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Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

The accurate assembly of the ribonucleoprotein (RNP) complex is fundamental for the replication and transcription of rhabdoviruses, which are known for their broad pathogenic impact. A novel 119‐amino‐acid protein, NLRP12‐119aa is identified, encoded by the circular RNA circNLRP12, that effectively disrupts the formation of rhabdovirus RNP complexes through two distinct mechanisms and significantly reduces their replication. NLRP12‐119aa exhibits a strong affinity for the conserved 18‐nucleotide sequence at the start of the leader RNA of rhabdoviruses VSV, SCRV, and RABV, outcompeting their native N protein interactions, thereby disrupting the assembly of RNP complexes and inhibiting viral replication. NLRP12‐119aa exerts anti‐rhabdoviral effects by directly binding to the viral N protein, leading to its destabilization and accelerated degradation, and consequently hindering the formation of the viral RNP complex. To assess the therapeutic potential of circNLRP12 against rhabdovirus infections, a zebrafish model of VSV infection is established and noted a substantial reduction in viral load after‐treatment with circNLRP12, as well as the recovery of spleen's to a normalized state from its previously enlarged and hemorrhagic state. Collectively, these findings elucidate a novel dual anti‐RNP assembly strategy mediated by NLRP12‐119aa, offering valuable insights for further exploration and clinical management of rhabdoviral infections. The NLRP12‐119aa protein exerts its antiviral effect by two distinct mechanisms. First, it competes with the N protein of rhabdoviruses for binding to the viral leRNA, thereby preventing the formation of the RNP complex. Second, NLRP12‐119aa directly interacts with the rhabdovirus N protein, destabilizing it and promoting its degradation, which ultimately disrupts the assembly of the viral RNP complex.

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Rhabdoviruses are a family of enveloped negative-sense single-stranded RNA viruses infecting a variety of hosts. Recently, two vertically transmitted salmon louse (Lepeophtheirus salmonis) rhabdoviruses (LsRV) have been identified. The prevalence of these viruses was measured along the Norwegian coast and found to be close to 100%, and with the present lack of suitable cell lines to propagate these viruses, it is challenging to obtain material to study their host impact and infection routes. Thus, virus free lice strains were established from virus infected lice carrying one or both LsRVs by treating them with N protein dsRNA twice during development. The viral replication of the N protein was specifically down-regulated following introduction of virus-specific dsRNA, and virus-free lice strains were maintained for several generations. A preliminary study on infection routes suggested that the LsRV-No9 is maternally transmitted, and that the virus transmits from males to females horizontally. The ability to produce virus free strains allows for further studies on transmission modes and how these viruses influences on the L.salmonis interaction with its salmonid host. Moreover, this study provides a general fundament for future studies on how vertically transmitted rhabdoviruses influence the biology of their arthropod hosts.

Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. We have found that two sigma viruses (Rhabdoviridae) recently discovered in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for the sigma virus of Drosophila melanogaster, we find that both males and females can transmit these viruses to their offspring. Males transmit lower viral titers through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. In natural populations of D. obscura in the United Kingdom, we found that 39% of flies were infected and that the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data we estimate that the virus has swept across the United Kingdom within the past ∼11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on our lab estimates of transmission rates, we show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations at rates consistent with those measured in the field. Therefore, as predicted by our simulations, the virus has undergone an extremely rapid and recent increase in population size. In light of this and earlier studies of a related virus in D. melanogaster, we conclude that vertically transmitted rhabdoviruses may be common in insects and that these host–parasite interactions can be highly dynamic.

Deep sequencing was used to discover a novel rhabdovirus (Bas-Congo virus, or BASV) associated with a 2009 outbreak of 3 human cases of acute hemorrhagic fever in Mangala village, Democratic Republic of Congo (DRC), Africa. The cases, presenting over a 3-week period, were characterized by abrupt disease onset, high fever, mucosal hemorrhage, and, in two patients, death within 3 days. BASV was detected in an acute serum sample from the lone survivor at a concentration of 1.09 × 10(6) RNA copies/mL, and 98.2% of the genome was subsequently de novo assembled from ≈ 140 million sequence reads. Phylogenetic analysis revealed that BASV is highly divergent and shares less than 34% amino acid identity with any other rhabdovirus. High convalescent neutralizing antibody titers of >1:1000 were detected in the survivor and an asymptomatic nurse directly caring for him, both of whom were health care workers, suggesting the potential for human-to-human transmission of BASV. The natural animal reservoir host or arthropod vector and precise mode of transmission for the virus remain unclear. BASV is an emerging human pathogen associated with acute hemorrhagic fever in Africa.

We sequenced a novel rhabdovirus, Tupavirus delphini (dolphin tupavirus), from the brain of a stranded dead Atlantic white-sided dolphin with severe encephalitis in Canada. In situ hybridization linked presence of the virus to the animal's brain pathology and death. Our findings underscore the importance of monitoring marine mammals for unexpected pathogens.

Rhabdoviruses infect a large number of plant species and cause significant crop diseases. They have a negative-sense, single-stranded unsegmented or bisegmented RNA genome. The number of plant-associated rhabdovirid sequences has grown in the last few years in concert with the extensive use of high-throughput sequencing platforms. Here, we report the discovery of 27 novel rhabdovirus genomes associated with 25 different host plant species and one insect, which were hidden in public databases. These viral sequences were identified through homology searches in more than 3000 plant and insect transcriptomes from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) using known plant rhabdovirus sequences as the query. The identification, assembly and curation of raw SRA reads resulted in sixteen viral genome sequences with full-length coding regions and ten partial genomes. Highlights of the obtained sequences include viruses with unique and novel genome organizations among known plant rhabdoviruses. Phylogenetic analysis showed that thirteen of the novel viruses were related to cytorhabdoviruses, one to alphanucleorhabdoviruses, five to betanucleorhabdoviruses, one to dichorhaviruses and seven to varicosaviruses. These findings resulted in the most complete phylogeny of plant rhabdoviruses to date and shed new light on the phylogenetic relationships and evolutionary landscape of this group of plant viruses. Furthermore, this study provided additional evidence for the complexity and diversity of plant rhabdovirus genomes and demonstrated that analyzing SRA public data provides an invaluable tool to accelerate virus discovery, gain evolutionary insights and refine virus taxonomy.

Superinfection exclusion (SIE) is a finely tuned virus–virus interaction mechanism closely linked to the viral infection cycle. However, the mechanistic basis of SIE remains incompletely understood in plant viruses, particularly among negative‐sense, single‐stranded RNA viruses. In this study, we first describe the development of an efficient reverse genetics system for the plant nucleorhabdovirus Physostegia chlorotic mottle virus (PhCMoV) by codon optimisation of the large polymerase coding sequence. Using fluorescently tagged variants of PhCMoV, as well as three additional closely or distantly related plant rhabdoviruses, we found that each rhabdovirus displayed homotypic SIE. Moreover, two closely related alphanucleorhabdoviruses, PhCMoV and eggplant mottled dwarf virus, also exhibited mutual exclusion. Loss‐ and gain‐of‐function reverse genetics analyses identified the rhabdovirus matrix (M) protein as the central SIE effector: M‐deficient mutant viruses lost exclusion capacity, whereas ectopically expressed heterologous M proteins conferred SIE against otherwise compatible, distantly related rhabdoviruses. Additional functional assays demonstrated that the ability of rhabdovirus M proteins to suppress cognate and noncognate viral RNA synthesis correlated with the intra‐ and interspecies SIE capacity. The widespread occurrence of SIE across distinct plant rhabdoviruses underscores its importance for understanding the viral replication cycle and highlights its practical relevance for the development of novel virus control strategies. Superinfection exclusion occurs between variants of the same plant rhabdoviruses or between closely related viruses, which is mediated through repression of viral RNA synthesis by the matrix protein.

Bats are natural reservoir hosts of highly virulent pathogens such as Marburg virus, Nipah virus, and SARS coronavirus. However, little is known about the role of bat ectoparasites in transmitting and maintaining such viruses. The intricate relationship between bats and their ectoparasites suggests that ectoparasites might serve as viral vectors, but evidence to date is scant. Bat flies, in particular, are highly specialized obligate hematophagous ectoparasites that incidentally bite humans. Using next-generation sequencing, we discovered a novel ledantevirus (mononegaviral family Rhabdoviridae , genus Ledantevirus ) in nycteribiid bat flies infesting pteropodid bats in western Uganda. Mitochondrial DNA analyses revealed that both the bat flies and their bat hosts belong to putative new species. The coding-complete genome of the new virus, named Kanyawara virus (KYAV), is only distantly related to that of its closest known relative, Mount Elgon bat virus, and was found at high titers in bat flies but not in blood or on mucosal surfaces of host bats. Viral genome analysis indicates unusually low CpG dinucleotide depletion in KYAV compared to other ledanteviruses and rhabdovirus groups, with KYAV displaying values similar to rhabdoviruses of arthropods. Our findings highlight the possibility of a yet-to-be-discovered diversity of potentially pathogenic viruses in bat ectoparasites.