Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia

Background Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. Methods We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. Results A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. Conclusion We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.

Molecular and Phylogenetic Characterisation of a Highly Divergent Novel Parvovirus (Psittaciform Chaphamaparvovirus 2) in Australian Neophema Parrots

Parvoviruses under the genus Chaphamaparvovirus (subfamily Hamaparvovirinae) are highly divergent and have recently been identified in many animals. However, the detection and characterisation of parvoviruses in psittacine birds are limited. Therefore, this study reports a novel parvovirus, tentatively named psittaciform chaphamaparvovirus 2 (PsChPV-2) under the genus Chaphamaparvovirus, which was identified in Australian Neophema birds. The PsChPV-2 genome is 4371 bp in length and encompasses four predicted open-reading frames, including two major genes, a nonstructural replicase gene (NS1), and a structural capsid gene (VP1). The NS1 and VP1 genes showed the closest amino acid identities of 56.2% and 47.7%, respectively, with a recently sequenced psittaciform chaphamaparvovirus 1 from a rainbow lorikeet (Trichoglossus moluccanus). Subsequent phylogenetic analyses exhibited that the novel PsChPV-2 is most closely related to other chaphamaparvoviruses of avian origin and has the greatest sequence identity with PsChPV-1 (60.6%). Further systematic investigation is warranted to explore the diversity with many avian-associated parvoviruses likely to be discovered.

Search for RNA aptamers against non-structural protein of SARS-CoV-2: Design using molecular dynamics approach

Background Recent outbreak of deadly Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) urges the scientist to identify the potential vaccine or drug to control the disease. SARS-CoV-2 with its single stranded RNA genome (length ~ 30 kb) is enveloped with active spike proteins. The genome is non-segmental with 5’-cap and 3’-poly tail and acts as a mRNA for the synthesis of replicase polyproteins. The replicase gene lying downstream to 5’-end encodes for non-structural protein, which in turn pose multiple functions ranging from envelope to nucleocapsid development. This study aims to identify the highly stable, effective and less toxic single strand RNA-based aptamers against non-structural protein 10 (NSP10). NSP10 is the significant activator of methyltransferase enzymes (NSP14 and NSP16) in SARS-CoV-2. Inhibiting the activation of methyltransferase leads to partial viral RNA capping or lack of capping, which makes the virus particles susceptible to host defence system. Results In this study, we focused on designing RNA aptamers through computational approach, docking of protein-aptamer followed by molecular dynamics simulation to perceive the binding stability of complex. Docking study reveals the high binding affinity of three aptamers namely RNA-053, 001, 010 to NSP10 with the HADDOCK score of − 88.5 ± 7.0, − 87.7 ± 11.5, − 86.1 ± 12 respectively. Molecular Dynamics suggests high conformational stability between the aptamer and the protein. Among the screened aptamers two aptamers maintained at least 3-4 intermolecular H-bonds throughout the simulation period. Conclusions The study identifies the potential aptamer candidate against less investigated but significant antiviral target i.e., NSP10/NSP16 interface complex.

Ancient Viral Integrations in Marsupials: A Potential Antiviral Defence

Marsupial viruses are understudied compared to their eutherian mammal counterparts, although they may pose severe threats to vulnerable marsupial populations. Genomic viral integrations, termed endogenous viral elements (EVEs) could protect the host from infection. It is widely known past viral infections and EVEs play an active role in antiviral defence in invertebrates and plants. This study aimed to characterise actively transcribed EVEs in Australian marsupial species, because they may play an integral role in cellular defence against viruses. This study screened publicly available RNA sequencing datasets (n=35) and characterised 200 viral transcripts from thirteen Australian marsupial species. Of the 200 transcripts, 188 originated from either Bornaviridae, Filoviridae or Parvoviridae EVEs. The other 12 transcripts were from putative active infections from members of the Herpesviridae and Anelloviridae, and Hepadnaviridae. EVE transcripts (n=188) were mapped to marsupial genomes (where available, n=5/13) to identify the genomic insertion sites. Of the 188 transcripts, 117 mapped to 39 EVEs within the koala, bare-nosed wombat, tammar wallaby, brushtail possum and Tasmanian devil genomes. The remaining eight animals had no available genome (transcripts n=71). Every marsupial have Bornaviridae, Filoviridae and Parvoviridae EVEs, a trend widely observed in eutherian mammals. Whilst eutherian bornavirus EVEs are predominantly nucleoprotein-derived, marsupial bornavirus EVEs demonstrate a surprising replicase gene bias. We predicted these widely distributed EVEs were conserved within marsupials from ancient germline integrations, as many were over 65 million years old. One bornavirus replicase EVE, present in six marsupial genomes, was estimated to be 160 million years old, predating the American-Australian marsupial split. We considered transcription of these EVEs through small non-coding RNA as an ancient viral defence. Consistent with this, in koala small RNA sequence datasets we detected Bornaviridae replicase and Filoviridae nucleoprotein produced piRNA. These were enriched in testis tissue, suggesting they could protect marsupials from vertically transmitted viral integrations.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90–80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5′-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses

Recent outbreaks of zoonotic coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have caused tremendous casualties and great economic shock. Although some repurposed drugs have shown potential therapeutic efficacy in clinical trials, specific therapeutic agents targeting coronaviruses have not yet been developed. During coronavirus replication, a replicase gene cluster, including RNA-dependent RNA polymerase (RdRp), is alternatively translated via a process called -1 programmed ribosomal frameshift (−1 PRF) by an RNA pseudoknot structure encoded in viral RNAs. The coronavirus frameshifting has been identified previously as a target for antiviral therapy. In this study, the frameshifting efficiencies of MERS-CoV, SARS-CoV and SARS-CoV-2 were determined using an in vitro −1 PRF assay system. Our group has searched approximately 9689 small molecules to identify potential −1 PRF inhibitors. Herein, we found that a novel compound, 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline (KCB261770), inhibits the frameshifting of MERS-CoV and effectively suppresses viral propagation in MERS-CoV-infected cells. The inhibitory effects of 87 derivatives of furo[2,3-b]quinolines were also examined showing less prominent inhibitory effect when compared to compound KCB261770. We demonstrated that KCB261770 inhibits the frameshifting without suppressing cap-dependent translation. Furthermore, this compound was able to inhibit the frameshifting, to some extent, of SARS-CoV and SARS-CoV-2. Therefore, the novel compound 2-(5-acetylthiophen-2yl)furo[2,3-b]quinoline may serve as a promising drug candidate to interfere with pan-coronavirus frameshifting.

Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

Brome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

First report of banana bunchy top virus in banana (Musa spp.) and its eradication in Togo

Banana (including plantain; Musa spp.) is a vegetatively propagated semi-perennial crop in fields and backyard gardens in Togo. Banana bunchy top disease (BBTD), caused by banana bunchy top virus (BBTV, genus Babuvirus) is the most economically important viral disease, infection of which causes severe stunting and production losses of 90-100% within two seasons. The virus is spread by banana aphid, Pentalonia nigronervosa, and through vegetative propagation from infected sources. BBTV occurrence was first reported in West Africa in 2011 with confirmation in Republic of Benin and in Nigeria in 2012 . A regional alliance (www.bbtvalliance.org) has been established for BBTV surveillance through frequent surveys in countries neighboring those affected, such as Togo. The surveys conducted in September 2018 in banana growing areas in Togo revealed plants with typical symptoms (severe stunting, bunchy growth with shortened petioles with chlorotic streaks and yellow leaf margins) in three banana fields. Locations were Tsévié, Préfecture de Zio, (6.44°N, 1.21028°E), Lilicope, Préfecture de Zio in Maritime region (6.56583°N, 1.18639°E), and Amoutchou, Préfecture de l’Ogou in Plateaux region (7.3775°N, 1.17472°E). Leaf samples were collected from symptomatic (N=8) and asymptomatic plants (N=30) and used for DNA extraction followed by a polymerase chain reaction (PCR) for BBTV detection to amplify ~240 bp sequence of DNA-R encoding for core replicase gene. All samples from symptomatic plants (N=8) tested positive and asymptomatic plants were negative. To ascertain virus identity the 240-bp PCR product was purified and sequenced in both directions. A BLAST search of the sequence (NCBI GenBank Acc.# MK073116) revealed 99% identity with DNA-R sequences of BBTV isolates from Africa (e.g., JQ437549-Benin, JN290301-Nigeria). Further analysis of the 240-bp nucleotide sequence with Maximum-likelihood phylogenetic analysis using MEGA-X software has grouped the BBTV isolate with sub-Saharan African sub-clade of the South Pacific group. To further confirm the virus identity, two samples from symptomatic (PCR positive) and asymptomatic (PCR negative) plants from Tsévié were tested by TAS-ELISA using BBTV ELISA reagent set (Cat. No. SRA24700-1000, Agdia, France) following the manufacturers’ protocol. Only samples from two symptomatic plants that were positive in PCR reacted positively in TAS-ELISA; asymptomatic plants were negative. BBTV was not observed in any of the 22 locations surveyed as a follow-up in banana producing areas. To our knowledge, this is the first report of BBTV infecting banana in Togo. The plants detected in the three sites were eradicated in the follow-up action implemented by the alliance team together with the Direction de la Protection des Végétaux of Togo. Follow-up surveys were conducted in the same regions in 2019 and 2020 to ensure disease-free status in these sites and other banana producing regions in Togo. Efforts have been made to raise awareness about BBTD recognition, diagnosis, and eradication. To the best of our knowledge this is the first case of rapid detection and eradication of BBTD in sub-Saharan Africa. This study illustrates the importance of regular surveillance for early detection of invasive virus threats and the value of rapid eradication to contain viruses before spread and establishment in a new territory.

Construction of an Agrobacterium Mediated RNAI Genetic Transformation Vector Targeting the Replicase Gene of Indian cassava mosaic virus and Evaluation of Their Transformation Ability in Cassava Immature Leaf Lobes

Cassava mosaic virus is one of the major problems affecting cassava industry in India. Currently there are no effective strategy to completely protect cassava from cassava mosaic viruses. In order to attain cassava mosaic virus resistance RNAi vectors targeting the replicase gene of Indian cassava mosaic virus is constructed in this study. Their efficiency to transform cassava immature leaf lobes were also studied here. Replicase gene of Indian cassava mosaic virus in Tamilnadu are cloned and sequenced. Conserved domains are identified and sub cloned to CSIRO RNAi vector system and transformation studies are done in immature cassava leaves. Two different RNAi vectors were constructed, utilizing a conserved 440bp of 5’ end of ICMV Rep (AC1) gene which also corresponds to a part of AC4 gene, and functions as a viral RNAi suppressor protein. The partial Rep gene of ICMV was cloned in sense and anti-sense orientations in the RNAi intermediate vector, pHANNIBAL. After cloning into pHANNIBAL, the cloned RNAi gene cassettes of ICMV is released and cloned into the binary vector, pART27, which contains the kanamycin-resistance gene as a plant selectable marker. In order to use hygromycin as a selection agent in cassava genetic transformation, RNAi–Rep gene cassettes of ICMV were cloned into pCAMBIA1305.2. These constructs were named pICR1 and pICR2 respectively. The Genetic transformation studies in cassava leaves done using pICR2 vector could generate PCR positive plants. An agrobacterium mediated replicase RNAi vector is developed and that can be transformed into cassava immature leaf lobes. Their efficiency to silence the Indian cassava mosaic virus should be studied further.