Some properties of a hitherto undescribed filamentous virus of the grapevine

An apparently new, non mechanically-transmissible clostero-like virus, for which the name ''grapevine leafroll-associated virus 7('') (GLRaV-7) is proposed, was found in Albanian grapevine accessions. Virus particles were filamentous, had conspicuous cross banding and a length of 1500-1700 nm. Virions had coat protein subunits with an estimated M(r) of ca. 37 kDa and a ssRNA genome with size of ca. 19.5 kb as deduced from the estimate of dsRNA (ca. 19.5 kbp) extracted from grapevine tissues. A virus-specific antiserum was raised, which decorated virions at a dilution of 1:1000. This antiserum did not recognize particles of any of the six grapevine leafroll-associated clostero-like viruses (GLRaV-1 to -6) known to date, nor of grapevine trichovirus A (GVA) and B (GVB). Grapevine indicators graft-inoculated with material from accessions containing GLRaV-7 reacted with mild leafroll-like symptoms. In a survey in which 2226 vines from 30 different countries were examined by ELISA, GLRaV-7 was found in 141 plants from Albania, Greece, Hungary, Egypt, and Italy

Studies on ''corky rugose wood'' of grapevine and on the diagnosis of grapevine virus B

Vines affected by corky rugose wood (CRW), a field syndrome characterized by pronounced cork production by the scion of several grapevine varieties just above the graft union, contain a number of filamentous and isometric phloem-limited viruses, such as grapevine leafroll-associated virus 1, 2, and 3 (GLRaV-1, GLRaV-2, GLRaV-3), grapevine virus A and B (GVA and GVB), and grapevine fleck virus (GFkV). However, the same viruses, with the exception of GVB, are widely represented also in vines with rugose wood without excessive corkyness. Although GVB was found in all vines indexing positive in LN 33 for corky bark disease, iis occurrence in CRW-affected vines was not consistent enough to suggest that it may have a determining role in the induction of this syndrome. Monoclonal antibodies to GVB raised previously were characterized and their possible use for reliable detection of GVB in field-grown vines investigated in detail. A triple antibody sandwich ELISA protocol that under our experimental conditions afforded consistent and repeatable results, was based on the use of crude cortical scraping extracts from mature canes collected in autumn, antibodies from a polyclonal antiserum for plate coating (trapping) and a monoclonal antibody for antigen detection

Deep sequencing of dsRNAs recovered from mosaic-diseased pigeonpea reveals the presence of a novel emaravirus: pigeonpea sterility mosaic virus 2

Deep-sequencing analysis of double-stranded RNA extracted from a mosaic-diseased pigeonpea plant (Cajanus cajan L., family Fabaceae) revealed the complete sequence of six emaravirus-like negative-sense RNA segments of 7009, 2229, 1335, 1491, 1833 and 1194 nucleotides in size. In the order from RNA1 to RNA6, these genomic RNAs contained ORFs coding for the RNA-dependent RNA polymerase (RdRp, p1 of 266 kDa), the glycoprotein precursor (GP, p2 of 74.5 kDa), the nucleocapsid (NC, p3 of 34.9 kDa), and the putative movement protein (MP, p4 of 40.7 kDa), while p5 (55 kDa) and p6 (27 kDa) had unknown functions. All RNA segments showed distant relationships to viruses of the genus Emaravirus, and in particular to pigeonpea sterility mosaic virus (PPSMV), with which they shared nucleotide sequence identity ranging from 48.5 % (RNA3) to 62.5 % (RNA1). In phylogenetic trees constructed from the sequences of the proteins encoded by RNA1, RNA2 and RNA3 (p1, p2 and p3), this new viral entity showed a consistent grouping with fig mosaic virus (FMV) and rose rosette virus (RRV), which formed a cluster of their own, clearly distinct from PPSMV-1. In experimental greenhouse trials, this novel virus was successfully transmitted to pigeonpea and French bean seedlings by the eriophyid mite Aceria cajani. Preliminary surveys conducted in the Hyderabad region (India) showed that the virus in question is widespread in pigeonpea plants affected by sterility mosaic disease (86.4 %) but is absent in symptomless plants. Based on molecular, biological and epidemiological features, this novel virus is the second emaravirus infecting pigeonpea, for which the provisional name pigeonpea sterility mosaic virus 2 (PPSMV-2) is proposed

Deep sequencing of Pigeonpea sterility mosaic virus discloses five RNA segments related to emaraviruses

The sequences of five viral RNA segments of Pigeonpea sterility mosaic virus (PPSMV), the agent of sterility mosaic disease (SMD) of pigeonpea (Cajanus cajan, Fabaceae), were determined using the Deep sequencing technology. Each of the five RNAs encodes a single protein on the negative-sense strand with an open reading frame (ORF) of 6885, 1947, 927, 1086, and 1422 nts, respectively. In order, from RNA1 to RNA5, these ORFs encode the RNA-dependent RNA polymerase (p1, 267.9 kDa), a putative glycoprotein precursor (p2, 74.3 kDa), a putative nucleocapsid protein (p3, 34.6 kDa), a putative movement protein (p4, 40.8 kDa), while p5 (55 kDa) has an unknown function. All RNA segments of PPSMV showed the highest identity with orthologs of fig mosaic virus (FMV) and rose rosette virus (RRV). In phylogenetic trees constructed with the amino acid sequences of p1, p2 and p3, PPSMV clustered consistently with other emaraviruses, close to clades comprising members of other genera of the family Bunyaviridae. Based on the molecular characteristics unveiled in this study and the morphological and epidemiological features similar to other emaraviruses, PPSMV seems to be the seventh species to join the list of emaraviruses known to date and accordingly, its classification in the genus Emaravirus seems now legitimate

Production of monoclonal antibodies to Grapevine virus D and contribution to the study of its aetiological role in grapevine diseases

Six stable hybridoma cell lines secreting monoclonal antibodies (MAbs) to Grapevine virus D (GVD) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14, In ELISA all MAbs detected the virus in Nicotiana leaf extracts or cortical shavings from mature grapevine canes, The use of a polyclonal antiserum for coating plates and of monoclonal antibodies and antimouse-conjugated antibodies for antigen detection, gave highly efficient and reproducible results for identification of GVD in field-grown grapevines. The reliability of the ELISA kit was confirmed by GVD-transmission tests to herbaceous hosts, using in vitro explants as inoculum, 223 vines affected by one or more of the 4 syndroms of the rugose wood complex (Kober stem grooving, Corky bark, LN stem grooving and Rupestris stem pitting) were tested in ELISA for the detection of Grapevine virus A (GVA), Grapevine virus B (GVB) and GVD and by Western blot for the detection of Grapevine rupestris stem pitting associated virus (GRSPaV). The possible cause-effect relationship between GVA and KSG, GVB and Co, and GRSPaV and RSP was confirmed, but no consistent association was found between GVD and any of the 4 above syndromes, Intriguingly, a reduction in the expression of stem pitting symptoms in V. rupestris (from 90 % to 75 %) and of stem grooving symptoms in Kober 5BB (from 95 % to 70 %) was observed when vitiviruses and GRSPaV were contemporarily present in the same indicator. Preliminary data of a survey involving 676 grapevine samples showed a high incidence (31 %) of GVD, regardless of the geographical origin of samples.

Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus

Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80–200 nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276 bp and 360 bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276 bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature

Molecular detection of Grapevine fleck virus-like viruses

Molecular reagents have been developed for virus-specific and simultaneous (virus-non-specific) detection of Grapevine fleck virus (GFkV) and allied viruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV). Degenerate primers designed on nucleotide sequences of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) domains of the GFkV genome, were able to give amplification products of the expected size from total nucleic acid extracts of:vines infected with GFkV, GAMaV, and GRGV;a Californian grapevine accession infected by a marafi-like virus;Greek grapevine accessions infected by an unidentified agent that induced symptoms reminiscent of those elicited by GAMaV in Vitis rupestris.Degenerate primers designed on the nucleotide sequence of the helicase (HEL) domain of the GFLV genome recognized all the above viruses except for GAMaV and the unidentified Greek viral agent. RD primer set worked well also with crude grapevine cortical scrapings, thus constituting a useful universal reagent for the non-specific molecular identification of GFkV-like viruses in Vitis . The marafi-like virus from California was amplified by all sets of primers, but was recognized only by the GRGV-specific probe, suggesting that it is a likely isolate of GRGV: Likewise, the unidentified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that call for further investigations to establish its taxonomic position. While GRGV was identified, though with a very low incidence, in some 11 southern Italian grapevine cultivars, no evidence was obtained for infection by GAMaV in any of 50 cultivars analyzed.

Worldwide diffusion of Fig latent virus 1 in fig accessions and its detection by serological and molecular tools

A virus with filamentous particles ca. 700 nm long, denoted Fig latent virus 1 (FLV-1) is widespread in Apulian (southern Italy) fig orchards, in trees showing or not mosaic symptoms and in symptomless seedlings. The virus was transmitted by sap inoculation to a very restricted range of herbaceous hosts without inducing apparent symptoms and was transmitted through fig seeds to a very high percentage (80 to 100 %). It was successfully purified from root tissues of infected figs. A virus-specific antiserum raised in rabbits, proved useful for its detection in fig leaf dips by immunosorbent electron microscopy (ISEM), Western Blot, dot immuno-binding (DIBA), ELISA. The viral genome structure resembles that of members of the genus Trichovirus in the family Flexiviridae. Keywords: fig latent virus, Trichovirus, serology, ISEM, Western blot, DIBA, ELIS

2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

Correction to: 2021 Taxonomic update of phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales. Archives of Virology (2021) 166:3567–3579. https://doi.org/10.1007/s00705-021-05266-wIn March 2021, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by four families (Aliusviridae, Crepuscuviridae, Myriaviridae, and Natareviridae), three subfamilies (Alpharhabdovirinae, Betarhabdovirinae, and Gammarhabdovirinae), 42 genera, and 200 species. Thirty-nine species were renamed and/or moved and seven species were abolished. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.This work was supported in part through Laulima Government Solutions, LLC prime contract with the US National Institute of Allergy and Infectious Diseases (NIAID) under Contract No. HHSN272201800013C. J.H.K. performed this work as an employee of Tunnell Government Services (TGS), a subcontractor of Laulima Government Solutions, LLC under Contract No. HHSN272201800013C. This work was also supported in part with federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Contract No. 75N91019D00024, Task Order No. 75N91019F00130 to I.C., who was supported by the Clinical Monitoring Research Program Directorate, Frederick National Lab for Cancer Research. This work was also funded in part by Contract No. HSHQDC-15-C-00064 awarded by DHS S&T for the management and operation of The National Biodefense Analysis and Countermeasures Center, a federally funded research and development center operated by the Battelle National Biodefense Institute (V.W.); and NIH contract HHSN272201000040I/HHSN27200004/D04 and grant R24AI120942 (N.V., R.B.T.). S.S. acknowledges partial support from the Special Research Initiative of Mississippi Agricultural and Forestry Experiment Station (MAFES), Mississippi State University, and the National Institute of Food and Agriculture, US Department of Agriculture, Hatch Project 1021494. Part of this work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (FC001030), the UK Medical Research Council (FC001030), and the Wellcome Trust (FC001030).S

Taxonomy of the family Arenaviridae and the order Bunyavirales: update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future