Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae.
ABSTRACT: The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3' poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5' and 541 nt at 3' termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98-99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate.
Project description:Chronic bee paralysis virus (CBPV) is the etiological agent of chronic paralysis, an infectious and contagious disease in adult honeybees. CBPV is a positive single-stranded RNA virus which contains two major viral RNA fragments. RNA 1 (3674 nt) and RNA 2 (2305 nt) encode three and four putative open reading frames (ORFs), respectively. RNA 1 is thought to encode the viral RNA-dependent RNA polymerase (RdRp) since the amino acid sequence derived from ORF 3 shares similarities with the RdRP of families Nodaviridae and Tombusviridae. The genomic organization of CBPV and in silico analyses have suggested that RNA 1 encodes non-structural proteins, while RNA 2 encodes structural proteins, which are probably encoded by ORFs 2 and 3. In this study, purified CBPV particles were used to characterize virion proteins by mass spectrometry. Several polypeptides corresponding to proteins encoded by ORF 2 and 3 on RNA 2 were detected. Their role in the formation of the viral capsid is discussed.
Project description:Mycoviruses are widespread in nature and often occur with dsRNA and positive-stranded RNA genomes. Recently, strong evidence from RNA sequencing analysis suggested that negative-stranded (-)ssRNA viruses could infect fungi. Here we describe a (-)ssRNA virus, Sclerotinia sclerotiorum negative-stranded RNA virus 1 (SsNSRV-1), isolated from a hypovirulent strain of Sclerotinia sclerotiorum. The complete genome of SsNSRV-1 is 10,002 nt with six ORFs that are nonoverlapping and linearly arranged. Conserved gene-junction sequences that occur widely in mononegaviruses, (A/U)(U/A/C)UAUU(U/A)AA(U/G)AAAACUUAGG(A/U)(G/U), were identified between these ORFs. The analyses 5' and 3' rapid amplification of cDNA ends showed that all genes can be transcribed independently. ORF V encodes the largest protein that contains a conserved mononegaviral RNA-dependent RNA polymerase (RdRp) domain. Putative enveloped virion-like structures with filamentous morphology similar to members of Filoviridae were observed both in virion preparation samples and in ultrathin hyphal sections. The nucleocapsids are long, flexible, and helical; and are 22 nm in diameter and 200-2,000 nm in length. SDS/PAGE showed that the nucleocapsid possibly contains two nucleoproteins with different molecular masses, ?43 kDa (p43) and ?41 kDa (p41), and both are translated from ORF II. Purified SsNSRV-1 virions successfully transfected a virus-free strain of S. sclerotiorum and conferred hypovirulence. Phylogenetic analysis based on RdRp showed that SsNSRV-1 is clustered with viruses of Nyamiviridae and Bornaviridae. Moreover, SsNSRV-1 is widely distributed, as it has been detected in different regions of China. Our findings demonstrate that a (-)ssRNA virus can occur naturally in fungi and enhance our understanding of the ecology and evolution of (-)ssRNA viruses.
Project description:The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.
Project description:Cherry virus A (CVA) (Capillovirus, Betaflexiviridae) is widely present in cherry-growing areas. We obtained the complete genome of a CVA isolate (CVA-TA) using small RNA deep sequencing, followed by overlapping reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). The newly identified 5'-untranslated region (5'-UTR) from CVA-TA may form additional hairpin and loop structures to stabilize the CVA genome.
Project description:We report the complete genome sequence of a novel virus, tentatively named "actinidia seed-borne latent virus" (ASbLV), isolated from Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3' poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.
Project description:Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.
Project description:BACKGROUND: Porcine sapovirus was first identified in the United States in 1980, hitherto, several Asian countries have detected this virus. In 2008, the first outbreak of gastroenteritis in piglets caused by porcine sapovirus in China was reported. The complete genome of the identified SaV strain Ch-sw-sav1 was sequenced and analyzed to provide gene profile for this outbreak. METHODS: The whole genome of Ch-sw-sav1 was amplified by RT-PCR and was sequenced. Sequence alignment of the complete genome or RNA dependent RNA polymerase (RdRp) gene was done. 3' end of ORF2 with 21-nt nucleotide insertion was further analyzed using software. RESULTS: Sequence analysis indicated that the genome of Ch-sw-sav1 was 7541 nucleotide long with two ORFs, excluding the 17 nucleotides ploy (A) at the 3' end. Phylogenetic analysis based on part of RdRp gene of this strain showed that it was classified into subgroup GIII. Sequence alignment indicated that there was an inserted 21-nt long nucleotide sequence at the 3' end of ORF2. The insertion showed high antigenicity index comparing to other regions in ORF2. CONCLUSION: Ch-sw-sav1 shared similar genetic profile with an American PEC strain except the 21-nt nucleotide at the 3' end of ORF2. The insert sequence shared high identity with part gene of Sus scrofa clone RP44-484M10.
Project description:Complete and partial genome sequences of two isolates of an unusual new plant virus, designated Donkey orchid symptomless virus (DOSV) were identified using a high-throughput sequencing approach. The virus was identified from asymptomatic plants of Australian terrestrial orchid Diuris longifolia (Common donkey orchid) growing in a remnant forest patch near Perth, western Australia. DOSV was identified from two D. longifolia plants of 264 tested, and from at least one plant of 129 Caladenia latifolia (pink fairy orchid) plants tested. Phylogenetic analysis of the genome revealed open reading frames (ORF) encoding seven putative proteins of apparently disparate origins. A 69-kDa protein (ORF1) that overlapped the replicase shared low identity with MPs of plant tymoviruses (Tymoviridae). A 157-kDa replicase (ORF2) and 22-kDa coat protein (ORF4) shared 32% and 40% amino acid identity, respectively, with homologous proteins encoded by members of the plant virus family Alphaflexiviridae. A 44-kDa protein (ORF3) shared low identity with myosin and an autophagy protein from Squirrelpox virus. A 27-kDa protein (ORF5) shared no identity with described proteins. A 14-kDa protein (ORF6) shared limited sequence identity (26%) over a limited region of the envelope glycoprotein precursor of mammal-infecting Crimea-Congo hemorrhagic fever virus (Bunyaviridae). The putative 25-kDa movement protein (MP) (ORF7) shared limited (27%) identity with 3A-like MPs of members of the plant-infecting Tombusviridae and Virgaviridae. Transmissibility was shown when DOSV systemically infected Nicotiana benthamiana plants. Structure and organization of the domains within the putative replicase of DOSV suggests a common evolutionary origin with 'potexvirus-like' replicases of viruses within the Alphaflexiviridae and Tymoviridae, and the CP appears to be ancestral to CPs of allexiviruses (Alphaflexiviridae). The MP shares an evolutionary history with MPs of dianthoviruses, but the other putative proteins are distant from plant viruses. DOSV is not readily classified in current lower order virus taxa.
Project description:Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5'- and 3'-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.