Proteins Of Rhabdoviruses Research Articles

Compensatory mutations in the matrix protein of viral hemorrhagic septicemia virus (VHSV) genotype IVa in response to artificial mutation of two amino acids (D62A E181A)

The matrix (M) protein of rhabdoviruses locates between the inner line of the viral envelope and the nucleocapsids core and plays an important role in viral replication. In the present study, we aimed to rescue a mutant of VHSV genotype IVa that has artificial mutations in the M protein (M-D62A E181A). However, most rescued recombinant viruses unexpectedly showed non-targeted secondary mutations in the M protein. Therefore, this study was conducted to know whether the targeted artificial mutation can lead to specific non-targeted secondary mutations in the M protein and whether the secondary mutations are compensatory for the targeted artificial mutations. Experiments were conducted to rescue three kinds of M protein mutants (rVHSV-M-D62A, -E181A, and -D62A E181A), and rVHSV-M-E181A and rVHSV-M-D62A E181A without the secondary mutations were rescued only from IRF-9 gene-knockout EPC cells. Recombinant VHSVs having only targeted mutation(s) (rVHSV-M-D62A, -E181A, and -D62A E181A) showed slower CPE progression and retarded growth compared to rVHSV-wild. Although the sites of secondary mutations were changed in every transfection experiment to generate recombinant VHSVs, the positions of the secondary mutations were not random. Some amino acid residues in the M protein showed more frequent mutations than others, and the changed amino acid residues were always the same. EPC cells infected with rVHSV-M-D62A E181A showed significantly higher type I interferon response and NF-κB activity, and the inhibitory activity against type I interferon response and NF-κB activity in other recombinant VHSVs having secondary mutations in M gene were similar to those of rVHSV-wild. In conclusion, the present results showed that VHSV actively responded to the artificial mutation of M protein through the secondary mutations, and those secondary mutations occurred when the artificial mutations were deleterious to viral replication and protein stability. Furthermore, most secondary mutations in recombinant viruses compensated for the deleterious effect of the engineered mutations.

Two Novel Rhabdoviruses Related to Hypervirulence in a Phytopathogenic Fungus.

Rhabdoviruses are ubiquitous and diverse viruses that propagate owing to bidirectional interactions with their vertebrate, arthropod, and plant hosts, and some of them could pose global health or agricultural threats. However, rhabdoviruses have rarely been reported in fungi. Here, two newly identified fungal rhabdoviruses, Rhizoctonia solani rhabdovirus 1 (RsRhV1) and RsRhV2, were discovered and molecularly characterized from the phytopathogenic fungus Rhizoctonia solani. The genomic organizations of RsRhV1 and RsRhV2 are 11,716 and 11,496 nucleotides (nt) in length, respectively, and consist of five open reading frames (ORFs) (ORFs I to V). ORF I, ORF IV, and ORF V encode the viral nucleocapsid (N), glycoprotein (G), and RNA polymerase (L), respectively. The putative protein encoded by ORF III has a lower level of identity with the matrix protein of rhabdoviruses. ORF II encodes a hypothetical protein with unknown function. Phylogenetic trees based on multiple alignments of N, L, and G proteins revealed that RsRhV1 and RsRhV2 are new members of the family Rhabdoviridae, but they form an independent evolutionary branch significantly distinct from other known nonfungal rhabdoviruses, suggesting that they represent a novel viral evolutionary lineage within Rhabdoviridae. Compared to strains lacking rhabdoviruses, strains harboring RsRhV2 and RsRhV1 showed hypervirulence, suggesting that RsRhV1 and RsRhV2 might be associated with the virulence of R. solani. Taken together, this study enriches our understanding of the diversity and host range of rhabdoviruses. IMPORTANCE Mycoviruses have been attracting an increasing amount of attention due to their impact on important medical, agricultural, and industrial fungi. Rhabdoviruses are prevalent across a wide spectrum of hosts, from plants to invertebrates and vertebrates. This study molecularly characterized two novel rhabdoviruses from four Rhizoctonia solani strains, based on their genomic structures, transcription strategy, phylogenetic relationships, and biological impact on their host. Our study makes a significant contribution to the literature because it not only enriches the mycovirus database but also expands the known host range of rhabdoviruses. It also offers insight into the evolutionary linkage between animal viruses and mycoviruses and the transmission of viruses from one host to another. Our study will also help expand the contemporary knowledge of the classification of rhabdoviruses, as well as providing a new model to study rhabdovirus-host interactions, which will benefit the agriculture and medical areas of human welfare.

A cytorhabdovirus phosphoprotein forms mobile inclusions trafficked on the actin/ER network for viral RNA synthesis.

As obligate parasites, plant viruses usually hijack host cytoskeletons for replication and movement. Rhabdoviruses are enveloped, negative-stranded RNA viruses that infect vertebrates, invertebrates, and plants, but the mechanisms of intracellular trafficking of plant rhabdovirus proteins are largely unknown. Here, we used Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, as a model to investigate the effects of the actin cytoskeleton on viral intracellular movement and viral RNA synthesis in a mini-replicon (MR) system. The BYSMV P protein forms mobile inclusion bodies that are trafficked along the actin/endoplasmic reticulum network, and recruit the N and L proteins into viroplasm-like structures. Deletion analysis showed that the N terminal region (aa 43–55) and the remaining region (aa 56–295) of BYSMV P are essential for the mobility and formation of inclusions, respectively. Overexpression of myosin XI-K tails completely abolishes the trafficking activity of P bodies, and is accompanied by a significant reduction of viral MR RNA synthesis. These results suggest that BYSMV P contributes to the formation and trafficking of viroplasm-like structures along the ER/actin network driven by myosin XI-K. Thus, rhabdovirus P appears to be a dynamic hub protein for efficient recruitment of viral proteins, thereby promoting viral RNA synthesis.

A dual-functional priming-capping loop of rhabdoviral RNA polymerases directs terminalde novoinitiation and capping intermediate formation

The L proteins of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus (RABV), possess an unconventional mRNA capping enzyme (GDP polyribonucleotidyltransferase, PRNTase) domain with a loop structure protruding into an active site cavity of the RNA-dependent RNA polymerase (RdRp) domain. Here, using complementary VSV and RABV systems, we show that the loop governs RNA synthesis and capping during the dynamic stop-start transcription cycle. A conserved tryptophan residue in the loop was identified as critical for terminal de novo initiation from the genomic promoter to synthesize the leader RNA and virus replication in host cells, but not for internal de novo initiation or elongation from the gene-start sequence for mRNA synthesis or pre-mRNA capping. The co-factor P protein was found to be essential for both terminal and internal initiation. A conserved TxΨ motif adjacent the tryptophan residue in the loop was required for pre-mRNA capping in the step of the covalent enzyme-pRNA intermediate formation, but not for either terminal or internal transcription initiation. These results provide insights into the regulation of stop-start transcription by the interplay between the RdRp active site and the dual-functional priming-capping loop of the PRNTase domain in non-segmented negative strand RNA viruses.

5'-Phospho-RNA Acceptor Specificity of GDP Polyribonucleotidyltransferase of Vesicular Stomatitis Virus in mRNA Capping.

The GDP polyribonucleotidyltransferase (PRNTase) domain of the multifunctional L protein of rhabdoviruses, such as vesicular stomatitis virus (VSV) and rabies virus, catalyzes the transfer of 5'-phospho-RNA (pRNA) from 5'-triphospho-RNA (pppRNA) to GDP via a covalent enzyme-pRNA intermediate to generate a 5'-cap structure (GpppA). Here, using an improved oligo-RNA capping assay with the VSV L protein, we showed that the Michaelis constants for GDP and pppAACAG (VSV mRNA-start sequence) are 0.03 and 0.4 μM, respectively. A competition assay between GDP and GDP analogues in the GpppA formation and pRNA transfer assay using GDP analogues as pRNA acceptors indicated that the PRNTase domain recognizes the C-2-amino group, but not the C-6-oxo group, N-1-hydrogen, or N-7-nitrogen, of GDP for the cap formation. 2,6-Diaminopurine-riboside (DAP), 7-deazaguanosine (7-deaza-G), and 7-methylguanosine (m7G) diphosphates efficiently accepted pRNA, resulting in the formation of DAPpppA, 7-deaza-GpppA, and m7GpppA (cap 0), respectively. Furthermore, either the 2'- or 3'-hydroxyl group of GDP was found to be required for efficient pRNA transfer. A 5'-diphosphate form of antiviral ribavirin weakly inhibited the GpppA formation but did not act as a pRNA acceptor. These results indicate that the PRNTase domain has a unique guanosine-binding mode different from that of eukaryotic mRNA capping enzyme, guanylyltransferase. IMPORTANCE mRNAs of nonsegmented negative-strand (NNS) RNA viruses, such as VSV, possess a fully methylated cap structure, which is required for mRNA stability, efficient translation, and evasion of antiviral innate immunity in host cells. GDP polyribonucleotidyltransferase (PRNTase) is an unconventional mRNA capping enzyme of NNS RNA viruses that is distinct from the eukaryotic mRNA capping enzyme, guanylyltransferase. In this study, we studied the pRNA acceptor specificity of VSV PRNTase using various GDP analogues and identified chemical groups of GDP as essential for the substrate activity. The findings presented here are useful not only for understanding the mechanism of the substrate recognition with PRNTase but also for designing antiviral agents targeting this enzyme.

Complete genome sequence of Menghai rhabdovirus, a novel mosquito-borne rhabdovirus from China.

Menghai rhabdovirus (MRV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, in August 2010. Whole-genome sequencing of MRV was performed using an Ion PGM™ Sequencer. We found that MRV is a single-stranded, negative-sense RNA virus. The complete genome of MRV has 10,744 nt, with short inverted repeat termini, encoding five typical rhabdovirus proteins (N, P, M, G, and L) and an additional small hypothetical protein. Nucleotide BLAST analysis using the BLASTn method showed that the genome sequence most similar to that of MRV is that of Arboretum virus (NC_025393.1), with a Max score of 322, query coverage of 14%, and 66% identity. Genomic and phylogenetic analyses both demonstrated that MRV should be considered a member of a novel species of the family Rhabdoviridae.

Specific Cleavage of the Nucleoprotein of Fish Rhabdovirus.

Siniperca chuatsi rhabdovirus (SCRV) is one of myriad rhabdoviruses recorded in fish. Preliminary data show that inhibition of the SCRV nucleoprotein (N) could significantly reduce the progeny virus titers in infected Epithelioma papulosum cyprinid (EPC) cells. Here, the authors propose that cleavage of the viral 47-kDa N protein is caspase-mediated based on caspase inhibition experiments, transient expression in EPC transfection, and analysis of cleavage sites. Cleavage of the SCRV N protein in culture was prevented by a pan-caspase inhibitor, z-VAD-FMK (z-Val-Ala-DL-Asp-fluoromethyl ketone). Subsequently, N was transiently expressed in EPC cells, the results of which indicated that the specific cleavage of N also occurred in the cells transfected with N-GFP plasmid. Several truncated fragments of the N gene were constructed and transiently transfected into EPC cells. Immunoblotting results indicated that D324 and D374 are the cleavage sites of N by caspases. The authors also found that z-VAD-FMK could inhibit the cytopathic effect in SCRV-infected EPC cells but not affect the production of infectious progeny, suggesting that the caspase-mediated cleavage of N protein is not required for in vitro SCRV replication. To the authors' knowledge, this is the first report on the cleavage of rhabdovirus proteins.

Super-Resolution Microscopy of Cells Expressing Rhabdovirus Proteins

Interferon (IFN)-mediated immunity is a central mode of defense against viral infection and evasion of this immune response is critical to the pathogenicity of viruses. IFN-antagonist proteins have recently been shown to interact with host microtubules (MTs) demonstrating a novel mechanism for subverting the IFN response (1). Using super-resolution fluorescence microscopy we have imaged the association of the IFN-antagonist protein with host cell MTs and the consequent changes to the architecture of the cell cytoskeleton.Super resolution imaging was achieved using the dSTORM approach on a home-built set-up. dSTORM imaging of MT architecture in cells expressing Rhabdovirus proteins has allowed previously unobservable changes caused by the IFN-antagonist proteins to be detected. Transfected cells COS7 cells labeled with Alexa 647 (Figure - left panel) show a high degree of bundling and abnormal curvature of the microtubule skeleton. In contrast, healthy control cells (Figure - center panel) show normal microtubule architecture.(1) K.G. Lieu, A. Brice, L. Wiltzer, B. Hirst, D.A. Jans, D. Blondel and G.W. Moseley, Journal of Virology, (2013) doi: 10/1128/JVI.00989-13.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Niakha virus: A novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal

Members of the family Rhabdoviridae have been assigned to eight genera but many remain unassigned. Rhabdoviruses have a remarkably diverse host range that includes terrestrial and marine animals, invertebrates and plants. Transmission of some rhabdoviruses often requires an arthropod vector, such as mosquitoes, midges, sandflies, ticks, aphids and leafhoppers, in which they replicate. Herein we characterize Niakha virus (NIAV), a previously uncharacterized rhabdovirus isolated from phebotomine sandflies in Senegal. Analysis of the 11,124nt genome sequence indicates that it encodes the five common rhabdovirus proteins with alternative ORFs in the M, G and L genes. Phylogenetic analysis of the L protein indicate that NIAV′s closest relative is Oak Vale rhabdovirus, although in this analysis NIAV is still so phylogenetically distinct that it might be classified as distinct from the eight currently recognized Rhabdoviridae genera. This observation highlights the vast, and yet not fully recognized diversity, of this family.

An ELISA for detection of trout antibodies to viral haemorrhagic septicemia virus using recombinant fragments of their viral G protein

An enzyme linked immunosorbent assay (ELISA) method to study serum antibodies to viral haemorrhagic septicemia virus (VHSV) was designed by using recombinant fragments of their G protein. By using this fragment-ELISA, we describe the binding of antibodies against recombinant G fragments of 45–445 amino acids present in VHSV-hyperimmunized trout sera. Fragments were designed by taking into account their tridimensional pH-dependent structure and functional domains. Sera were obtained from hyperimmunized trout following 4–5 intraperitoneal injections of VHSV antigens by using Freund's or saponin adjuvants. Sera from different hyperimmunized trout differed quantitatively rather than qualitatively in their recognition of solid-phase frg11 (56–110), frg12 (65–109), frg13 (97–167), frg14 (141–214), frg15 (65–250), frg16 (252–450) and G (G21–465) by Western blot and ELISA. However, titres were higher when using frg11, frg15 or frg16, rather than G21–465, suggesting higher accessibility to G epitopes. Further knowledge of the antigenicity of the G protein of rhabdoviruses by using fragments might be used to improve current vaccines. On the other hand, they might be used to dissect the trout antibody response to VHSV infections, to complement in vitro neutralizing assays, and/or to quantitate anti-VHSV antibodies in VHSV-infected/vaccinated trout, other fish and/or other body fluids such as mucus.

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.

VSV in 3D

![Figure][1] CREDIT: GE ET AL. Rhabdoviruses are a family of negative-stranded RNA viruses that includes rabies virus, which have a characteristic bullet shape. Though structures of individual rhabdovirus proteins have been reported, how these are organized into a bullet shape has remained

Cryo-EM Model of the Bullet-Shaped Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV) is a bullet-shaped rhabdovirus and a model system of negative-strand RNA viruses. Through direct visualization by means of cryo-electron microscopy, we show that each virion contains two nested, left-handed helices: an outer helix of matrix protein M and an inner helix of nucleoprotein N and RNA. M has a hub domain with four contact sites that link to neighboring M and N subunits, providing rigidity by clamping adjacent turns of the nucleocapsid. Side-by-side interactions between neighboring N subunits are critical for the nucleocapsid to form a bullet shape, and structure-based mutagenesis results support this description. Together, our data suggest a mechanism of VSV assembly in which the nucleocapsid spirals from the tip to become the helical trunk, both subsequently framed and rigidified by the M layer.

Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.

Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

Rhabdovirus assembly and budding

Rhabdoviruses are a diverse, widely-distributed group of enveloped viruses that assemble and bud from the plasma membrane of host cells. Recent advances in the identification of domains on both the envelope glycoprotein and the matrix protein of rhabdoviruses that contribute to virus assembly and release have allowed us to refine current models of rhabdovirus budding and to describe in better detail the interplay between both viral and cellular components involved in the budding process. In this review we discuss the steps involved in rhabdovirus assembly beginning with genome encapsidation and the association of nucleocapsid-matrix protein pre-assembly complexes with the inner leaflet of the plasma membrane, how condensation of these complexes may occur, how microdomains containing the envelope glycoprotein facilitate bud site formation, and how multiple forms of the matrix protein may participate in virion extrusion and release.

Complete nucleotide sequence of the hirame rhabdovirus, a pathogen of marine fish

Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed for the hirame rhabdovirus (HIRRV) CA-9703 strain from Korea, and the DNA was sequenced. The 3′-end of genomic RNA was cloned by poly(A)-tailing of the genomic RNA before reverse transcription, and the 5′-end of the genome was cloned by poly(G)- or poly(C)-tailing of the first strand, followed by PCR. The remainder of the genomic DNA was cloned by reverse transcription-polymerase chain reaction using primers that were based on the published rhabdovirus sequences. The complete genome of HIRRV CA-9703 strain comprises 11,034 nucleotides and encodes six genes in the order of: 3′-leader, N, P, M, G, NV, L, and 5′-trailer. These genes are separated by conserved sequences or gene junctions, with one-nucleotide gene spacers. The first 16 of the 19 nucleotides at the ends of the HIRRV genome are complementary, and the first four nucleotides at the 3′-ends of the HIRRV, infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), and snakehead rhabdovirus (SHRV) genomes are identical. The HIRRV proteins share the highest amino acid sequence homology (ranging from 72% to 92%) with the proteins of IHNV, of all the known fish rhabdoviruses, and the highest sequence homology with respect to the L protein was shared among HIRRV, IHNV, VHSV, and SHRV. Although there were differences in the degrees of relatedness, phylogenetic trees that were derived from multiple sequence alignments of the rhabdovirus proteins showed similar patterns of relationship among these viruses, in which fish Novirhabdoviruses formed a separate clade from spring viremia of carp virus (SVCV), unassigned fish rhabdovirus that was closer to mammalian rhabdoviruses.

First Record of Barley yellow striate mosaic virus Affecting Wheat Summer-Nurseries in Syria.

A limited survey to identify virus diseases affecting wheat in summer nurseries in agricultural stations in southern Syria was conducted during October 2002. A total of 94 bread and durum wheat samples with symptoms suggestive of virus infection (stripping, stunting, and yellowing) were collected. All samples were tested for the presence of four viruses by tissue-blot immunoassay (2) at the Virology Laboratory of ICARDA, Aleppo, Syria using the following polyclonal antibodies: Barley stripe mosaic virus (BSMV); Barley yellow dwarf virus-PAV (BYDV-PAV) and Wheat streak mosaic virus (WSMV) from the Virology Laboratory at ICARDA; and Barley yellow striate mosaic virus (BYSMV) isolated from Italy (BYSMV-Italy) and provided by M. Conti, Instituto di Fitovirologia applicata, Turino, Italy. Serological results obtained indicated that BYSMV was the most commonly encountered virus (78.7%) followed by BYDV-PAV (22.3%), whereas, BSMV and WSMV were not detected in any of the samples tested. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, purified BYSMV preparations were observed to contain a 47-kDa structural protein typical of the N protein of Rhabdoviruses that reacted strongly with three BYSMV antisera (BYSMV-Italy, BYSMV-Lebanon [4], and BYSMV-Morocco [1]). Samples that reacted with BYSMV antisera were transmitted from wheat to wheat, barley, and oat plants by the planthopper Laodelphax striatella (Fallen) (Hemiptera: family Delphacidae) in a persistent manner, and the major symptoms of BYSMV on cereal crops were stripping and stunting. BYDV-PAV has been reported from Syria earlier (3) but to our knowledge, this is the first report of BYSMV affecting wheat in Syria.

First Record of Barley yellow striate mosaic virus and Cereal yellow dwarf virus-RPV Infecting Wheat in Uzbekistan.

A preliminary survey to identify virus diseases affecting wheat in Uzbekistan was conducted during May 2001. The survey covered 12 wheat fields from 2 cereal-growing regions (Tashkent-Angren and Tashkent-Samarkand). A total of 250 wheat samples with symptoms suggestive of virus infection were collected and tested for the presence of nine viruses by tissue-blot immunoassay (TBIA) (1) at the Virology Laboratory of ICARDA, Aleppo, Syria, using the following antisera: monoclonal antibodies for Cereal yellow dwarf virus-RPV (CYDV-RPV) (ATCC PVAS-669 [American Type Culture Collection, Manassas, VA]) and Barley yellow dwarf virus-MAV (BYDV-MAV) (ATCC PVAS-673); and polyclonal antibodies for BYDV-SGV and BYDV-RMV (3); BYDV-PAV, Barley stripe mosaic virus, and Wheat streak mosaic virus (from Virology Laboratory, ICARDA); Wheat dwarf virus (provided by J. Vacke, Research Institute of Crop Production, Prague, Czeck Republic); and Barley yellow striate mosaic virus (BYSMV) isolated from Lebanon (2). The most common virus present was BYDV-PAV (detected in 12% of the 250 samples tested), followed by BYDV-SGV (10.8%), BYSMV (5.6%), BYDV-RMV (2.4%), BYDV-MAV (2%), and CYDV-RPV (1.2%). CYDV-RPV was detected in three fields; one field was 50 km southeast of Tashkent, and the other two fields were between Tashkent and Samarkand. The majority of BYSMV-positive samples originated from the same field, ≈40 km northeast of Samarkand. Field symptoms of BYSMV-infected plants included yellow flag leaf and stunting. All samples that produced a positive reaction to BYSMV-Lebanon antiserum were tested against four other rhabdovirus antisera: BYSMV-Italy, BYSMV-Morocco, Cereal chlorotic mottle virus, and American wheat striate mosaic virus. Serological tests showed that 100% of the samples reacted strongly with BYSMV-Italy and BYSMV-Morocco. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by western blots, extracts from BYSMV-infected plants were found to contain 66- and 47-kDa structural proteins, typical of G and N proteins of rhabdoviruses, both of which reacted strongly with BYSMV-Italy antiserum. To our knowledge, this is the first report of BYSMV and CYDV-RPV in Uzbekistan.

Nucleotide sequence of the coat protein gene of Lettuce big-vein virus.

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M(r) of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M(r) of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7.3 kb (ss-1) and 6.6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.