Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Rescue of a plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies.

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.

How do filovirus filaments bend without breaking?

Viruses of the Mononegavirales have helical nucleocapsids containing a single-stranded negative-sense RNA genome complexed with the nucleoprotein and several other virus-encoded proteins. This RNA-protein complex acts as the template for replication and transcription during infection. Recent structural data has advanced our understanding of how these functions are achieved in filoviruses, which include dangerous pathogens such as Ebola virus. Polyploid filoviruses package multiple genome copies within strikingly long filamentous viral envelopes, which must be flexible to avoid breakage of the 19kb non-segmented genomic RNA. We review how the structure of filoviruses and paramyxoviruses permits this morphological flexibility in comparison to rhabdoviruses that have short, bullet-shaped virions with relatively rigid envelopes.

Molecular and cellular aspects of rhabdovirus entry.

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.

RNA splicing in a new rhabdovirus from Culex mosquitoes.

Among members of the order Mononegavirales, RNA splicing events have been found only in the family Bornaviridae. Here, we report that a new rhabdovirus isolated from the mosquito Culex tritaeniorhynchus replicates in the nuclei of infected cells and requires RNA splicing for viral mRNA maturation. The virus, designated Culex tritaeniorhynchus rhabdovirus (CTRV), shares a similar genome organization with other rhabdoviruses, except for the presence of a putative intron in the coding region for the L protein. Molecular phylogenetic studies indicated that CTRV belongs to the family Rhabdoviridae, but it is yet to be assigned a genus. Electron microscopic analysis revealed that the CTRV virion is extremely elongated, unlike virions of rhabdoviruses, which are generally bullet shaped. Northern hybridization confirmed that a large transcript (approximately 6,500 nucleotides [nt]) from the CTRV L gene was present in the infected cells. Strand-specific reverse transcription-PCR (RT-PCR) analyses identified the intron-exon boundaries and the 76-nt intron sequence, which contains the typical motif for eukaryotic spliceosomal intron-splice donor/acceptor sites (GU-AG), a predicted branch point, and a polypyrimidine tract. In situ hybridization exhibited that viral RNAs are primarily localized in the nucleus of infected cells, indicating that CTRV replicates in the nucleus and is allowed to utilize the host's nuclear splicing machinery. This is the first report of RNA splicing among the members of the family Rhabdoviridae.

Identification and characterization of structural proteins of orchid fleck virus.

Orchid fleck virus (OFV) has a bipartite negative-sense RNA genome with sequence similarities to plant rhabdoviruses. The non-enveloped bullet-shaped particles of OFV are similar to those of the internal ribonucleoprotein (RNP)-M protein structure of rhabdoviruses, but they are about half the size of typical plant rhabdoviruses. Purified preparations contained intact bullet-shaped and filamentous particles. The filamentous particles showed a tightly coiled coil structure or a coiled structure with a helical twist, which resembles the RNP complex of rhabdoviruses. OFV bullet-shaped particles were structurally stable in solutions containing 2% Triton X-100 and 0.8 M NaCl. Western blot analyses revealed that the bullet-shaped particles contained N, P and M proteins, while filamentous particles contained mainly N and P proteins. In addition, a small amount of the L protein was detected in both types of particles. Thus, the structural proteins of OFV have properties similar to those of rhabdoviruses, except that the particles are non-enveloped and are relatively resistant to detergent-treatment under high-salt conditions.

Truncated particles produced in fish surviving infectious hematopoietic necrosis virus infection: mediators of persistence?

Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that produces an acute, lethal infection in rainbow trout (Oncorhynchus mykiss). Fish that survive infection cease to produce detectable infectious virus at approximately 46 days after infection, yet there is evidence that survivor fish continue to harbor virus particles (B. S. Drolet, P. P. Chiou, J. Heidel, and J. C. Leong, J. Virol. 69:2140-2147, 1995). In an effort to determine the biological function of these particles, the kidneys and livers from IHNV survivors were harvested and divided into samples for nested reverse transcriptase PCR analysis and explant culture. Sequences for the IHNV nucleoprotein and polymerase genes were detected in 50 and 89%, respectively, of the organs from survivor fish. When explant tissue cultures were infected with purified standard IHNV, the liver tissues from survivor fish produced up to 10-fold less virus than naive control fish liver tissues. In addition, immunosorbent electron microscopy analysis of the supernatant media from the cultured explants of survivor fish revealed truncated particles, whereas the control tissue supernatants contained only standard viral particles. These results suggest that the truncated IHNV particles observed in persistently infected fish are defective interfering particles that may mediate virus persistence.

Vesicular stomatitis virus M protein may be inside the ribonucleocapsid coil.

Vesicular stomatitis virus is an enveloped virus with an external glycoprotein G and a nucleocapsid that form, together with the M protein, a tight helically coiled structure: the skeleton. Negative staining and immunoelectron microscopy studies on skeleton preparations were performed to determine the localization of the M protein. These studies have resulted in a new model for the structure of rhabdoviruses in which the nucleocapsid is wound around a core containing the M protein. This model predicts contact between M and lipid only at the extreme ends of the skeleton, which is confirmed by skeleton-liposome binding studies.

Manitoba virus, a new rhabdovirus isolated from Culex tarsalis mosquitoes collected in Manitoba, Canada.

A rhabdovirus, Mn 936-77, was isolated from a pool of two Culex tarsalis collected on August 16, 1977, from Morris, Manitoba. Isolate Mn 936-77 was not pathogenic for suckling Swiss white mice inoculated by the intracerebral route. The virus propagated in three vertebrate cell lines (Vero, primary chick embryo, mouse neuroblastoma), but apparently not in Aedes albopictus C6/36 cells. Isolate Mn 936-77 did not react by amplified enzyme-linked immunosorbant assay with 230 viruses of proven or possible arbovirus etiology or by immunofluorescence with 88 members of the family Rhabdoviridae. Isolate Mn 936-77 appears to be a newly discovered virus for which the name Manitoba virus is proposed.

A new virus isolate from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps.

A new virus was isolated from infectious hypodermal and hematopoietic necrosis virus (IHHNV)-infected penaeid shrimps. The virus was isolated from two species of penaeid shrimps obtained from three different sources employing a previously developed cell-culture assay. Electron-microscopical studies of both purified virus and infected cells showed bullet-shaped particles identifying it as a rhabdovirus.

The free nucleocapsids of the viral haemorrhagic septicaemia virus contain two antigenically related nucleoproteins.

A protein of 34 kDa (Nx) was induced in vitro by the infection of fish cell cultures with the rhabdovirus agent of viral haemorrhagic septicaemia (VHS) of the trout. This protein only appeared as a major component in concentrated or intracellular labeled VHS virus but not in purified VHS or in the related infectious haematopoietic necrosis virus. That Nx protein is antigenically related to the nucleoprotein of purified virus was shown by its reaction with four anti-nucleoprotein monoclonal antibodies (at least 3 of them reacting non competitively against different epitopes) and by immunoprecipitation with polyvalent international reference sera. The Nx protein was shown to be specifically associated with free non-infective particles isolated by ultracentrifugation which were confirmed to be nucleocapsids by electron microscopy.

Isolation and characterization of a tupaia rhabdovirus.

Liver tumor biopsies of a 9-year-old moribund Tupaia (tree shrew) were explanted and cultured in vitro. The cell cultures degenerated spontaneously. A virus was isolated from the cell-free supernatant of these cultures and subsequent electron microscopy revealed rhabdovirus-like particles. Negative staining showed typical bullet-shaped particles 125-220 nm in length with a diameter of 68 nm studded with a dense layer of surface projections 9-11 nm in length. One end of the virion was flat, the other end was open; a distinct ribonucleoprotein (RNP) core was visible. The pitch of the RNP was 4.5 nm. Virus was assembled and matured by budding primarily into regions of dilated endoplasmic reticulum. The dimensions of the virion also were determined in ultrathin sections: the diameter and length of the virion were 52 and 125-255 nm, respectively, those of the RNP core were 39 and 120-240 nm. Only tupaia embryonic fibroblasts and kidney cells were susceptible to the rhabdovirus. The virus, when plaque-assayed on tupaia embryonic fibroblasts, grew to a titer of 1 X 10(6) PFU.

Serological relationships between the nucleocapsids of some planthopper-borne rhabdoviruses of cereals.

Leaves infected with barley yellow striate mosaic virus (BYSMV) from Italy, wheat chlorotic streak virus (WCSV) from France, northern cereal mosaic virus (NCMV) from Japan, maize sterile stunt virus (MSSV) from Australia or Shiraz maize rhabdovirus (SMRV) from Iran were homogenized in buffered 1% Nonidet P-40, releasing intact nucleocapsids. These (except SMRV) were trapped on electron microscope grids using appropriate antisera and tested by decoration with serial dilutions of antisera to BYSMV, NCMV, MSSV, Moroccan wheat rhabdovirus (MWRV), wheat rosette stunt virus (WRSV) from China, and maize mosaic virus (MMV) from Venezuela. The results suggest that BYSMV and NCMV, though related, are distinct viruses; MWRV and WCSV are strains of BYSMV; MSSV is intermediate between BYSMV and NCMV but may be considered a strain of BYSMV; WRSV is a strain of NCMV; and MMV and SMRV are unrelated to each other and to the other viruses tested. SMRV was morphologically quite different from BYSMV, NCMV, WCSV and MSSV, all of which had the same morphology.