirus, and Solendovirus, respectively.

First complete genome sequence of garlic virus X infecting Allium sativum- G282 from India.

The present report communicates the first full genome sequencing of the Garlic virus X from northern India. The total genome size of Garlic virus X (MK503771) reported in this study is 8458 bp ssRNA. The full genome sequence analysis showed the close relationship of Garlic virus X from India to that of from China, Korea, Australia and Spain. The full genome sequence based study of Indian Garlic virus X reveals the geographical relationship of this virus in India and global origin which may assists in development of control strategy for this virus.

Complete genome sequence and genetic organization of a Garlic virus D infecting garlic (Allium sativum) from northern India.

The present paper describes first full genome sequence of the Garlic virus D (GarV-D) from northern India with a genome size of 8425 bp long ssRNA. The infected leaves and bulbs of garlic variety Yamuna Safed (G-282) plants suspected for GarV-D infection were collected with the aim to identify contagion virus during March, 2018. The total RNA was extracted from the pooled garlic plants using TRIzol reagent and sequenced using an Illumina HiSeq 2000 platform. BLASTn search in the NCBI database identified contagion as GarV-D (MK518067). It shared 83.63-85.83% nucleotide sequence identities with other (GarV-D) isolates from Argentina (KF550407, KF555653, KR819505) and 83.15% with isolates from China (MF795136, MF363012). Keywords: Allium sativum; Allexivirus; Garlic virus D; India.

Changes in salivary output after induction at high-altitude areas and its effects on dental caries among Indian Army troops.

BACKGROUND: This study was aimed to evaluate the changes in salivary output and its effect on dental caries among Indian troops after 6 months of stay at high-altitude area (HAA). METHODS: All troops undergo mandatory dental checkup during acclimatization phase before deployment at HAA. Two thousand troops who fulfilled inclusion and exclusion criteria were selected, and consent for the study was obtained. Stimulated and unstimulated salivary samples were collected, the decayed, missing, and filled teeth (DMFT) index was evaluated, and required dental treatment was completed. The same salivary samples were collected after 6 months (on deinduction) to evaluate the salivary output. The DMFT index was re-evaluated to check the initiation of caries. RESULTS: The mean values of unstimulated and stimulated saliva at the initial visit were 4.105 and 17.03 gm, respectively, whereas the mean values of unstimulated and stimulated saliva after 180 days were 3.034 and 15.831 gm, respectively. Salivary flow was found to be significantly decreased after 6 months both in unstimulated and stimulated saliva. The mean DMFT at the time of induction of the study was 6.18 ± 3.03, and on deinduction, it was 7.22 ± 3.45 with p < 0.001, which was highly statistically significant. CONCLUSION: A decrease in body fluids and changed sympathetic and parasympathetic at HAA lead to decreased salivary secretions. Low water intake, high carbohydrate solid diet, negligible supply of fresh food, difficulty in maintenance of oral hygiene, and overall stress due to loneliness are all contributory factors for an increase in dental caries. It clearly demonstrates that prolonged stay at HAA affects salivary volume outflow, both stimulated and unstimulated, which has a corresponding effect on new caries.

An isothermal based recombinase polymerase amplification assay for rapid, sensitive and robust indexing of citrus yellow mosaic virus.

Management of viral diseases relies on definite and sensitive detection methods. Citrus yellow mosaic virus (CYMV), a double stranded DNA virus of the genus Badnavirus, causes yellow mosaic disease in citrus plants. CYMV is transmitted through budwood and requires a robust and simplified indexing protocol for budwood certification programme. The present study reports development and standardization of an isothermal based recombinase polymerase amplification (RPA) assay for a sensitive, rapid, easy, and cost-effective method for detection and diagnosis of CYMV. Two different oligonucleotide primer sets were designed from ORF III (coding for polyprotein) and ORF II (coding for virion associated protein) regions of CYMV to perform amplification assays. Comparative evaluation of RPA, PCR and immuno-capture recombinase polymerase amplification (IC-RPA) based assays were done using purified DNA and plant crude sap. CYMV infection was efficiently detected from the crude sap in RPA and IC-RPA assays. The primer set used in RPA was specific and did not show any cross-amplification with banana streak MY virus (BSMYV), another Badnavirus species. The results from the present study indicated that RPA assay can be used easily in routine indexing of citrus planting material. To the best of our knowledge, this is the first report on development of a rapid and simplified isothermal detection assay for CYMV and can be utilized as an effective technique in quarantine and budwood certification process.

Simultaneous Detection of Brown Rot- and Soft Rot-Causing Bacterial Pathogens from Potato Tubers Through Multiplex PCR.

Ralstonia solanacearum (Smith) Yabuuchi et al. and Erwinia carotovora subsp. carotovora (Jones) Bergey et al. (Pectobacterium carotovorum subsp. carotovorum) are the two major bacterial pathogens of potato causing brown rot (wilt) and soft rot diseases, respectively, in the field and during storage. Reliable and early detection of these pathogens are keys to avoid occurrence of these diseases in potato crops and reduce yield loss. In the present study, multiplex polymerase chain reaction (PCR) protocol was developed for simultaneous detection of R. solanacearum and E. carotovora subsp. carotovora from potato tubers. A set of oligos targeting the pectatelyase (pel) gene of E. carotovora subsp. carotovora and the universal primers based on 16S r RNA gene of R. solanacearum were used. The standardized multiplex PCR protocol could detect R. solanacearum and E. carotovora subsp. carotovora up to 0.01 and 1.0 ng of genomic DNA, respectively. The protocol was further validated on 96 stored potato tuber samples, collected from different potato-growing states of India, viz. Uttarakhand, Odisha, Meghalaya and Delhi. 53.1 % tuber samples were positive for R. solanacearum, and 15.1 % of samples were positive for E. carotovora subsp. carotovora, and both the pathogens were positive in 26.0 % samples when BIO-PCR was used. This method offers sensitive, specific, reliable and fast detection of two major bacterial pathogens from potato tubers simultaneously, particularly pathogen-free seed certification in large scale.

N-terminal in coat protein of Garlic virus X is indispensible for its serological detection.

Conserved coat protein region of plant viruses is often used as source of antigen for production of polyclonal antibodies for broad-based detection of closely related viruses. Antigenic region in coat protein is located either on N-terminal, and/or C-terminal or in the middle of coat protein. A study was undertaken to determine if antigenic region resides in N-terminal in Garlic virus X (GarV-X) of Allexivirus. In allexiviruses, N-terminal of coat protein region (1-57 amino acids) was highly variable. A complete coat protein of 27 kDa and a truncated protein without N-terminal (20 kDa) of GarV-X were expressed in pET expression vector and confirmed in western blotting using anti-His antisera. These expressed proteins were purified and used for antisera production. Specific and strong reaction was obtained for antisera generated against GarV-X full CP and GarV-X was detected in field-grown allium crops viz., onion, garlic, leek, and bunching onion and chives in ELISA. Antisera against GarV-X CPΔ1-61 (truncated CP) did not show reaction for GarV-X detection in immunoassay. Epitope mapping also indicated N-terminal as major antigenic determinant region with highest antigenic signal score. Our studies confirm that antigenic signals or epitopes reside in the N-terminal region of GarV-X which can be synthesized and used for production of monoclonal antibodies for specific detection purposes.

A study to evaluate whether CTR increases refractive unpredictability between predicted and actual IOL position.

BACKGROUND: The surgical management of cataract associated with extensive zonular loss presents a challenge for ophthalmic surgeon. Capsular Tension Ring (CTR) is commonly being used to stabilize the capsular bag in patients with zonular dialysis. CTR helps to avoid capsular collapse and vitreous presentation in AC during surgery and maintains the capsular bag, allowing the circular contour of the capsular bag, allowing intra ocular lens to be easily placed in the bag. The aim of the study was to know if there is any shift of IOL following use of CTR ring. METHOD: We did a Ultrabiomicroscopy (UBM) examination to find out shift in PCIOL in cases in which CTR ring and compared it with cases without CTR ring. RESULT: It was found out through UBM in this study that there is actually a posterior shift of PCIOL after use of CTR ring leading to hypermetropic correction needed after surgery. CONCLUSION: It is suggested that posterior shift of IOL following use of CTR should be kept in mind and the IOL implanted should be of + 1.0 to 2.0 D more than that calculated preoperatively.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India.

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL-1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL-1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

The genome sequence of an isolate of Indian citrus ringspot virus infecting the sweet orange in India.

Whole-genome sequencing of an isolate of Mandarivirus infecting the sweet orange [Citrus sinensis (L) Blanco] in the western part of India (Pune) was done. The single-stranded positive-sense RNA genome of Indian citrus ringspot virus (ICRSV) Pune has 7,560 nucleotides (nt), excluding a poly(A) tail, comprised of 27.98% (2,115 nt) A, 32.12% (2,428 nt) C, 19.68% (1,488 nt) G, and 20.22% (1,529 nt) T residues. The genome, organized into six open reading frames (ORFs), shares 97.7% sequence identity with the complete genome of the ICRSV K1 isolate (AF406744.1) infecting the kinnow (Citrus reticulate Blanco, a hybrid between King and Willow mandarins) in north India. The ICRSV Pune genome formed a complex secondary structure with a large number of unpaired cytosine-rich regions, and recombination analysis highlighted potential recombination in the ICRSV genome.

Broadening the host range and genetic diversity of waikaviruses.

Waikaviruses are monopartite, positive sense, single-stranded RNA viruses that cause economically important plant diseases. Despite their importance, waikaviruses are poorly understood and only ten members are currently recognized. The present study on Sequence Read Archive (SRA)-based data-driven virus discovery (DDVD) identified 22 putative new waikaviruses, nearly doubling the number of known waikaviruses, in SRA libraries of diverse plant species, from ferns to trees. Besides, a highly divergent secoviral sequence with distinct genome features was identified in a wheat transcriptome. Other significant findings of the study include identification of a new waikavirus in a library derived from diseased water chestnut sample wherein a caulimovirus was reported, prediction of coiled-coils in hypothetical protein region of waikaviral polyprotein alignment and phylogenetic clustering of tree-infecting waikaviruses. The study not only reiterates the importance of DDVD in unveiling hitherto hidden viral sequences in plant SRA libraries but also deepens our understanding of waikaviral diversity.

Characterization of a recombinant tomato leaf curl New Delhi Virus (ToLCNDV) in a perennial medicinal climber host (Ipomoea cairica (L.) Sweet).

During the year 2020-2021, a disease syndrome very commonly observed in railway creepers (Ipomoea cairica (L.) Sweet) was taken into consideration from Gorakhpur Province (UP East region). Whitefly, a common vector for plant-related viral diseases was observed for wide transmission of disease. DNA from 17 infected leaf samples was isolated and analyzed through PCR using specific primers designed for the detection of Begomoviruses. Further amplification of isolated DNA fragments supporting a begomovirus infection with an estimated size of (2.7 kb). RCA of the isolated DNA sample was carried out using ϕ29 DNA polymerase by digesting it through a set of restriction endonucleases (such as BamHI, Kpn1, HindIII, EcoRI) obtaining the best result through KpnI. The amplified segment was cloned into pUC 18vectors. The obtained sequences were matched using the NCBI BLAST database showing the highest sequence similarity of 98.7% with ToLCNDV of snake gourd (Accession no. KY780199), isolates of CP genes sequence in Varanasi. ToLCNDV, a begomovirus of bipartite nature was first reported to be from Tomato (Solanaceae), later affecting certain members of the Cucurbitaceae family in India and adjacent countries. The obtained DNA sequence was submitted at NCBI with the name ToLCNDV-IP (GenBank Accession no. OM777194). The phylogenetic analysis clubbed the current isolate ToLCNDV-IP with Asian isolates. All European isolates were clubbed in separate clades indicating two distinct groups of ToLCNDV isolates. ToLCNDV-IP isolate was found to be an intralineage recombinant between two Asian isolates viz ToLCNDV isolate from Papaya (India) and ToLCNDV isolate from Tomato (Pakistan). This study shows the association of recombinant ToLCNDV infection in a new host Ipomoea cairica for the first time in India.