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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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Myxovirus resistance protein 1 (MX1) and MX2 are homologous, dynamin-like large GTPases, induced upon interferon exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus, by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with a GTPase domain and a carboxy-terminal stalk domain, connected by a bundle signaling element. These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging, and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against influenza A virus, Vesicular Stomatitis Virus, and La Crosse virus. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse, and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities but does modify MX1 protein subcellular localization. The discovery of this essential and highly conserved residue defines this region as key for antiviral activity and may reveal insights as to the mechanism(s) of action of MX1 proteins., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A novel rhabdovirus was isolated from the serum of a healthy Japanese wild boar ( Sus scrofa leucomystax ) and identified using the rapid determination system for viral nucleic acid sequences (RDV), next-generation sequencing, and electron microscopy. The virus was tentatively named wild boar rhabdovirus 1 (WBRV1). Phylogenetic analysis of the entire genome sequence indicated that WBRV1 is closely related to Tupaia rhabdovirus (TRV), which was isolated from cultured cells of hepatocellular carcinoma tissue of tree shrew. TRV has not been assigned to any genus of Rhabdoviridae till date. Analysis of the L gene indicated that WBRV1 belongs to the genus Vesiculovirus. These observations suggest that both TRV and WBRV1 belong to a new genus of Rhabdoviridae. Next-generation genome sequencing of WBRV1 revealed 5 open reading frames of 1329, 765, 627, 1629, and 6336 bases in length. The WBRV1 gene sequences are similar to those of other rhabdoviruses. Epizootiological analysis of a population of wild boars in Wakayama prefecture in Japan indicated that 6.5% were positive for the WBRV1 gene and 52% were positive for WBRV1-neutralizing antibodies. Furthermore, such viral neutralizing antibodies were found in domestic pigs in another prefecture. WBRV1 was inoculated intranasally and intraperitoneally into SCID and BALB/c mice and viral RNA was detected in SCID mice, suggesting that WBRV1 can replicate in immunocompromised mice. These results indicate this novel virus is endemic in wild animals and livestock in Japan. [ABSTRACT FROM AUTHOR]

The rhabdovirus matrix (M) protein is a multifunctional virion protein that plays major role in virus assembly and budding, virus-induced inhibition of host gene expression and cytopathic effects observed in infected cells. The myriad roles played by this protein in the virus biology make it a critical player in viral pathogenesis. Therefore, discerning the interactions of this protein with host can greatly facilitate our understanding of virus infections, ultimately leading to both improved therapeutics and insight into cellular processes. Chandipura virus (CHPV; Family Rhabdoviridae , Genus Vesiculovirus ) is an emerging rhabdovirus responsible for several outbreaks of fatal encephalitis among children in India. The present study aims to screen the human fetal brain cDNA library for interactors of CHPV M protein using yeast two-hybrid system. Ten host protein interactors were identified, three of which were further validated by affinity pull down and protein interaction ELISA. The study identified novel human host interactors for CHPV which concurred with previously described associations in other human viruses. [ABSTRACT FROM AUTHOR]

Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus. [ABSTRACT FROM AUTHOR]

Neuroinvasive viral infections invade the nervous system, often eliciting serious disease and death. Members of four viral families are both neuroinvasive and capable of transmitting progeny virions or virion components within the long neuronal extensions known as axons. Axons provide physical structures that enable viral infection to spread within the host while avoiding extracellular immune responses. Technological advances in the analysis of in vivo neural circuits, neuronal culturing, and live imaging of fluorescent fusion proteins have enabled an unprecedented view into the steps of virion assembly, transport, and egress involved in axonal spread. In this review we summarize the literature supporting anterograde (axon to cell) spread of viral infection, describe the various strategies of virion transport, and discuss the effects of spread on populations of neuroinvasive viruses. [ABSTRACT FROM AUTHOR]

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Thaumetopoea pityocampa (pine processionary moth) is one of the most important pine pests in the forests of Mediterranean countries, Central Europe, the Middle East and North Africa. Apart from causing significant damage to pinewoods, T. pityocampa occurrence is also an issue for public and animal health, as it is responsible for dermatological reactions in humans and animals by contact with its irritating hairs. High throughput sequencing technologies have allowed the fast and cost-effective generation of genetic information of interest to understand different biological aspects of non-model organisms as well as the identification of potential pathogens. Using these technologies, we have obtained and characterized the transcriptome of T. pityocampa larvae collected in 12 different geographical locations in Turkey. cDNA libraries for Illumina sequencing were prepared from four larval tissues, head, gut, fat body and integument. By pooling the sequences from Illumina platform with those previously published using the Roche 454-FLX and Sanger methods we generated the largest reference transcriptome of T. pityocampa. In addition, this study has also allowed identification of possible viral pathogens with potential application in future biocontrol strategies. [ABSTRACT FROM AUTHOR]

The advent of reverse genetic approaches to manipulate the genomes of both positive (+) and negative (−) sense RNA viruses allowed researchers to harness these genomes for basic research. Manipulation of positive sense RNA virus genomes occurred first largely because infectious RNA could be transcribed directly from cDNA versions of the RNA genomes. Manipulation of negative strand RNA virus genomes rapidly followed as more sophisticated approaches to provide RNA-dependent RNA polymerase complexes coupled with negative-strand RNA templates were developed. These advances have driven an explosion of RNA virus vaccine vector development. That is, development of approaches to exploit the basic replication and expression strategies of RNA viruses to produce vaccine antigens that have been engineered into their genomes. This study has led to significant preclinical testing of many RNA virus vectors against a wide range of pathogens as well as cancer targets. Multiple RNA virus vectors have advanced through preclinical testing to human clinical evaluation. This review will focus on RNA virus vectors designed to express heterologous genes that are packaged into viral particles and have progressed to clinical testing. [ABSTRACT FROM AUTHOR]

Effective oncolytic virus (OV) therapy is dependent on the ability of replication-competent viruses to kill infected cancer cells. We previously showed that human pancreatic ductal adenocarcinoma (PDAC) cell lines are highly heterogeneous in their permissiveness to vesicular stomatitis virus (VSV), in part due to differences in type I interferon (IFN) signaling. Here, using 10 human PDAC cell lines and three different VSV recombinants (expressing ΔM51 or wild type matrix protein), we examined cellular and viral factors affecting VSV-mediated apoptosis activation in PDACs. In most cell lines, VSVs activated both extrinsic and intrinsic apoptosis pathways, and VSV-ΔM51 primarily activated the type II extrinsic pathway. In cells with defective IFN signaling, all VSV recombinants induced robust apoptosis, whereas VSV-ΔM51 was a more effective apoptosis activator in PDACs with virus-inducible IFN signaling. Three cell lines constitutively expressing high levels of IFN-stimulated genes (ISGs) were resistant to apoptosis under most experimental conditions, even when VSV replication levels were dramatically increased by Jak inhibitor I treatment. Two of these cell lines also poorly activated apoptosis when treated with Fas activating antibody, suggesting a general defect in apoptosis. [ABSTRACT FROM AUTHOR]

Koolpinyah virus (KOOLV) isolated from healthy Australian cattle and Yata virus (YATV) isolated from a pool of Mansonia uniformis mosquitoes in the Central African Republic have been tentatively identified as rhabdoviruses. KOOLV was shown previously to be related antigenically to kotonkon virus, an ephemerovirus that has caused an ephemeral fever-like illness in cattle in Nigeria, but YATV failed to react antigenically with any other virus tested. Here we report the complete genome sequences of KOOLV (16,133 nt) and YATV (14,479 nt). Each has a complex genome organisation, with multiple genes, including a second non-structural glycoprotein (G NS ) gene and a viroporin (α1) gene, between the G and L genes as is characteristic of ephemeroviruses. Based on an analysis of genome organisation, sequence identity and cross-neutralisation, we demonstrate that both KOOLV and YATV should be classified as two new species in the genus Ephemerovirus . [ABSTRACT FROM AUTHOR]

Lyssavirus surveillance in bats was performed in Bangladesh during 2003 and 2004. No virus isolates were obtained. Three serum samples (all from Pteropus giganteus, n = 127) of 288 total serum samples, obtained from bats in 9 different taxa, neutralized lyssaviruses Aravan and Khujand. The infection occurs in bats in Bangladesh, but virus prevalence appears low.

Most pathogens utilize mucosal surfaces to enter and propagate within the host. As one of the main pathogens of fatal and highly contagious diseases, rhabdoviruses are distributed widely in nature affecting both human and animals. Therefore, local mucosal immune responses, most effectively induced by mucosal vaccines, act as frontline immunity to block the pathogens at its initial replication sites. However, the underlying regulatory mechanisms of mucosal immunity triggered by mucosal vaccine remains unclear. Herein, a rhabdoviruses glycoprotein-based mucosal vaccine (G131c) was used to elucidate the regulatory mechanism of local mucosal immunity in zebrafish, a typical immunological model. Firstly, we verified the strong immunoprotection of G131c mucosal vaccine. Furthermore, the delivery kinetics of G131c was evaluated in vivo , indicating the effective uptake of vaccines by mucosal tissues through immersion vaccination. Importantly, we demonstrate immersion with G131c vaccine could activate antigen presenting cells (APCs) at the local mucosal sites, and then arose robust local mucosal and systemic immune responses. More critically, we found that G131c mediated these immune effects by interacting with Toll-like receptor 2 (TLR2) and activating downstream MAPK and NF-κB signaling pathways. Thus, our findings provide previously unappreciated evidence that rhabdovirus glycoprotein could interact with TLR2 and then activate the APCs in local mucosal sites. This study provides a comprehensive perspective on the mechanism of TLR2-mediated mucosal immunity in the early vertebrates. A scheme showing the potential mechanism of TLR2-mediated local mucosal immunity in teleost fish. After immersion vaccination with SVCV G131c subunit vaccine, G131c antigen are taken up by antigen presenting cells (APCs) at the local mucosal tissues/sites. G131c antigen induces APCs activation by interacting with TLR2 and presented to T cells. Antigen-specific CD4-T cells then produce cytokines to activate B cells. Activated B cells start proliferating and differentiating into plasma cells to locally produce antigen-specific IgT, which will be transported by polymeric immunoglobulin receptor (pIgR) into mucus or other sites. [Display omitted] • Immersion vaccination with SVCV G131c subunit vaccine elicits strong immunoprotection. • The kinetics of G131c subunit vaccine in fish via immersion immunization was evaluated. • Immersion vaccination with G131c induces robust mucosal and systemic immune responses. • TLR2-mediated mucosal immune priming boosts anti-rhabdoviral immunity after immersion vaccination. [ABSTRACT FROM AUTHOR]

More than two thirds of emerging viruses are of zoonotic origin, and among them RNA viruses represent the majority. Ceratopogonidae (genus Culicoides) are well-known vectors of several viruses responsible for epizooties (bluetongue, epizootic haemorrhagic disease, etc.). They are also vectors of the only known virus infecting humans: the Oropouche virus. Female midges usually feed on a variety of hosts, leading to possible transmission of emerging viruses from animals to humans. In this context, we report here the analysis of RNA viral communities of Senegalese biting midges using next-generation sequencing techniques as a preliminary step toward the identification of potential viral biohazards. Sequencing of the RNA virome of three pools of Culicoides revealed the presence of a significant diversity of viruses infecting plants, insects and mammals. Several novel viruses were detected, including a novel Thogotovirus species, related but genetically distant from previously described tick-borne thogotoviruses. Novel rhabdoviruses were also detected, possibly constituting a novel Rhabdoviridae genus, and putatively restricted to insects. Sequences related to the major viruses transmitted by Culicoides, i.e., African horse sickness, bluetongue and epizootic haemorrhagic disease viruses were also detected. This study highlights the interest in monitoring the emergence and circulation of zoonoses and epizooties using their arthropod vectors.

The emergence of viruses in Coffee ( Coffea arabica and Coffea canephora ), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a member of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. [ABSTRACT FROM AUTHOR]

A rhabdovirus was isolated in cell culture inoculated with tissue material from diseased grayling, Thymallus thymallus (L.), originating from a fish farm affected by a mortality episode in Poland. Diagnostics tests showed that the virus was not related to novirhabdoviruses known in Europe, nor to vesiculovirus-like species, except perch rhabdovirus ( PRhV) with which it shared moderate serological relations. However, RT- PCR with PRhV probes gave negative results. To identify the virus, a random-priming sequence-independent single primer amplification was adopted. Surprisingly, two of the obtained sequences exhibited a high identity (>99%) with hirame rhabdovirus ( HIRRV), a novirhabdovirus usually found in fish in marine Asiatic countries, for instance Japan, China and Korea. The full-length sequence of the phosphoprotein gene (P) demonstrated a higher identity of the present isolate with HIRRV from China compared with the Korean isolate. An identical viral sequence was also found in brown trout, Salmo trutta trutta L., affected by mortalities in a second farm in the same region, after a likely contamination from the grayling farm. To our knowledge, this is the first report of HIRRV in Europe, and in two hosts from fresh water that have not been described before as susceptible species. [ABSTRACT FROM AUTHOR]

Abstract: The transcriptional strategy of orchid fleck virus (OFV), which has a two-segmented negative-strand RNA genome and resembles plant nucleorhabdoviruses, remains unexplored. In this study, the transcripts of six genes encoded by OFV RNA1 and RNA2 in the poly(A)-enriched RNA fraction from infected plants were molecularly characterized. All of the OFV mRNAs were initiated at a start sequence 3'-UU-5' with one to three non-viral adenine nucleotides which were added at the 5' end of each mRNA, whereas their 3' termini ended with a 5'-AUUUAAA(U/G)AAAA(A)n-3' sequence. We also identified the presence of polyadenylated short transcripts derived from the 3'-terminal leader regions of both genomic and antigenomic strands, providing the first example of plus- and minus-strand leader RNAs in a segmented minus-strand RNA virus. The similarity in the transcriptional strategy between this bipartite OFV and monopartite rhabdoviruses, especially nucleorhabdoviruses (family Rhabdoviridae) is additional support for their close relationship. [Copyright &y& Elsevier]

Lichens are symbiotic associations of fungi with green algae or cyanobacteria. They have arisen independently several times within the Ascomycota and Basidiomycota. This symbiosis became with time one of the most successful life forms on Earth. Outside of the symbiotic algae and fungi, there are endophytic fungi, other algae, and lichen-associated bacteria present within lichen thalli. Till now, no lichen-specific pathogens have been reported among bacteria and viruses. Around 15 dsRNA viruses are known from Eurotiomycetes and another dsRNA and reverse transcribed ssRNA viruses from Dothideomycetes containing some lichenized fungal lineages. Algal viruses have been identified from less than 1 % of known eukaryotic algal species but no virus has been found in Trebouxia or in Trentepohlia (Chlorophyta, Pleurastrophyceae, Pleurastrales), the most common green lichen photobionts. On the other hand, dsDNA viruses infecting related Chlorella algae are well known from freshwater phytoplankton. However, high-molecular weight dsRNA isolated from different lichen thalli indicated to us presence of ss or dsRNA viruses. A PCR-based search for viruses with genus-specific and species-specific primers resulted in amplification of genome segments highly identical with those of plant cytorhabdoviruses and with Apple mosaic virus (ApMV). The nucleotide sequence of the putative lichen cytorhabdovirus showed high identity (98 %) with Ivy latent cytorhabdovirus. The nucleotide sequences of six Apple mosaic virus isolates from lichens showed high similarity with ApMV isolates from apple and pear hosts. The lichen ApMV isolates were mechanically transmitted to an herbaceous host and detected positive in ELISA 14 days thereafter, which support its infectivity on plants. We prepared axenic cultures of photobionts identified as Trebouxia sp. from this ApMV-positive lichen samples. All these cultures were positive for ApMV in RT-PCR test. We suggest that lichens as a whole (or their photobionts, more specifically) could serve as reservoirs for viruses, despite the fact that the way of transmission between different organisms is not clear. We showed that lichens could harbour several viruses simultaneously, as the plant cytorhabdovirus and ApMV were detected in the same host, also. [ABSTRACT FROM AUTHOR]

Australian bat lyssavirus (ABLV) is a recently emerged rhabdovirus of the genus lyssavirus considered endemic in Australian bat populations that causes a neurological disease in people indistinguishable from clinical rabies. There are two distinct variants of ABLV, one that circulates in frugivorous bats (genus Pteropus) and the other in insectivorous microbats (genus Saccolaimus). Three fatal human cases of ABLV infection have been reported, the most recent in 2013, and each manifested as acute encephalitis but with variable incubation periods. Importantly, two equine cases also arose recently in 2013, the first occurrence of ABLV in a species other than bats or humans. Similar to other rhabdoviruses, ABLV infects host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion facilitated by its single fusogenic envelope glycoprotein (G). Recent studies have revealed that proposed rabies virus (RABV) receptors are not sufficient to permit ABLV entry into host cells and that the unknown receptor is broadly conserved among mammalian species. However, despite clear tropism differences between ABLV and RABV, the two viruses appear to utilize similar endocytic entry pathways. The recent human and horse infections highlight the importance of continued Australian public health awareness of this emerging pathogen. [ABSTRACT FROM AUTHOR]

Background Ticks are implicated as hosts to a wide range of animal and human pathogens. The full range of microbes harbored by ticks has not yet been fully explored. Methods As part of a viral surveillance and discovery project in arthropods, we used unbiased highthroughput sequencing to examine viromes of ticks collected on Long Island, New York in 2013. Results We detected and sequenced the complete genome of a novel rhabdovirus originating from a pool of Amblyomma americanum ticks. This virus, which we provisionally name Long Island tick rhabdovirus, is distantly related to Moussa virus from Africa. Conclusions The Long Island tick rhabdovirus may represent a novel species within family Rhabdoviridae. [ABSTRACT FROM AUTHOR]

Abstract: Flanders virus (FLAV) and Hart Park virus (HPV) are rhabdoviruses that circulate in mosquito–bird cycles in the eastern and western United States, respectively, and constitute the only two North American representatives of the Hart Park serogroup. Previously, it was suggested that FLAV is unique among the rhabdoviruses in that it contains two pseudogenes located between the P and M genes, while the cognate sequence for HPV has been lacking. Herein, we demonstrate that FLAV and HPV do not contain pseudogenes in this region, but encode three small functional proteins designated as U1–U3 that apparently arose by gene duplication. To further investigate the U1–U3 region, we conducted the first large-scale evolutionary analysis of a member of the Hart Park serogroup by analyzing over 100 spatially and temporally distinct FLAV isolates. Our phylogeographic analysis demonstrates that although FLAV appears to be slowly evolving, phylogenetically divergent lineages co-circulate sympatrically. [Copyright &y& Elsevier]

Abstract: Viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. A metagenomics approach was used to identify virus sequences in Australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. Sequence comparisons revealed strains of Liao Ning virus (Reovirus, Seadornavirus), previously detected only in China, livestock-infecting Stretch Lagoon virus (Reovirus, Orbivirus), two novel dimarhabdoviruses, named Beaumont and North Creek viruses, and two novel orthobunyaviruses, named Murrumbidgee and Salt Ash viruses. The novel virus proteomes diverged by ≥50% relative to their closest previously genetically characterized viral relatives. Deep sequencing also generated genomes of Warrego and Wallal viruses, orbiviruses linked to kangaroo blindness, whose genomes had not been fully characterized. This study highlights viral metagenomics in concert with traditional arbovirus surveillance to characterize known and new arboviruses in field-collected mosquitoes. Follow-up epidemiological studies are required to determine whether the novel viruses infect humans. [Copyright &y& Elsevier]

Highlights: [•] The zebrafish beta defensin 2 (zfBD2) possesses antiviral properties against the spring viraemia of carp (SVC) virus. [•] The immunomodulatory properties of the zfBD2 result in protection of adult zebrafish against SVCV in vivo. [•] Adjuvant-like properties of zfBD2 improve the efficacy of a DNA vaccine against SVCV. [•] Most likely, zfBD2 modulates the zebrafish immune system through an initial activation of the master regulator NF-κB. [Copyright &y& Elsevier]

Two new rabies-related viruses were discovered in Russia during 2002. Viruses were isolated from bats in Eastern Siberia near Baikal Lake and in the western Caucasus Mountains. After preliminary antigenic and genetic characterization, we found that both viruses should be considered as new putative lyssavirus genotypes.

To better understand the response of largemouth bass ( Micropterus salmoides ) to Micropterus salmoides rhabdovirus (MSRV) infection, we investigated the intestinal bacterial flora and transcriptome profile of fish at 72 hours post-infection (hpi). Total of 1574 differentially expressed genes (DEGs) were identified in largemouth bass spleen following MSRV infection, including 573 upregulated and 1001 downregulated genes. KEGG and GO enrichment analysis revealed that upregulated genes were enriched in certain antiviral related signaling pathway, including NOD-like receptor (NLR), RIG-I like receptors (RLR) and regulation of the interferon (IFN)-γ-mediated signaling pathway, whereas some immune-related DEGs enriched in focal adhesion (FA) and ECM-receptor interaction(ECM-RI) were downregulated, as well as genes associated with metabolic processes, such as peroxisome proliferator-activated receptors (PPAR), adipocytokine signaling pathway, Glycerolipid and Retinol metabolism. Furthermore, the principal component analysis (PCA) and phylogenetic analysis revealed that MSRV infection significantly affected the microbiota of largemouth bass intestine; the LEfSe analysis showed that relative abundances of Streptococcus were significantly increased, while the content of Akkermansia, Enterococcus and Lactobacillus were remarkably decreased in the fish intestine following MSRV infection. Additionally, a high correlation was determined between the expressions of interferon-related upregulated genes and the relative abundance of Streptococcus by redundancy analysis (RDA). These results collectively illustrated that intestinal microbiota composition might be associated with the immune-related gene expression in largemouth bass in response to MSRV infection., Competing Interests: Author SQ is employed by Huzhou Baijiayu Biotech Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fei, Yi, Zhang, Cheng, Zhang, Yu, Qian, Huang and Yang.)

Potato yellow dwarf virus (PYDV) is the type species of the genus Nucleorhabdovirus and, like all members of this genus, replication and morphogenesis occurs inside the nuclei of infected cells. Protein localization prediction algorithms failed to identify a nuclear localization signal (NLS) in PYDV nucleocapsid (N) protein, although PYDV-N has been shown to localize exclusively to the nucleus when expressed as a GFP:N fusion in plant cells. Deletion analysis using fragments of PYDV-N identified a karyophillic region in the carboxy-terminal 122 amino acids. Alanine-scanning mutagenesis was performed across this region in the context of the full-length N protein. Mutants were assayed for their ability to nuclear localize using live-cell imaging and a yeast-based assay. Two amino acid motifs, 419QKR421 and 432KR433 were shown to be essential for nuclear import and interaction with importin-α. Additional bimolecular fluorescence complementation (BiFC) showed that the PYDV-N NLS mutants cannot be ferried into the nucleus via interaction with PYDV-P or -M. In contrast, interaction with N NLS mutants appeared to retard the nuclear import of PYDV-P. GFP fused to aa 419-434 established that the PYDV-N-NLS can function outside the context of this protein. Taken together, it was determined that PYDV-N contains the bipartite NLS 419QKRANEEAPPAAQKR433.

Two novel rhabdoviruses were isolated from birds during surveillance for arboviral encephalitis in the northeastern United States. The first, designated Farmington virus, is a tentative new member of the Vesiculovirus genus. The second, designated Rhode Island virus, is unclassified antigenically, but its ultrastructure and size are more similar to those of some of the plant rhabdoviruses. Both viruses infect birds and mice, as well as monkey kidney cells in culture, but their importance for human health is unknown.

Durante los años 2004 y 2005 se observaron síntomas de la leprosis de los cítricos en los departamentos de Meta y Casanare. Para confirmar la causa de esta sintomatología, se colectaron hojas de naranja ‘Valencia’ con lesiones típicas en municipios de Casanare (Yopal y Aguazul) y Meta (Guamal, Villavicencio y Cumaral). Las muestras fueron procesadas identificar el virus por medio de microscopía electrónica de transmisión (MET) y transcripción reversa - reacción en cadena de la polimerasa (RT-PCR). Diez de las 21 muestras analizadas por MET resultaron positivas para el virus de la leprosis (Citrus Leprosis Virus, CiLV-C). En algunas muestras se observaron partículas baciliformes características del CiLV-C en el retículo endoplasmático y formas irregularles de viroplasmas en el citoplasma. Catorce de las 32 muestras procesadas por RT-PCR resultaron positivas al CiLV-C. Los análisis RT-PCR amplificaron fragmentos de cADN del tamaño esperado para las muestras colectadas en el Meta. Una de las ampliaciones fue secuenciada (GenBank accessión No. DQ272491) y la secuencia obtenida guarda identidad del 98% con la secuencia brasilera aislada de nucleótidos para CiLV-C (GenBank accessión No. AY289190.1). La secuencia obtenida y los primers fueron entregados al Instituto Colombiano Agropecuario —ICA— para el diagnóstico del virus CiLV-C en Colombia. Entre los ácaros recolectados en las plantas afectadas se identificó a Brevipalpus phoenicis (Geijskes) reconocido como principal vector de CiLV-C.

Abstract: Australian bat lyssavirus (ABLV) is a rhabdovirus of the lyssavirus genus capable of causing fatal rabies-like encephalitis in humans. There are two variants of ABLV, one circulating in pteropid fruit bats and another in insectivorous bats. Three fatal human cases of ABLV infection have been reported with the third case in 2013. Importantly, two equine cases also arose in 2013; the first occurrence of ABLV in a species other than bats or humans. We examined the host cell entry of ABLV, characterizing its tropism and exploring its cross-species transmission potential using maxGFP-encoding recombinant vesicular stomatitis viruses that express ABLV G glycoproteins. Results indicate that the ABLV receptor(s) is conserved but not ubiquitous among mammalian cell lines and that the two ABLV variants can utilize alternate receptors for entry. Proposed rabies virus receptors were not sufficient to permit ABLV entry into resistant cells, suggesting that ABLV utilizes an unknown alternative receptor(s). [Copyright &y& Elsevier]

Abstract: Spring viraemia of carp (SVC) is a disease of international importance that predominantly affects cyprinid fish and can cause significant mortality. In the United Kingdom (UK), SVC was first detected in 1977 with further cases occurring in fisheries, farms, wholesale and retail establishments throughout England and Wales (but not Scotland, where few cyprinid populations exist, nor Northern Ireland where SVC has never been detected) over the subsequent 30years. Following a control and eradication programme for the disease initiated in 2005, the UK was recognised free of the disease in 2010. This study compiles historic records of SVC cases in England and Wales with a view to understanding its routes of introduction and spread, and assessing the effectiveness of the control and eradication programme in order to improve contingency plans to prevent and control future disease incursions in the cyprinid fish sectors. Between 1977 and 2010 the presence of SVC was confirmed on 108 occasions, with 65 of the cases occurring in sport fisheries and the majority of the remainder occurring in the ornamental fish sector. The study found that throughout the history of SVC in the UK, though cases were widely distributed, their occurrence was sporadic and the virus did not become endemic. All evidence indicates that SVC was not able to persist under UK environmental conditions, suggesting that the majority of cases were a result of new introductions to the UK as opposed to within-country spread. The control and eradication programme adopted in 2005 was highly effective and two years after its implementation cases of SVC ceased. Given the non-persistent nature of the pathogen the most important aspect of the control programme focused on preventing re-introduction of the virus to the UK. Despite the effectiveness of these controls against SVC, this approach is likely to be less effective against more persistent pathogens such as koi herpesvirus, which are likely to require more stringent measures to prevent within-country spread. [Copyright &y& Elsevier]

Although some DNA vaccines have proved to be very efficient in field trials, their authorisation still remains limited to a few countries. This is in part due to safety issues because most of them contain viral regulatory sequences to driving the expression of the encoded antigen. This is the case of the only DNA vaccine against a fish rhabdovirus (a negative ssRNA virus), authorised in Canada, despite the important economic losses that these viruses cause to aquaculture all over the world. In an attempt to solve this problem and using as a model a non-authorised, but efficient DNA vaccine against the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV), we developed a plasmid construction containing regulatory sequences exclusively from fish origin. The result was an 'all-fish vector', named pJAC-G, containing 5′ and 3′ regulatory sequences of β-acting genes from carp and zebrafish, respectively. In vitro and in vivo, pJAC-G drove a successful expression of the VHSV glycoprotein G (G), the only antigen of the virus conferring in vivo protection. Furthermore, and by means of in vitro fusion assays, it was confirmed that G protein expressed from pJAC-G was fully functional. Altogether, these results suggest that DNA vaccines containing host-homologous gene regulatory sequences might be useful for developing safer DNA vaccines, while they also might be useful for basic studies. [ABSTRACT FROM AUTHOR]

Abstract: Viruses may remain infectious outside the host cell for considerable time and represent a source of accidental infection if not properly inactivated. In this study, the survival of vesicular stomatitis virus (VSV) in suspension and dried on surfaces was analyzed. In addition, the sensitivity of VSV to disinfectants and physicochemical changes was investigated. VSV showed a notable stability in suspension at 4°C with virus titers remaining high over several weeks. The presence of serum proteins had a stabilizing effect on virus infectivity, whereas elevated temperatures reduced survival times. VSV dried on polystyrene, glass or stainless steel surfaces remained infectious for at least 6 days at ambient temperature. VSV showed a remarkable resistance to extreme pH in particular in the alkaline range, but could be rapidly inactivated by heating at 55°C or higher. The virus was highly sensitive to inactivation by commonly used disinfectants such as aldehydes, alcohols, and detergents. The high stability of VSV on surfaces and in suspension may facilitate dissemination of the virus in livestock by contaminated feeding and water troughs, hands, and milking equipment. This knowledge on the sensitivity of VSV to disinfectants will help to set up appropriate hygiene measures. [Copyright &y& Elsevier]

Abstract: The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus (VSV), a prototypic nonsegmented negative strand (NNS) RNA virus classified into the Rhabdoviridae family, has been used to investigate the fundamental molecular mechanisms of NNS RNA viral mRNA synthesis and processing. In vitro studies on mRNA cap formation with the VSV L protein eventually led to the discovery of the unconventional mRNA capping pathway catalyzed by the guanosine 5′-triphosphatase and RNA:GDP polyribonucleotidyltransferase (PRNTase) activities. The PRNTase activity is a novel enzymatic activity, which transfers 5′-monophosphorylated (p-) RNA from 5′-triphosphorylated (ppp-) RNA to GDP to form 5′-capped RNA (GpppRNA) in a viral mRNA-start sequence-dependent manner. This unconventional capping (pRNA transfer) reaction with PRNTase can be experimentally distinguished from the conventional capping (GMP transfer) reaction with eukaryotic GTP:RNA guanylyltransferase (GTase) on the basis of the following differences in their substrate specificity for the cap formation: PRNTase uses GDP and pppRNA, but not ppRNA, whereas GTase employs GTP, but not GDP, and ppRNA. The pRNA transfer reaction with PRNTase proceeds through a covalent enzyme-pRNA intermediate with a phosphoamide bond. Hence, to prove the PRNTase activity, it is necessary to demonstrate the following consecutive steps separately: (1) the enzyme forms a covalent enzyme-pRNA intermediate, and (2) the intermediate transfers pRNA to GDP. This article describes the methods for in vitro transcription and capping with the recombinant VSV L protein, which permit detailed characterization of its enzymatic reactions and mapping of active sites of its enzymatic domains. It is expected that these systems are adaptable to rhabdoviruses and, by extension, other NNS RNA viruses belonging to different families. [Copyright &y& Elsevier]

Initiation is the primary target of translational control for all organisms. Regulation of eukaryotic translation is traditionally thought to occur through initiation factors and RNA structures. Here, we characterize a transcript-specific translation initiation mechanism that is mediated by the ribosome. By studying vesicular stomatitis virus (VSV), we identify the large ribosomal subunit protein rpL40 as requisite for VSV cap-dependent translation but not bulk cellular or internal ribosome entry site-driven translation. This requirement is conserved among members of the order Mononegavirales, including measles virus and rabies virus. Polysome analyses and in vitro reconstitution of initiation demonstrate that rpL40 is required for 80S formation on VSV mRNAs through a cis-regulatory element. Using deep sequencing, we further uncover a subset of cellular transcripts that are selectively sensitive to rpL40 depletion, suggesting VSV may have usurped an endogenous translation pathway. Together, these findings demonstrate that the ribosome acts as a translational regulator outside of its catalytic role during protein synthesis. [ABSTRACT FROM AUTHOR]

Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host shifts. And yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein, we uncovered viruses of flatworms (phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda, and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of neodermatans to a parasitic lifestyle. Most Neodermata viruses seem to codiversify with their host, with the exception of rhabdoviruses, which may switch hosts more often, based on phylogenetic relationships. Neodermata rhabdoviruses also have a position ancestral to vertebrate-associated rhabdo viruses, including lyssaviruses, suggesting that vertebrate-associated rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases. IMPORTANCE Little is known about the diversity of parasite-associated viruses and how these viruses may impact parasite fitness, parasite-host interactions, and virus evolution. The discovery of over a hundred viruses associated with a range of free-living and parasitic flatworms, including parasites of economic and clinical relevance, allowed us to compare the viromes of flatworms with contrasting lifestyles. The results suggest that flatworms acquired novel viruses after their transition to a parasitic lifestyle and highlight the possibility that they acquired viruses from their hosts and vice versa. An interesting example is the discovery of flatworm rhabdoviruses that have a position ancestral to rabies viruses and other vertebrate-associated rhabdoviruses, demonstrating that flatworm-associated viruses have emerged in a vertebrate host at least once in history. Therefore, parasitic flatworms may play a role in virus diversity and emergence. The roles that parasite-infecting viruses play in parasite-associated diseases remain to be investigated.

Honey bees are globally important pollinators threatened by many different pathogens, including viruses. We investigated the virome of honey bees collected at the end of the beekeeping season (August/September) in Czechia, a Central European country. Samples were examined in biological replicates to assess the homogeneity, stability, and composition of the virome inside a single hive. By choice of healthy workers from colonies, where Varroa destructor was under control, we could identify ubiquitous bee viruses. Deformed wing virus (DWV) was highly prevalent, even though the bees were healthy, without any noticeable disease signs. The overall virome composition (consisting of honey bee-, plant-, and bacterium-infecting viruses) was driven primarily by the hive and its location. However, honey bee-specific viruses showed an uneven distribution within the same hive. In addition, our results point to an unusual cooccurrence between two rhabdoviruses and reveal the presence of five distinct lineages of Lake Sinai viruses (LSVs) clustering with other LSV strains described globally. Comparison of our results with the virome of Australian honey bees, the last truly Varroa- and DWV-free population, showed a strong difference with respect to DWV and a set of diverse members of the Picornavirales , of which the latter were absent in our samples. We hypothesize that the occurrence of DWV introduced by Varroa strongly affects the virome structure despite the mite being under control. IMPORTANCE The Western honey bee, Apis mellifera, is a vital part of our ecosystem as well as cultural heritage. Annual colony losses endanger beekeeping. In this study, we examined healthy bees from the heart of Central Europe, where honey bee colonies have been commonly affected by varroosis over 5 decades. Our virome analysis showed the presence of ubiquitous viruses in colonies where the mite Varroa destructor was under control and no honey bee disease signs were observed. Compared to previous studies, an important part of our study was the analysis of multiple replicates from individual hives. Our overall results indicate that the virome structure (including bee-infecting viruses, plant-infecting viruses, and bacteriophages) is stable within hives; however, the bee-infecting viruses varied largely within interhive replicates, suggesting variation of honey bee viruses within individual bees. Of interest was the striking difference between the viromes of our 39 pools and 9 pools of honey bee viromes previously analyzed in Australia. It could be suggested that Varroa not only affects DWV spread in bee colonies but also affects diverse members of the Picornavirales , which were strongly decreased in Czech bees compared to the Varroa - and DWV-naive Australian bees.

Abstract: Kimberley virus (KIMV) is an arthropod-borne rhabdovirus that was isolated in 1973 and on several subsequent occasions from healthy cattle, mosquitoes (Culex annulirostris) and biting midges (Culicoides brevitarsis) in Australia. Malakal virus (MALV) is an antigenically related rhabdovirus isolated in 1963 from mosquitoes (Mansonia uniformis) in Sudan. We report here the complete genome sequences of KIMV (15442 nt) and MALV (15444 nt). The genomes have a similar organisation (3′-l-N-P-M-G-GNS-α1-α2-β-γ-L-t-5′) to that of bovine ephemeral fever virus (BEFV). High levels of amino acid identity in each gene, similar gene expression profiles, clustering in phylogenetic analyses of the N, P, G and L proteins, and strong cross-neutralisation indicate that KIMV and MALV are geographic variants of the same ephemerovirus that, like BEFV, occurs in Africa, Asia and Australia. [Copyright &y& Elsevier]