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.

Human Tibroviruses: Commensals or Lethal Pathogens?

Rhabdoviruses are a large and ecologically diverse family of negative-sense RNA viruses (Mononegavirales: Rhabdoviridae). These viruses are capable of infecting an unexpectedly wide variety of plants, vertebrates, and invertebrates distributed over all human-inhabited continents. However, only a few rhabdoviruses are known to infect humans: a ledantevirus (Le Dantec virus), several lyssaviruses (in particular, rabies virus), and several vesiculoviruses (e.g., Chandipura virus, vesicular stomatitis Indiana virus). Recently, several novel rhabdoviruses have been discovered in the blood of both healthy and severely ill individuals living in Central and Western Africa. These viruses-Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2-are members of the little-understood rhabdoviral genus Tibrovirus. Other than the basic genomic architecture, tibroviruses bear little resemblance to well-studied rhabdoviruses such as rabies virus and vesicular stomatitis Indiana virus. These three human tibroviruses are quite divergent from each other, and each of them clusters closely with tibroviruses currently known only from biting midges or healthy cattle. Seroprevalence studies suggest that human tibrovirus infections may be common but are almost entirely unrecognized. The pathogenic potential of this diverse group of viruses remains unknown. Although certain tibroviruses may be benign and well-adapted to humans, others could be newly emerging and produce serious disease. Here, we review the current knowledge of tibroviruses and argue that assessing their impact on human health should be an urgent priority.

Characterisation of a novel nucleorhabdovirus infecting alfalfa (Medicago sativa).

BACKGROUND: Nucleorhabdoviruses possess bacilliform particles which contain a single-stranded negative-sense RNA genome. They replicate and mature in the nucleus of infected cells. Together with viruses of three other genera of the family Rhabdoviridae, they are known to infect plants and can be transmitted by arthropod vectors, during vegetative propagation, or by mechanical means. In 2010, an alfalfa (Medicago sativa) plant showing virus-like symptoms was collected from Stadl-Paura, Austria and sent to Julius Kühn Institute for analysis. METHODS: Electron microscopy (EM) of leaf extracts from infected plants revealed the presence of rhabdovirus-like particles and was further used for ultrastructural analyses of infected plant tissue. Partially-purified preparations of rhabdovirus nucleocapsids were used for raising an antiserum. To determine the virus genome sequence, high throughput sequencing (HTS) was performed. RT-PCR primers were designed to confirm virus infection and to be used as a diagnostic tool. RESULTS: EM revealed bacilliform virions resembling those of plant-infecting rhabdoviruses. HTS of ribosomal RNA-depleted total RNA extracts revealed a consensus sequence consisting of 13,875 nucleotides (nt) and containing seven open reading frames (ORFs). Homology and phylogenetic analyses suggest that this virus isolate represents a new species of the genus Nucleorhabdovirus (family Rhabdoviridae). Since the virus originated from an alfalfa plant in Austria, the name alfalfa-associated nucleorhabdovirus (AaNV) is proposed. Viroplasms (Vp) and budding virions were observed in the nuclei of infected cells by EM, thus confirming its taxonomic assignment based on sequence data. CONCLUSIONS: In this study, we identified and characterised a new nucleorhabdovirus from alfalfa. It shared only 39.8% nucleotide sequence identity with its closest known relative, black currant-associated rhabdovirus 1. The virus contains an additional open reading frame (accessory gene) with unknown function, located between the matrix protein and the glycoprotein genes. Serological and molecular diagnostic assays were designed for future screening of field samples. Further studies are needed to identify other natural hosts and potential vectors.

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.

Development of Model Systems for Plant Rhabdovirus Research.

This chapter reviews the discoveries and initial characterizations (1930-1990) of three plant rhabdoviruses, sonchus yellow net virus, potato yellow dwarf virus, and lettuce necrotic yellows virus, that have become model systems for research on this group of enveloped negative-strand RNA plant viruses. We have used our personal perspectives to review the early historical studies of these viruses, the important technologies and tools, such as density gradient centrifugation, that were developed during the research, and to highlight the eminent scientists involved in these discoveries. Early studies on sites of virus replication, virion structure, physicochemical composition, and the use of protoplasts and vector insect cell culture for virus research are discussed, and differences between the nuclear and cytoplasmic lifestyles of plant rhabdoviruses are contrasted. Finally, we briefly summarize the genome organization and more recent developments culminating in the development of a reverse genetics system for plant negative-strand RNA viruses.

Complete study demonstrating the absence of rhabdovirus in a distinct Sf9 cell line.

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Developments in Plant Negative-Strand RNA Virus Reverse Genetics.

Twenty years ago, breakthroughs for reverse genetics analyses of negative-strand RNA (NSR) viruses were achieved by devising conditions for generation of infectious viruses in susceptible cells. Recombinant strategies have subsequently been engineered for members of all vertebrate NSR virus families, and research arising from these advances has profoundly increased understanding of infection cycles, pathogenesis, and complexities of host interactions of animal NSR viruses. These strategies also permitted development of many applications, including attenuated vaccines and delivery vehicles for therapeutic and biotechnology proteins. However, for a variety of reasons, it was difficult to devise procedures for reverse genetics analyses of plant NSR viruses. In this review, we discuss advances that have circumvented these problems and resulted in construction of a recombinant system for Sonchus yellow net nucleorhabdovirus. We also discuss possible extensions to other plant NSR viruses as well as the applications that may emanate from recombinant analyses of these pathogens.

A peculiar new virus-spermatozoon association in the bug Raphigaster nebulosa (Poda) (Heteroptera-Insecta).

The sperm of the heteropteran bug Raphigaster nebulosa (Poda) are of two types, differing in length and size of their flagella. The thicker sperm are shorter than the thinner ones and have large mitochondrial derivatives. The presence of virus particles associated with the plasma membrane of thinner sperm is described for the first time; thicker sperm are immune to virus infection. The fact that virus particles are present on thinner sperm only initiates considerations on the transmission of virus.

Genomic characterization and phylogenetic position of two new species in Rhabdoviridae infecting the parasitic copepod, salmon louse (Lepeophtheirus salmonis).

Several new viruses have emerged during farming of salmonids in the North Atlantic causing large losses to the industry. Still the blood feeding copepod parasite, Lepeophtheirus salmonis, remains the major challenge for the industry. Histological examinations of this parasite have revealed the presence of several virus-like particles including some with morphologies similar to rhabdoviruses. This study is the first description of the genome and target tissues of two new species of rhabdoviruses associated with pathology in the salmon louse. Salmon lice were collected at different Atlantic salmon (Salmo salar) farming sites on the west coast of Norway and prepared for histology, transmission electron microscopy and Illumina sequencing of the complete RNA extracted from these lice. The nearly complete genomes, around 11,600 nucleotides encoding the five typical rhabdovirus genes N, P, M, G and L, of two new species were obtained. The genome sequences, the putative protein sequences, and predicted transcription strategies for the two viruses are presented. Phylogenetic analyses of the putative N and L proteins indicated closest similarity to the Sigmavirus/Dimarhabdoviruses cluster, however, the genomes of both new viruses are significantly diverged with no close affinity to any of the existing rhabdovirus genera. In situ hybridization, targeting the N protein genes, showed that the viruses were present in the same glandular tissues as the observed rhabdovirus-like particles. Both viruses were present in all developmental stages of the salmon louse, and associated with necrosis of glandular tissues in adult lice. As the two viruses were present in eggs and free-living planktonic stages of the salmon louse vertical, transmission of the viruses are suggested. The tissues of the lice host, Atlantic salmon, with the exception of skin at the attachment site for the salmon louse chalimi stages, were negative for these two viruses.

Spread and morphological-structural properties of plant rhabdoviruses and similar pathogens in Basidiomycetes.

Long-term studies of spread of rhabdoviruses which indicated their harmfulness in different plant species under conditions of environmental factors were first discussed. Their harmfulness to different plant species under environmental conditions was shown. A comparative description of rhabdoviruses with similar pathogens of the mushrooms is carried out. Thus the main focus was on the morphology and structure of the pathogens. These data are extremely important in the study of distribution of the rhabdovirus on crops in different regions.

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.

[Plant rhabdoviruses with bipartite genomes].

Members of the family Rhabdoviridae (order Mononegavirales) have a broad range of hosts, including humans, livestock, fish, plants, and invertebrates. They have a nonsegmented negative-sense RNA as the genome. Orchid fleck virus (OFV) is distributed world-wide on several orchid plants and transmitted by the false spider mite, Brevipalpus californicus. Based on its virions morphology and cytopathic effects in the infected cells, OFV was tentatively placed as unassigned plant rhabdoviruses in the sixth ICTV Report. However, the molecular studies reveled that OFV has a unique two-segmented negative-sense RNA genome that resembles monopartite genomes of plant nucleorhabdoviruses. In this review, we describe the current knowledge on the genome structure and gene expression strategy of OFV, the possible mechanism of nuclear viroplasm formation, and the taxonomical consideration of the virus as well.

Spring viremia of carp virus in Minnehaha Creek, Minnesota.

Spring viremia of carp virus (SVCV) causes a highly contagious and serious disease of freshwater cyprinid fishes, generating significant economic and ecological impacts throughout the world. The SVCV is therefore listed as a notifiable pathogen by the International Organization for Animal Health. In June 2011, a significant mortality event of wild common carp Cyprinus carpio occurred in Minnehaha Creek near its confluence with Mississippi River Pool 2 in Minneapolis, Minnesota. Clinical signs of moribund fish included hemorrhagic lesions in the skin, eyes, and internal adipose tissue. The SVCV was isolated from pooled kidney and spleen of the fish. Rhabdovirus particles were seen upon examination of infected cell culture fluid by electron microscopy. The virus was confirmed to be SVCV subtype Ia by reverse transcription PCR and sequencing. This is the first report of SVCV within the state of Minnesota and the ninth documented case in North America.

Initial sequence characterization of the rhabdoviruses of squamate reptiles, including a novel rhabdovirus from a caiman lizard (Dracaena guianensis).

Rhabdoviruses infect a variety of hosts, including non-avian reptiles. Consensus PCR techniques were used to obtain partial RNA-dependent RNA polymerase gene sequence from five rhabdoviruses of South American lizards; Marco, Chaco, Timbo, Sena Madureira, and a rhabdovirus from a caiman lizard (Dracaena guianensis). The caiman lizard rhabdovirus formed inclusions in erythrocytes, which may be a route for infecting hematophagous insects. This is the first information on behavior of a rhabdovirus in squamates. We also obtained sequence from two rhabdoviruses of Australian lizards, confirming previous Charleville virus sequence and finding that, unlike a previous sequence report but in agreement with serologic reports, Almpiwar virus is clearly distinct from Charleville virus. Bayesian and maximum likelihood phylogenetic analysis revealed that most known rhabdoviruses of squamates cluster in the Almpiwar subgroup. The exception is Marco virus, which is found in the Hart Park group.

Complete genomic sequence and taxonomic position of eel virus European X (EVEX), a rhabdovirus of European eel.

Eel virus European X (EVEX) was first isolated from diseased European eel Anguilla anguilla in Japan at the end of seventies. The virus was tentatively classified into the Rhabdoviridae family on the basis of morphology and serological cross reactivity. This family of viruses is organized into six genera and currently comprises approximately 200 members, many of which are still unassigned because of the lack of molecular data. This work presents the morphological, biochemical and genetic characterizations of EVEX, and proposes a taxonomic classification for this virus. We provide its complete genome sequence, plus a comprehensive sequence comparison between isolates from different geographical origins. The genome encodes the five classical structural proteins plus an overlapping open reading frame in the phosphoprotein gene, coding for a putative C protein. Phylogenic relationship with other rhabdoviruses indicates that EVEX is most closely related to the Vesiculovirus genus and shares the highest identity with trout rhabdovirus 903/87.

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.

Characterization of Durham virus, a novel rhabdovirus that encodes both a C and SH protein.

The family Rhabdoviridae is a diverse group of non-segmented, negative-sense RNA viruses that are distributed worldwide and infect a wide range of hosts including vertebrates, invertebrates, and plants. Of the 114 currently recognized vertebrate rhabdoviruses, relatively few have been well characterized at both the antigenic and genetic level; hence, the phylogenetic relationships between many of the vertebrate rhabdoviruses remain unknown. The present report describes a novel rhabdovirus isolated from the brain of a moribund American coot (Fulica americana) that exhibited neurological signs when found in Durham County, North Carolina, in 2005. Antigenic characterization of the virus revealed that it was serologically unrelated to 68 other known vertebrate rhabdoviruses. Genomic sequencing of the virus indicated that it shared the highest identity to Tupaia rhabdovirus (TUPV), and as only previously observed in TUPV, the genome encoded a putative C protein in an overlapping open reading frame (ORF) of the phosphoprotein gene and a small hydrophobic (SH) protein located in a novel ORF between the matrix and glycoprotein genes. Phylogenetic analysis of partial amino acid sequences of the nucleoprotein and polymerase protein indicated that, in addition to TUPV, the virus was most closely related to avian and small mammal rhabdoviruses from Africa and North America. In this report, we present the morphological, pathological, antigenic, and genetic characterization of the new virus, tentatively named Durham virus (DURV), and discuss its potential evolutionary relationship to other vertebrate rhabdoviruses.

Genome of turbot rhabdovirus exhibits unusual non-coding regions and an additional ORF that could be expressed in fish cell.

Genomic sequence of Scophthalmus maximus rhabdovirus (SMRV) isolated from diseased turbot has been characterized. The complete genome of SMRV comprises 11,492 nucleotides and encodes five typical rhabdovirus genes N, P, M, G and L. In addition, two open reading frames (ORF) are predicted overlapping with P gene, one upstream of P and smaller than P (temporarily called Ps), and another in P gene which may encodes a protein similar to the vesicular stomatitis virus C protein. The C ORF is contained within the P ORF. The five typical proteins share the highest sequence identities (48.9%) with the corresponding proteins of rhabdoviruses in genus Vesiculovirus. Phylogenetic analysis of partial L protein sequence indicates that SMRV is close to genus Vesiculovirus. The first 13 nucleotides at the ends of the SMRV genome are absolutely inverse complementarity. The gene junctions between the five genes show conserved polyadenylation signal (CATGA(7)) and intergenic dinucleotide (CT) followed by putative transcription initiation sequence A(A/G)(C/G)A(A/G/T), which are different from known rhabdoviruses. The entire Ps ORF was cloned and expressed, and used to generate polyclonal antibody in mice. One obvious band could be detected in SMRV-infected carp leucocyte cells (CLCs) by anti-Ps/C serum via Western blot, and the subcellular localization of Ps-GFP fusion protein exhibited cytoplasm distribution as multiple punctuate or doughnut shaped foci of uneven size.

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.