Characterization of Sf9 cell clones with differential susceptibilities to Sf-rhabdovirus X+3.7 and Sf-rhabdovirus X- replication.

Our laboratory previously discovered a novel rhabdovirus in the Spodoptera frugiperda Sf9 insect cell line that was designated as Sf-rhabdovirus. Using limiting dilution, this cell line was found to be a mixed population of cells infected by Sf-rhabdovirus variants containing either the full length X accessory gene with a 3.7 kb internal duplication (designated as Sf-rhabdovirus X+3.7) or lacking the duplication and part of the X gene (designated as Sf-rhabdovirus X-), and cells that were negative for Sf-rhabdovirus. In this paper, we found that the Sf-rhabdovirus negative cell clones had sub-populations with different susceptibilities to the replication of Sf-rhabdovirus X+3.7 and X- variants: cell clone Sf9-13F12 was more sensitive to replication by both virus variants compared to Sf9-3003; moreover, Sf9-3003 showed more resistance to X+3.7 replication than to X- replication. RNA-Seq analysis indicated significant differentially expressed genes in the Sf9-13F12 and Sf9-3003 cell clones further supporting that distinct sub-populations of virus-negative cells co-exist in the parent Sf9 cell line.

ICTV Virus Taxonomy Profile: Filoviridae 2024.

Filoviridae is a family of negative-sense RNA viruses with genomes of about 13.1-20.9 kb that infect fish, mammals and reptiles. The filovirid genome is a linear, non-segmented RNA with five canonical open reading frames (ORFs) that encode a nucleoprotein (NP), a polymerase cofactor (VP35), a glycoprotein (GP1,2), a transcriptional activator (VP30) and a large protein (L) containing an RNA-directed RNA polymerase (RdRP) domain. All filovirid genomes encode additional proteins that vary among genera. Several filovirids (e.g., Ebola virus, Marburg virus) are pathogenic for humans and highly virulent. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Filoviridae, which is available at www.ictv.global/report/filoviridae.

A novel rhabdovirus detected in Anisakis larvae distributed in the coastal areas of Japan: Viral genome analysis and possible coevolutionary relationship between virus and host nematodes.

In the last decade, it has become evident that various RNA viruses infect helminths including Order Ascaridida. However, there is still no information available for viruses infecting Anisakis. We herewith demonstrate the presence of a novel rhabdovirus from Anisakis larvae detected by next-generation sequencing analysis and following RT-PCR. We determined the nearly all nucleotide sequence (12,376 nucleotides) of the viral genome composed of seven open reading frames, and we designated the virus as Suzukana rhabdo-like virus (SkRV). BLASTx search indicated that SkRV is a novel virus belonging to the subfamily Betanemrhavirus, rhabdovirus infecting parasitic nematodes of the Order Ascaridida. SkRV sequence was detectable only in the total RNA but not in the genomic DNA of Anisakis, ruling out the possibility of SkRV being an endogenous viral element incorporated into the host genomic DNA. When we individually tested Anisakis larvae obtained from Scomber japonicus migrating in the coastal waters of Japan, not all but around 40% were SkRV-positive. In the phylogenetic trees of Betanemrhavirus and of the host Ascaridida nematodes, we observed that evolutional distances of viruses were, to some extent, parallel with that of host nematodes, suggesting that viral evolution could have been correlated with evolution of the host. Although biological significance of SkRV on Anisakis larvae is still remained unknown, it is interesting if SkRV were somehow related to the pathogenesis of anisakiasis, because it is important matter of public health in Japan and European countries consuming raw marine fishes.

Rhabdovirus encoded glycoprotein induces and harnesses host antiviral autophagy for maintaining its compatible infection.

Macroautophagy/autophagy has been recognized as a central antiviral defense mechanism in plant, which involves complex interactions between viral proteins and host factors. Rhabdoviruses are single-stranded RNA viruses, and the infection causes serious harm to public health, livestock, and crop production. However, little is known about the role of autophagy in the defense against rhabdovirus infection by plant. In this work, we showed that Rice stripe mosaic cytorhabdovirus(RSMV) activated autophagy in plants and that autophagy served as an indispensable defense mechanism during RSMV infection. We identified RSMV glycoprotein as an autophagy inducer that interacted with OsSnRK1B and promoted the kinase activity of OsSnRK1B on OsATG6b. RSMV glycoprotein was toxic to rice cells and its targeted degradation by OsATG6b-mediated autophagy was essential to restrict the viral titer in plants. Importantly, SnRK1-glycoprotein and ATG6-glycoprotein interactions were well-conserved between several other rhabdoviruses and plants. Together, our data support a model that SnRK1 senses rhabdovirus glycoprotein for autophagy initiation, while ATG6 mediates targeted degradation of viral glycoprotein. This conserved mechanism ensures compatible infection by limiting the toxicity of viral glycoprotein and restricting the infection of rhabdoviruses.Abbreviations: AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy related; AZD: AZD8055; BiFC: bimolecular fluorescence complementation; BYSMV: barley yellow striate mosaic virus; Co-IP: co-immunoprecipitation; ConA: concanamycin A; CTD: C-terminal domain; DEX: dexamethasone; DMSO: dimethyl sulfoxide; G: glycoprotein; GFP: green fluorescent protein; MD: middle domain; MDC: monodansylcadaverine; NTD: N-terminal domain; OE: over expression; Os: Oryza sativa; PBS: phosphate-buffered saline; PtdIns3K: class III phosphatidylinositol-3-kinase; qRT-PCR: quantitative real-time reverse-transcription PCR; RFP: red fluorescent protein; RSMV: rice stripe mosaic virus; RSV: rice stripe virus; SGS3: suppressor of gene silencing 3; SnRK1: sucrose nonfermenting1-related protein kinase1; SYNV: sonchus yellow net virus; TEM: transmission electron microscopy; TM: transmembrane region; TOR: target of rapamycin; TRV: tobacco rattle virus; TYMaV: tomato yellow mottle-associated virus; VSV: vesicular stomatitis virus; WT: wild type; Y2H: yeast two-hybrid; YFP: yellow fluorescent protein.

Separation of spring viraemia of carp virus from large-volume samples using immunomagnetic beads.

A method for separation of spring viraemia of carp virus (SVCV) from large-volume samples using immunomagnetic beads (IMBs) coated with a polyclonal antibody against SVCV was developed. The optimum amount of IMBs was 2 mg in 100 mL. After IMB treatment, the detection limit of SVCV in reverse transcription quantitative PCR (RT-qPCR) was 103 times the 50% tissue culture infectious dose per mL in 100-mL samples. The concentration of viral RNA extracted from SVCV that had been separated using IMBs was 5.18 × 103-fold higher than that of the unseparated SVCV. When fish samples were tested, the concordance rates of the IMBs/RT-qPCR and RT-qPCR were 100% and 67.5%, respectively.

circMORC3-encoded novel protein negatively regulates antiviral immunity through synergizing with host gene MORC3.

The protein-coding ability of circRNAs has recently been a hot topic, but the role of protein-coding circRNAs in antiviral innate immunity of teleost fish has rarely been reported. Here, we identified a novel circRNA, termed circMORC3, derived from Microrchidia 3 (MORC3) gene in Miichthys miiuy. circMORC3 can inhibit the expression of antiviral cytokines. In addition, circMORC3 encodes a novel peptide with a length of 84 amino acids termed MORC3-84aa. MORC3-84aa not only significantly inhibited TRIF-mediated activation of IRF3 and NF-κB signaling pathways, but also effectively suppressed the expression of antiviral cytokines triggered by RNA virus Siniperca chuatsi rhabdovirus (SCRV). We found that MORC3-84aa directly interacted with TRIF and negatively regulated TRIF protein expression. In addition, host gene MORC3 attenuates SCRV-induced IFN and ISG expression. Mechanistically, MORC3-84aa promotes autophagic degradation of TRIF by enhancing K6-linked ubiquitination and inhibits TRIF-mediated activation of the type I interferon signaling pathway. And the host gene MORC3 not only repressed IRF3 protein expression but also inhibited IRF3 phosphorylation levels. Our study shows that circMORC3 and host gene MORC3 played a synergistic role in viral immune escape.

Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses.

Cytorhabdoviruses (genus Cytorhabdovirus, family Rhabdoviridae) are plant-infecting viruses with enveloped, bacilliform virions. Established members of the genus Cytorhabdovirus have unsegmented single-stranded negative-sense RNA genomes (ca. 10-16 kb) which encode four to ten proteins. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and genomic characterization of 93 novel viruses with genetic and evolutionary cues of cytorhabdoviruses. Strikingly, five unprecedented viruses with tri-segmented genomes were also identified. This finding represents the first tri-segmented viruses in the family Rhabdoviridae, and they should be classified in a novel genus within this family for which we suggest the name "Trirhavirus". Interestingly, the nucleocapsid and polymerase were the only typical rhabdoviral proteins encoded by those tri-segmented viruses, whereas in three of them, a protein similar to the emaravirus (family Fimoviridae) silencing suppressor was found, while the other predicted proteins had no matches in any sequence databases. Genetic distance and evolutionary insights suggest that all these novel viruses may represent members of novel species. Phylogenetic analyses, of both novel and previously classified plant rhabdoviruses, provide compelling support for the division of the genus Cytorhabdovirus into three distinct genera. This proposed reclassification not only enhances our understanding of the evolutionary dynamics within this group of plant rhabdoviruses but also illuminates the remarkable genomic diversity they encompass. This study not only represents a significant expansion of the genomics of cytorhabdoviruses that will enable future research on the evolutionary peculiarity of this genus but also shows the plasticity in the rhabdovirus genome organization with the discovery of tri-segmented members with a unique evolutionary trajectory.

Molecular characterization of a novel cytorhabdovirus infecting Plumbago indica L.

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3' leader-N-(P')-P-P3-M-G-P6-L-5' trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus.

Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication.

We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio's single-molecule real-time sequencing and Oxford's Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767-8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS.

Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

Identification, molecular characterization and phylogenetic analysis of a novel nucleorhabdovirus infecting Paris polyphylla var. yunnanensis.

A novel betanucleorhabdovirus infecting Paris polyphylla var. yunnanensis, tentatively named Paris yunnanensis rhabdovirus 1 (PyRV1), was recently identified in Yunnan Province, China. The infected plants showed vein clearing and leaf crinkle at early stage of infection, followed by leaf yellowing and necrosis. Enveloped bacilliform particles were observed using electron microscopy. The virus was mechanically transmissible to Nicotiana bethamiana and N. glutinosa. The complete genome of PyRV1 consists of 13,509 nucleotides, the organization of which was typical of rhabdoviruses, containing six open reading frames encoding proteins N-P-P3-M-G-L on the anti-sense strand, separated by conserved intergenic regions and flanked by complementary 3'-leader and 5'-trailer sequences. The genome of PyRV1 shared highest nucleotide sequence identity (55.1%) with Sonchus yellow net virus (SYNV), and the N, P, P3, M, G, and L proteins showed 56.9%, 37.2%, 38.4%, 41.8%, 56.7%, and 49.4% amino acid sequence identities with respective proteins of SYNV, suggesting RyRV1 belongs to a new species of the genus Betanucleorhabdovirus.

Plasticity, Paralogy, and Pseudogenization: Rhabdoviruses of Freshwater Mussels Elucidate Mechanisms of Viral Genome Diversification and the Evolution of the Finfish-Infecting Rhabdoviral Genera.

Viruses in the family Rhabdoviridae display remarkable genomic variation and ecological diversity. This plasticity occurs despite the fact that, as negative sense RNA viruses, rhabdoviruses rarely if ever recombine. Here, we describe nonrecombinatorial evolutionary processes leading to genomic diversification in the Rhabdoviridae inferred from two novel rhabdoviruses of freshwater mussels (Mollusca: Bivalvia: Unionida). Killamcar virus 1 (KILLV-1) from a plain pocketbook (Lampsilis cardium) is closely related phylogenetically and transcriptionally to finfish-infecting viruses in the subfamily Alpharhabdovirinae. KILLV-1 offers a novel example of glycoprotein gene duplication, differing from previous examples in that the paralogs overlap. Evolutionary analyses reveal a clear pattern of relaxed selection due to subfunctionalization in rhabdoviral glycoprotein paralogs, which has not previously been described in RNA viruses. Chemarfal virus 1 (CHMFV-1) from a western pearlshell (Margaritifera falcata) is closely related phylogenetically and transcriptionally to viruses in the genus Novirhabdovirus, the sole recognized genus in the subfamily Gammarhabdovirinae, representing the first known gammarhabdovirus of a host other than finfish. The CHMFV-1 G-L noncoding region contains a nontranscribed remnant gene of precisely the same length as the NV gene of most novirhabdoviruses, offering a compelling example of pseudogenization. The unique reproductive strategy of freshwater mussels involves an obligate parasitic stage in which larvae encyst in the tissues of finfish, offering a plausible ecological mechanism for viral host-switching. IMPORTANCE Viruses in the family Rhabdoviridae infect a variety of hosts, including vertebrates, invertebrates, plants and fungi, with important consequences for health and agriculture. This study describes two newly discovered viruses of freshwater mussels from the United States. One virus from a plain pocketbook (Lampsilis cardium) is closely related to fish-infecting viruses in the subfamily Alpharhabdovirinae. The other virus from a western pearlshell (Margaritifera falcata) is closely related to viruses in the subfamily Gammarhabdovirinae, which until now were only known to infect finfish. Genome features of both viruses provide new evidence of how rhabdoviruses evolved their extraordinary variability. Freshwater mussel larvae attach to fish and feed on tissues and blood, which may explain how rhabdoviruses originally jumped between mussels and fish. The significance of this research is that it improves our understanding of rhabdovirus ecology and evolution, shedding new light on these important viruses and the diseases they cause.

Complete genome sequence of cnidium virus 2, a novel cytorhabdovirus isolated from Cnidium officinale in South Korea.

High-throughput sequencing identified a cytorhabdovirus, tentatively named "cnidium virus 2" (CnV2), in Cnidium officinale, and Sanger sequencing confirmed the genome sequence. CnV2 is 13,527 nucleotides in length and contains seven open reading frames in the order 3'-N-P-3-4-M-G-L-5', separated by intergenic regions. The full-length nucleotide sequence of CnV2 shares 19.4-53.8% identity with other known cytorhabdovirus genome sequences. The N, P, P3, M, G, and L proteins share 15.8-66.7%, 11-64.3%, 11.1-80.5%, 10.8-75.3%, 12.3-72.1%, and 20-72.7% amino acid sequence identity, respectively, with the cognate deduced protein sequences from known cytorhabdoviruses. CnV2 is related to other members of the genus Cytorhabdovirus, with sambucus virus 1 being the closest relative. Thus, CnV2 should be classified as a new member in the genus Cytorhabdovirus of the family Rhabdoviridae.

Gene network modeling and pathway analysis of maize transcriptomes in response to Maize Iranian mosaic virus.

Maize Iranian mosaic virus (MIMV, family Rhabdoviridae) is one of the factors limiting cereal production in Iran. In the present study, we sought to find critical genes and key pathways involved in MIMV infection and analyzed gene networks, pathways and promoters using transcriptome data. We determined the hub genes involved in pathways related to the proteasome and ubiquitin. The results showed the important role of the cellular endoplasmic reticulum in MIMV infection. Network cluster analysis confirmed the result of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The discovered miRNAs belonged to miR166, miR167, miR169, miR395, miR399, miR408 and miR482 families, which are involved in various pathogenicity or resistance processes against MIMV or other viruses. The results of this study provide a list of hub genes, important pathways and new insights for the future development of virus-resistant transgenic crops and clarify the basic mechanism of plant response.

Complete genome sequence of daphne virus 1, a novel cytorhabdovirus infecting Daphne odora.

A novel cytorhabdovirus was identified in Daphne odora in South Korea using high-throughput sequencing. The virus, tentatively named "daphne virus 1" (DV1), has a full-length genome sequence of 13,206 nucleotides with a genome organization comparable to that of unsegmented plant rhabdoviruses and contains seven antisense putative genes in the order 3'-leader-N-P'-P-P3-M-G-L-5'-trailer. The coding region of the genome is flanked by a 3' leader and a 5' trailer sequence, 261 and 151 nucleotides long, respectively. The DV1 genome shares 33.74%-57.44% nucleotide sequence identity with other cytorhabdoviruses. The DV1-encoded proteins share the highest amino acid sequence identity with homologues from Asclepias syriaca virus 1. Phylogenetic analysis showed that DV1 clustered with representative cytorhabdoviruses. We propose classifying DV1 in a new species within the genus Cytorhabdovirus, family Rhabdoviridae.

Identification and molecular characterization of a novel cytorhabdovirus from rose plants (Rosa chinensis Jacq.).

A novel negative-sense single-stranded RNA virus, tentatively named "rose-associated cytorhabdovirus" (RaCV), was identified by high-throughput sequencing. RaCV is 16,067 nucleotides in length and contains eight open reading frames (ORFs 1-8) encoding a nucleocapsid protein (N), a putative phosphoprotein (P), a putative P3 protein (P3), a putative P4 protein (P4), a putative matrix protein (M), a glycoprotein (G), a putative P7 protein (P7), and an RNA-dependent RNA polymerase (L), respectively. The coding genes are flanked by a 3' leader sequence (228 nt) and a 5' trailer sequence (251 nt) and are separated by conserved intergenic junctions (3'-AUUCUUUUUG(N)nCUN-5'). Phylogenetic analysis showed that RaCV clustered with yerba mate virus A (YmVA) within the cytorhabdovirus clade, and it exhibited low a degree of nt sequence similarity (<40% identity) to other rhabdoviruses. Amino acid sequence comparisons between the putative proteins of RaCV and the corresponding proteins of other cytorhabdoviruses showed that the sequence identity levels were far below the species demarcation cutoff of 80% for cytorhabdoviruses. These results suggest that RaCV should be classified as a new member of the genus Cytorhabdovirus.

Transgene-Free Genome Editing in Nicotiana benthamiana with CRISPR/Cas9 Delivered by a Rhabdovirus Vector.

The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems have become the most widely adopted genome editing platform owing to their unprecedented simplicity, efficiency, and versatility. Typically, the genome editing enzyme is expressed in plant cells from an integrated transgene delivered by either Agrobacterium-mediated or biolistic transformation. Recently, plant virus vectors have emerged as promising tools for the in planta delivery of CRISPR/Cas reagent. Here, we provide a protocol for CRISPR/Cas9-mediated genome editing in the model tobacco plant Nicotiana benthamiana using a recombinant negative-stranded RNA rhabdovirus vector. The method is based on infection of N. benthamiana with a Sonchus yellow net virus (SYNV)-based vector that carries the Cas9 and guide RNA expression cassettes to target specific genome loci for mutagenesis. With this method, mutant plants free of foreign DNA can be obtained within 4-5 months.

Monitoring for Anguillicoloides crassus, Anguillid herpesvirus 1, aquabirnavirus EVE and rhabdovirus EVEX in the European eel population of southern Spain.

European eel is critically endangered in Europe. Among other stressors, pathogens are well-known to harm eels' fitness. One hundred and eighty-two eels were captured in three Eel Management Units in Andalucía (SE Spain) and analysed for Anguillicoloides crassus, Anguillid herpesvirus 1 (AngHV1), the rhabdovirus Eel Virus European X (EVEX) and the aquabirnavirus Eel Virus European (EVE). A. crassus adults and preadults were isolated and morphometrically identified, and the eel swimbladders were artificially digested to count A. crassus larvae. Also, eel tissues were examined by PCRs for the presence of viruses. EVEX and EVE were not detected in any of the eels. The estimated prevalence (95% confidence limits) was 71 (64-78)% for A. crassus and 35 (28-42)% for AngHV-1, varying these prevalences significantly between and within EMUs. Moreover, A. crassus prevalence was highest in smaller eels, in sites closest to the sea and eels sampled in the autumn. By contrast, AngHV-1 prevalence was highest in biggest eels, in sites far from the sea and sampled in the summer or winter. However, in mixed effects logistic models including site as a random variable, the risk of infection was associated with distance to the sea in both A. crassus and AngHV-1 infections and also to winter sampling in the case of AngHV-1 and not to other variables. These results are evidence that both pathogens are highly endemic in eels from Andalusian habitats. Further studies are needed to better understand the risk factors associated with these pathogens on eel populations.

A Novel, Divergent Member of the Rhabdoviridae Family Infects Strawberry.

Strawberry (Fragaria × ananassa) is the most important berry crop worldwide and viruses pose a constant threat to the industry. In this communication, we describe a novel virus in the family Rhabdoviridae referred to as strawberry virus 3 (StrV-3). The virus does not show significant homology when compared with recognized rhabdoviruses and, therefore, the establishment of a new genus should be considered. A triplex reverse-transcription PCR test was developed and successfully employed in a survey of the National Clonal Germplasm Repository Fragaria collection. A CRISPR-Cas-based protocol was also developed and shown to detect the virus in as little as 1 fg of total RNA, a protocol to be used in the detection of the virus in candidate G1 plants. The strawberry aphid (Chaetosiphon fragaefolii) was evaluated-alas, unsuccessfully-as a potential vector of the virus. This work broadens our understanding of the family Rhabdoviridae and assists in the quest of releasing plant material free of viruses.