Biological and Molecular Characterization of Olive latent virus 1
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Olive latent virus 1 (OLV-1) belongs to the Necrovirus genus, Tombusviridae family and is pathogenic to olive, citrus and tulip plants. It is easily mechanically transmissible to indicator plants causing necrotic lesions and can be transmitted through the soil into the plant roots in the absence of biological vectors. Infected cells contain virus aggregates, inclusions made up of excess of viral coded peptides and extensive vesiculation in the cytoplasm. The virions are isometric with ca. 30 nm, possess a monopartite single-stranded positive-sense RNA genome sized 3700 nt with 5 open reading frames (ORFs) and small inter cistronic regions. ORF 1 encodes a polypeptide with a molecular weight of 23 kDa and the read through of its amber stop codon results in ORF 1 RT that encodes the virus RNA dependent RNA polymerase with 82 kDa. ORF2 and ORF3 encode two small peptides, with 8 kDa and 6 kDa, respectively, which appear to be involved in the virus cell-to-cell movement. ORF 4 is located in the 3′-terminal and encodes a protein with 30 kDa identified as the viral coat protein. The complete genomic sequences of two well characterized OLV-1 isolates (obtained from citrus and olive) are similar, revealing an overall nucleotide sequence identity of 95%. The electrophoretic profile of the dsRNAs recovered from infected tissues exhibits three major species with ca. 3.7, 1.5, and 1.3 kbp. Application of molecular techniques based on PCR and on dot blot hybridization has been successfully used for routine diagnosis of OLV-1 infections.Keywords:
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The complete nucleotide sequence of RNA2 of Helicoverpa armigera stunt virus (HaSV), a member of the Tetraviridae, was determined by characterization of cloned cDNA and PCR products and direct sequencing of genomic RNA. The capped, positive sense, single-stranded RNA is 2478 nucleotides in length and has two overlapping open reading frames (ORFs) likely to be cistrons which are situated between terminal non-coding regions of 282 and 168 bases, 5′ and 3′, respectively. Extensive secondary structure of the RNA strand is indicated, including a tRNA-like structure at the 3′ terminus which is the first such structure discerned in an animal virus. The first ORF encodes a 17 kDa PEST protein (p17) of unknown function while the second ORF encodes the 71 kDa coat protein precursor (p71) that is cleaved at an Asn-Phe site into the 64 kDa and 7 kDa coat proteins. The precursor coat protein is 66% identical to that of another tetravirus, the Nudaurelia ω virus, with most of the difference residing in a 165 amino acid region located in the middle of the sequence. Despite the extensive similarity, no serological relationship was observed between the two viruses, suggesting that the dissimilar region is exposed on the capsid exterior. Expression in bacteria of the two RNA2 gene products shows they are likely to be expressed by a leaky scan-through mechanism. Bacterial expression of p71 did not produce virus-like particles while expression of p17 produced large arrays of mostly hollow, hexagonal tube-like structures.
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Olive latent virus 2 (OLV2), a virus with particle shapes resembling those of alfalfa mosaic alfamovirus (AMV), has four major RNA species, two of which (RNA3 and RNA4) were completely sequenced. RNA3 was a bicistronic molecule containing two clear-cut ORFs, one of which (ORF1) coded for a 36.5 kDa polypeptide with conserved motifs of the '30K superfamily' movement proteins and the other (ORF2) encoded a 20 kDa polypeptide identified as the viral coat protein. RNA4, which was a little smaller than RNA3 (2078 nt versus 2438 nt), also differed from RNA3 in a few positions, but its in vitro translation did not produce any protein. By contrast, an additional RNA, 1042 nt in size with strong sequence homology with RNA3 and RNA4, was identified in RNA extracts from infected plants. This RNA, which may be a subgenomic form of RNA3 carrying the coat protein cistron, is apparently encapsidated to a very small extent, or not at all. Comparative computer-assisted analysis of virus-coded protein sequences disclosed homologies suggesting that OLV2 may belong to the family Bromoviridae, but as an entity separated from the currently known genera.
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An unusual novel plant virus provisionally named goji berry chlorosis virus (GBCV) was isolated from goji berry plants (Lycium chinense Miller) showing chlorosis symptoms and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 10,100 ribonucleotides and contains six open reading frames (ORFs). Electron microscopy showed that the viral genome is packaged as a filamentous particle with an average length of approximately 850 nm. Phylogenetic analysis and amino acid similarity analysis of the encoded ORFs revealed that this new virus could be classified in an intermediate position between the families Benyviridae and Virgaviridae. The GBCV 200-kDa replicase (ORF1) is more similar to benyvirus replicases than to virgavirus replicases, while its 17-kDa coat protein (CP, ORF2) is more closely related with virgavirus CPs than benyvirus CPs. ORF3 was predicted to produce a C-terminally extended protein from ORF2 via frameshifting. While ORF4 (45-kDa), ORF5 (44-kDa), and ORF6 (16-kDa) have no apparent sequence homology with other known viruses, ORF5 is predicted to encode a movement protein (MP) that is phylogenetically related to the furovirus MP and ORF6 was experimentally proven to encode a viral suppressor of RNA silencing. These unusual characteristics suggest that GBCV may represent an evolutionary link between the families Benyviridae and Virgaviridae and indicate the existence of a novel, unidentified virus group.
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