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.

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 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.

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.

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.

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.

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.

Revisiting the amalgaviral landscapes in plant transcriptomes expands the host range of plant amalgaviruses.

Plant amalgaviruses are monopartite, double-stranded RNA viruses, capable of vertical transmission through seeds. An attempt to revisit plant transcriptome-assembled contigs for amalgaviral sequences identified 40 putative novel amalgaviruses in 35 plant species, nearly doubling the number of plant amalgaviruses. Of the 35 plant species, 33 are reported to host amalgaviruses for the first time, including a pteridophytic and two gymnospermic species. Coding-complete genomes of all identified viruses were recovered and the putative +1 programmed ribosomal frameshift (PRF) sites were determined. Genomes of 35 identified amalgaviruses contained the conserved +1 PRF motif 'UUU_CGN', while variant versions were predicted in five genomes. Phylogenetic analysis grouped pteridophyte- and gymnosperm-infecting amalgaviruses together in divergent sub-clades while few of the related angiosperm-infecting amalgaviruses infect members of the same plant family, reiterating the co-evolution of plant amalgaviruses and their hosts. The current study paves way for further studies on understanding biological properties of identified viruses.

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.

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.

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.

First report of natural infection by two potyviruses on amaryllis (Hippeastrum hybridum) plants from India.

Amaryllis plants showing the chlorotic stripes and yellowing of the leaves were collected. Electron microscopy of the infected plants revealed flexuous filamentous particles and disease incidence was recorded up to 64%. The presence of virus was confirmed using potyvirus genus and species-specific primers using Reverse transcription PCR. Blastn analysis showed that the samples were infected with leek yellow stripe virus (LYSV) and onion yellow dwarf virus (OYDV) the of genus Potyvirus, family Potyviridae. To the best of our knowledge, this is the first report of an association between LYSV (MZ203474 to MZ203478) and OYDV (MZ203479-MZ203482) with amaryllis. The result also showed that infected Amaryllis plants may act as a reservoir of LYSV and OYDV for economically important Allium crops.

Discovery of putative novel viruses in the transcriptomes of endangered plant species native to India and China.

Viruses are abundant entities that infect almost every living organism. In recent years, Next Generation Sequencing coupled with bioinformatic analyses is widely adopted for identification of known and unknown viruses in a plant sample. In the present study, nine putative novel viruses were discovered from public domain transcriptome datasets of five endangered plant species by de novo assembly of reads using CLC and SPAdes followed by BLAST analysis. Of the identified viruses, ten coding-complete and five partial genomic segments were recovered. Based on phylogeny and BLAST analysis, the identified viruses were putatively assigned to various plant viral genera except dactylorhiza hatagirea benylike virus that probably represents a new group of plant virus. The methodology followed can be adopted for the discovery of novel viruses in plant species with little genomic information. Viral genome sequences recovered in the study will serve as a valuable resource for further characterization of identified viruses.

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.

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses.

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3' polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

The role of enzymatic activities of antiviral proteins from plants for action against plant pathogens.

Antiviral proteins (AVPs) from plants possess multiple activities, such as N-glycosidase, RNase, DNase enzymatic activity, and induce pathogenesis-related proteins, salicylic acid, superoxide dismutase, peroxidase, and catalase. The N-glycosidase activity releases the adenine residues from sarcin/ricin (S/R) loop of large subunit of ribosomes and interfere the host protein synthesis process and this activity has been attributed for antiviral activity in plant. It has been shown that AVP binds directly to viral genome-linked protein of plant viruses and interfere with protein synthesis of virus. AVPs also possess the RNase and DNase like activity and may be targeting nucleic acid of viruses directly. Recently, the antifungal, antibacterial, and antiinsect properties of AVPs have also been demonstrated. Gene encoding for AVPs has been used for the development of transgenic resistant crops to a broad range of plant pathogens and insect pests. However, the cytotoxicity has been observed in transgenic crops using AVP gene in some cases which can be a limiting factor for its application in agriculture. In this review, we have reviewed various aspects of AVPs particularly their characteristics, possible mode of action and application.

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.

Robust Virome Profiling and Whole Genome Reconstruction of Viruses and Viroids Enabled by Use of Available mRNA and sRNA-Seq Datasets in Grapevine (Vitis vinifera L.).

Next-generation sequencing (NGS) based virome analyses of mRNA and sRNA have recently become a routine approach for reliable detection of plant viruses and viroids. In the present study we identified the viral/viroidal spectrum of several Indian grapevine cultivars and reconstructed their whole genomes using the publically available mRNAome and sRNAome datasets. Twenty three viruses and viroids (including two variants of grapevine leafroll associated virus 4) were identified from two tissues (fruit peels and young leaves) of three cultivars among which nine unique grapevine viruses and viroids were identified for the first time in India. Irrespective of the assemblers and tissues used, the mRNA based approach identified more acellular pathogens than the sRNA based approach across cultivars. Further, the mRNAome was on par with the whole transcriptome in viral identification. Through de novo assembly of transcriptomes followed by mapping against reference genome, we reconstructed 19 complete/near complete genomes of identified viruses and viroids. The reconstructed viral genomes included four larger RNA genomes (>13 kb), a DNA genome (RG grapevine geminivirus A), a divergent genome (RG grapevine virus B) and a genome for which no reference is available (RG grapevine virus L). A large number of SNPs detected in this study ascertained the quasispecies nature of viruses. Detection of three recombination events and phylogenetic analyses using reconstructed genomes suggested the possible introduction of viruses and viroids into India from several continents through the planting material. The whole genome sequences generated in this study can serve as a resource for reliable indexing of grapevine viruses and viroids in quarantine stations and certification programs.

Development of RT-PCR degenerate primers for the detection of two mandariviruses infecting citrus cultivars in India.

Indian citrus ringspot virus (ICRSV) and Citrus yellow vein clearing virus (CYVCV) are the mandariviruses infecting various citrus cultivars in India and around the world. In the fields, it was observed that citrus plants infected by both the viruses and frequently expressed only ringspot symptoms. The ICRSV-specific polyclonal-antibody used in immuno-sorbent electron microscopy (ISEM) and enzyme linked immuno-sorbent assay (ELISA) could detect only ICRSV in mixed infections. Therefore, the conserved sequences of the RNA dependent RNA polymerase (RdRP) gene of the alphaflexiviruses were exploited for developing a RT-PCR based assay for detection of both the mandariviruses simultaneously, if present. A degenerate primer pair was designed to amplify a ∼435bp fragment by multiple alignments of the RdRP gene sequences of the members of genera Mandarivirus, Potexvirus and Allexivirus. The developed RT-PCR assay was validated for detecting both, CYVCV and ICRSV in mixed infections as well as in single virus-infected citrus plants. The presence of ICRSV or CYVCV or both of them together in such plants were confirmed by using primer pair specific to each of these viruses. Further, the identity of the amplicons was confirmed by sequencing and the virus species were determined with BLASTN analysis. The degenerate primers also amplified the corresponding target sequences of an allexivirus and a potexvirus from the respective infected garlic/ onion and tobacco plants. The use of the degenerate primers for the detection of these virus species of the genus Mandarivirus will be useful in citrus certification programmes.