Genomic characterization of a novel dsRNA virus detected in the phytopathogenic fungus Verticillium dahliae Kleb

Four novel double-stranded RNA segments were detected in a Verticillium dahliae Kleb. strain (V. dahliae isolate 0-21), a causal fungal agent of Verticillium wilt disease of cotton. Each dsRNA genome segment contains a single large open reading frame (ORF) that encodes a distinctive protein with modest levels of sequence similarities to the corresponding putative proteins in the genus Chrysovirus. These include an RNA-dependent RNA polymerase (RdRp), a coat protein, an undefined replication-related protein and an ovarian tumor domain peptidase. Phylogenetic analysis of the four putative proteins unanimously indicated that they are evolutionarily related to viruses in Chrysovirus. The 5'- and 3'-untranslated regions of the four dsRNAs share highly similar internal sequence and contain conserved sequence stretches of UGAUAAAAAA(/U)UG(/U)AAAAA- (in the 5'-UTR) and -UUUACUACU (in the 3'-UTR), indicating that they have a common virus origin. Indeed, isometric virus-like particles (VLPs) with a diameter of approximately 34nm were extracted from the fungal mycelia, and the four dsRNA segments were also detected in the virus-like particle (VLP) fraction. These results suggest that the mycovirus with four different dsRNA genome segments from the fungal isolate 0-21 is a new member of the genus Chrysovirus. We named the virus Verticillium dahliae chrysovirus 1 (VdCV1).     

A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix

Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family.     

A novel dsRNA element isolated from the Aspergillus foetidus mycovirus complex

Aspergillus foetidus virus (AfV) contains at least two icosahedral particle types named AfV-fast (-F) and AfV-slow (-S), based on relative electrophoretic mobility. AfV-F is a quadripartite double-stranded RNA (dsRNA) virus, and AfV-S contains AfV-S1, which is a member of the genus Victorivirus in the family Totiviridae, and AfV-S2, which may be a satellite RNA or satellite virus and is described here. Analysis of the complete AfV-S2 nucleotide sequence reveals it to be significantly similar to an unclassified RNA from the fungus Rosellinia necatrix and distantly related to the RNA-dependent RNA polymerases of several single-stranded RNA genomes.     

The complete nucleotide sequence of a novel partitivirus isolated from the plant pathogenic fungus Verticillium albo-atrum

We have characterized the bisegmented genome of a novel double-stranded RNA (dsRNA) virus isolated from the plant pathogenic fungus Verticillium albo-atrum. We determined that its larger segment (dsRNA1) was 1747 base pairs in length and potentially encoded an RNA-dependent RNA polymerase of 539 amino acids, whereas the smaller segment (dsRNA2) was 1517 base pairs long and was predicted to encode a capsid protein of 435 amino acids. Homology searches and phylogenetic analysis confirmed that, as expected from its dsRNA banding profile, the identified virus was a new member of the family Partitiviridae, and we have therefore designated it V. a lbo- a trum partitivirus 1 (VaaPV1). This is the first report of a mycovirus identified in V. albo-atrum.     

Complete sequence of the genome of two dsRNA viruses from Discula destructiva

Complete nucleotide sequences were determined for the four dsRNA segments present in isolate 247 of Discula destructiva from South Carolina. The largest dsRNA (dsRNA 1) was 1787 bp in length with a single open reading frame (ORF) that coded for a putative RNA-dependent RNA polymerase (RdRp). The dsRNA 2 was 1585 bp in length with a single ORF that coded for a putative viral coat protein. Both the dsRNA 3 (1178 bp in length) and dsRNA 4 (308 bp) contained single ORFs. However, neither the nucleotide sequence nor the sequence of the putative translation products, showed any similarity with sequences currently available from GenBank. Although distinct, all 4 dsRNAs showed conserved nucleotides at both the 5′ and 3′ termini. Sequences of the two dsRNAs in an isolate of D. destructiva (331 originating from Idaho) were similar in length to, and shared similarity with, the dsRNA 1 and dsRNA 2 of isolate 247. However, although the putative RdRps of isolates 247 and 331 are closely related, the putative viral coat proteins coded for by the respective dsRNA 2s are distinct. Thus, the dsRNAs in the two fungal isolates appeared to originate from distinct, but related viruses, which we have named D. destructiva virus 1 and D. destructiva virus 2, respectively. Phylogenetic analysis indicated that the two viruses were most closely related to Fusarium solani virus 1 and should be considered members of the genus Partitivirus. Another isolate of D. destructiva (272.1) contains a 12 kb dsRNA in addition to the 4 dsRNAs found in isolate 247. Partial sequence of this 12 kb molecule showed a relationship to other large dsRNA molecules isolated from plants.     

Complete genome sequence and organization of a novel virus from the rice false smut fungus Ustilaginoidea virens

In this study, three dsRNA segments from the rice false smut fungus Ustilaginoidea virens, the causal agent of a serious disease in rice, with molecular size ranging from 1.3 to 5 Kb, were isolated and named as dsRNA-L, dsRNA-M, and dsRNA-S. The complete nucleotide sequences of dsRNA-M and dsRNA-S were determined and analyzed. The dsRNA-M putatively encodes an RNA-dependent RNA polymerase, which is similar to that of the partitiviruses in the family Partitiviridae. Although the protein encoded by dsRNA-S showed less similarity to the typical coat protein of the virus in the family Partitiviridae, the structural analysis results indicated that the dsRNA-S might function as the capsid protein. We propose that the virus is Ustilaginoidea virens partitivirus 2-Uv0901, a new member, but distantly related to the newly proposed genus Gammapartitivirus with a distinct sequence pattern of capsid protein.     

Complete genome sequence of a novel calicivirus from a goose

A novel goose calicivirus (GoCV) was sequenced. The 8013-nt-long genome was organized into two open reading frames that were in the same frame and separated by 3 nucleotides. This feature is similar to what has been observed in turkey calicivirus (TuCV). Comparison of GoCV with other caliciviruses showed that it shared the highest amino acid sequence identities of 62, 38, and 52 % in the nonstructural protein, VP1, and VP2, respectively, with TuCV. Phylogenetic analysis based on the amino acid sequences of nonstructural protein and VP1 demonstrated that GoCV was most closely related to but distinct from TuCV. Thus, GoCV was identified as a novel member in the proposed genus Nacovirus.     

Development of a double monoclonal antibody sandwich ELISA test for Verticillium dahliae Kleb.

Verticillium dahliae causes wilt diseases in a wide range of horticultural and field crops in many parts of Turkey. In the Aegean region it has been a serious problem in cotton and vegetables for many years. More recently, it has become a major problem for olive growers in relatively newly established orchards. The fact that the only possible control methods of Verticillium wilt disease is the use of healthy and pathogen free propagating material has directed us to produce monoclonal antibodies against V. dahliae in order to apply rapid, sensitive and reliable serological detection methods. Verticillium spp. isolates were obtained from olive plantations, as well as from cotton and tomato fields in the Aegean and Marmara Regions. All suspected isolates were obtained as V. dahliae after determining microscopic morphological characteristics and pathogenicity tests on cotton seedlings. Immunizing antigens were prepared by three different methods including surface washing system, czapek dox agar and gel filtration methods. BALB/c mice were immunized with each antigenic form. Lymph node, spleen and bone marrow cells were used as sources of B-lymphocytes and 8D2 (IgM) and 7D6 (IgG1) were obtained from the spleen and lymph node fusion. The monoclonal antibodies were purified and immunoglobulin types were identified. 8D2 monoclonal antibody gave positive reaction with the V.dahliae isolates from olive, cotton, tomato and watermelon; however, it didn't give any cross reactivity with other epiphytic fungi. 7D6 antibody displayed cross-reactions with a few fungi. The monoclonal antibody (8D2) was conjugated with horseradish peroxidase (HRP). These monoclonal antibodies were characterized for use in the development of diagnostic kits based on double-monoclonal antibody sandwich ELISA test system for detecting V. dahliae in Turkish isolates. In this test, the first antibody was used as capture antibody and the second one was used for detection of antigens.     

Complete nucleotide sequence and genome organization of a dsRNA partitivirus infecting Pleurotus ostreatus

The nucleotide sequences of the genomic dsRNA mycovirus infecting Pleurotus ostreatus (P. ostreatus virus 1; PoV1) were determined and compared to the sequences of the other mycoviruses belonging to partitiviruses and totivirues. PoV1 dsRNA-1 and dsRNA-2 had genomes of 2296 and 2223 nucleotides, respectively. The purified virus preparations contained isometric particles of 28-30 nm in diameter, and also the same two dsRNAs were isolated from purified virus preparations. The sequences of PoV1 dsRNA-1 and dsRNA-2 had GC contents of 48.4 and 51.5%, respectively. dsRNA-1 had 78 and 97 nucleotides of 5'- and 3'-untranslated region (UTR) while dsRNA-2 had 114 and 198 nucleotides of 5'- and 3'-UTR, respectively. Computer analysis of putative open reading frame (ORF) shows that dsRNA-1 and dsRNA-2 contain a single ORF encoding proteins of 82.2 and 71.1 kDa that show high sequence identity with RNA-dependent RNA polymerase and capsid protein of partitiviruses, respectively. When compared to other dsRNA mycoviruses in a phylogenetic analysis they were found to form a distinct virus clade with partitiviruses, and were more distantly related to totiviruses.     

Complete genome sequence of a novel sea otterpox virus

Members of the Poxviridae family are large, double-stranded DNA viruses that replicate in the cytoplasm of their host cells. The subfamily Chordopoxvirinae contains viruses that infect a wide range of vertebrates including marine mammals within the Balaenidae, Delphinidae, Mustelidae, Odobenidae, Otariidae, Phocidae, and Phocoenidae families. Recently, a novel poxvirus was found in a northern sea otter pup (Enhydra lutris kenyoni) that stranded in Alaska in 2009. The phylogenetic relationships of marine mammal poxviruses are not well established because of the lack of complete genome sequences. The current study sequenced the entire sea otterpox virus Enhydra lutris kenyoni (SOPV-ELK) genome using an Illumina MiSeq sequencer. The SOPV-ELK genome is the smallest poxvirus genome known at 127,879 bp, is 68.7% A+T content, is predicted to encode 132 proteins, and has 2546 bp inverted terminal repeats at each end. Genetic and phylogenetic analyses based on the concatenated amino acid sequences of 7 chorodopoxvirus core genes revealed the SOPV-ELK is 52.5–74.1% divergent from other known chordopoxviruses and is most similar to pteropoxvirus from Australia (PTPV-Aus). SOPV-ELK represents a new chordopoxvirus species and may belong to a novel genus. SOPV-ELK encodes eight unique genes. While the function of six predicted genes remains unknown, two genes appear to function as novel immune-modulators. SOPV-ELK-003 appears to encode a novel interleukin-18 binding protein (IL-18 BP), based on limited sequence and structural similarity to other poxviral IL-18 BPs. SOPV-ELK-035 appears to encode a novel tumor necrosis factor receptor-like (TNFR) protein that may be associated with the depression of the host’s antiviral response. Additionally, SOPV-ELK-036 encodes a tumor necrosis factor-like apoptosis-inducing ligand (TRAIL) protein that has previously only been found in PTPV-Aus. The SOPV-ELK genome is the first mustelid poxvirus and only the second poxvirus from a marine mammal to be fully sequenced. Sequencing of the SOPV-ELK genome is an important step in unraveling the position of marine mammal poxviruses within the larger Poxviridae phylogenetic tree and provides the necessary sequence to develop molecular tools for future diagnostics and epidemiological studies.