Complementary DNA cloning and expression of the papaya ringspot potyvirus sequences encoding capsid protein and a nuclear inclusion-like protein in Escherichia coli

Three cDNA clones that express viral gene products in Escherichia coli JM83 were derived from a watermelon mosaic virus-1 strain of papaya ringspot virus (PRSV-W). DNAs complementary to portions of the viral RNA were inserted into the pUC8 and pUC9 plasmids, and the expressed polypeptides were fusion products with the amino terminus of beta-galactosidase. Clones W1-77 and W2-1 expressed fusion products with apparent molecular weights of 40,000 (40K) and 14K, respectively, which were serologically related to PRSV capsid protein. A 52K product serologically related to a 54K nuclear inclusion protein of tobacco etch virus was produced by clone W1-18. The sequences encoding the capsid and 57K nuclear inclusion-like proteins of PRSV were physically mapped to adjacent positions through Southern blot analyses of clones W1-77 and W1-18.     

Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs

Summary The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genusTombusvirus andCarmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Database under the accession number X85215.     

Molecular evidence that zucchini yellow fleck virus is a distinct and variable potyvirus related to papaya ringspot virus and Moroccan watermelon mosaic virus

Summary. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) infects cultivated or wild cucurbits in the Mediterranean basin and occasionally causes severe damage in crops. Biological and serological data tend to indicate that ZYFV is related to other cucurbit-infecting potyviruses, mainly papaya ringspot virus (PRSV) and Moroccan watermelon mosaic virus (MWMV). In order to establish unambiguously the taxonomic status of ZYFV, the sequence of the 3′ part of the genome – encompassing the CP coding region – of two ZYFV strains originating from Italy and France was obtained and compared with other potyviruses. The results obtained indicate that ZYFV belongs to a distinct potyvirus species, related to but different from PRSV and MWMV.     

Genetic variation of papaya ringspot virus in Venezuela

Summary The genetic variation of papaya ringspot virus (PRSV) in Venezuela was estimated by single strand conformation and nucleotide sequence analyses of two genomic regions of twenty-six isolates. These analyses showed that mutation, virus movement, selection, mixed infections and recombination contributed to shape the genetic variation observed. Phylogenetic analysis indicated that Venezuelan isolates were within a clade composed of isolates from the Americas and Australia. The genetic diversity of these isolates was sufficiently large that it must be taken into account when designing control strategies such as transgenic resistance and cross-protection. Correspondence: Edgloris Marys, Instituto Venezolano Investigaciones Científicas (IVIC), Centro de Microbiología y Biología Celular, Laboratorio de Biotecnología y Virología Vegetal, Apartado Postal 21827, Caracas 1020-A, Venezuela     

Nucleotide sequence of the capsid protein gene of papaya leaf-distortion mosaic potyvirus

Summary The DNA complementary to the 3-terminal 1 404 nucleotides [excluding the poly(A) tail] of papaya leaf-distortion mosaic potyvirus (PLDMV) RNA was cloned and sequenced. The sequence starts within a long open reading frame (ORF) of 1 195 nucleotides and is followed by a 3 non-coding region of 209 nucleotides. Capsid protein (CP) is encoded at the 3 terminus of the ORF. The CP contains 293 residues and has a M_r of 33 277. The CP of PLDMV exhibits 49 to 59% sequence similarity at the amino acid level to the CPs of papaya ringspot potyvirus (PRSV) and other potyviruses. This result is consistent with the absence of a serological relationship between PLDMV and PRSV or other potyviruses. The results support the assignment of PLDMV as a distinct member of the genusPotyvirus.Sequence data from this article has been deposited with the EMBL/GenBank/DDBJ databases under accession no. D50082.     

Intraviral homology and subgenomic RNAs of pepper ringspot virus

The Tobraviruses constitute a group of rod-shaped, bipartite, plus-stranded RNA viruses. We report on homologies between the two viral genomic RNA molecules of pepper ringspot virus (PRV) and on the subgenomic components generated from them during infection. It has previously been shown that the 3'-terminal 459 nucleotides of PRV RNA-1 and RNA-2 are identical. Here it is shown that there is a second homology between RNA-1 and a region of RNA-2, located at least 240 nucleotides downstream from its 5' terminus. In agreement with strains in another Tobravirus group, we observe three subgenomic components in extracts of plants infected with PRV. Two of these, RNAs-1a (1.6 kb) and -1b (0.8 kb), are derived from RNA-1 (the larger genomic RNA). They are probably mRNAs for 29- and 16-kDa nonstructural virus proteins. The smaller genomic RNA, RNA-2, generates the subgenomic component RNA-2a (1.3-1.45 kb) which is probably an efficient capsid protein mRNA. Previously reported subgenomic components of 2.8 and 1.1 kb are shown to be electrophoretic artifacts. RNA-2a, but not 1a or 1b, is present in virion RNA preparations, indicating that it is the only subgenomic species to be encapsidated.     

Complete genome of Hainan papaya ringspot virus using small RNA deep sequencing

Small RNA deep sequencing allows for virus identification, virus genome assembly, and strain differentiation. In this study, papaya plants with virus-like symptoms collected in Hainan province were used for deep sequencing and small RNA library construction. After in silicon subtraction of the papaya sRNAs, small RNA reads were used to in the viral genome assembly using a reference-guided, iterative assembly approach. A nearly complete genome was assembled for a Hainan isolate of papaya ringspot virus (PRSV-HN-2). The complete PRSV-HN-2 genome (accession no.: KF734962) was obtained after a 15-nucleotide gap was filled by direct sequencing of the amplified genomic region. Direct sequencing of several random genomic regions of the PRSV isolate did not find any sequence discrepancy with the sRNA-assembled genome. The newly sequenced PRSV-HN-2 genome shared a nucleotide identity of 96 and 94 % to that of the PRSV-HN (EF183499) and PRSV-HN-1 (HQ424465) isolates, and together with these two isolates formed a new PRSV clade. These data demonstrate that the small RNA deep sequencing technology provides a viable and rapid mean to assemble complete viral genomes in plants.     

Sequential encapsidation of heterologous RNAs with papaya mosaic virus protein.

Papaya mosaic virus protein encapsidates heterologous RNAs under conditions normally restricted to the encapsidation of homologous RNA if the heterologous RNAs are first initiated with protein under conditions of nonspecificity. This means that initiation, but not maturation, controls the specificity of encapsidation.     

Polymorphism of genomic RNAs within rotavirus serotypes and subgroups

Summary The genome of human rotaviruses is composed of 11 segments of double stranded RNA. Analysis by polyacrylamide gel electrophoresis of the genomic RNA from a number of strains of the same serotype has demonstrated a significant polymorphism in the migration pattern. However, two strains differing from each other in serotype were found to have apparently identical RNA migration patterns. It is concluded that serotype cannot be deduced from RNA migration patterns.Degrees of polymorphism have been observed within each of three known human rotavirus serotypes.With 5 Figures     

RNAs of influenza C virus strains

Summary Up to 9 RNA segments with molecular weights (MW) 2.3–9.4×10⁵ were-found in an influenza C virus by polyacrylamide gel electrophoresis at 50° C and three different strains. All contained RNA of the same mobility. Possible coding assignments of the RNA segments is discussed.With 2 Figures     

Molecular characterization of a severe isolate of papaya ringspot virus in Mexico and its relationship with other isolates

The virus most often reported in papaya (Carica papaya L.) is papaya ringspot (PRSV). The aim of this work was the molecular genomic characterization of a Mexican severe isolate of PRSV-P “Mex-VrPO” (isolate from the State of Veracruz in Paso de Ovejas) as well as its comparison with other isolates from other world regions. The linear, assembled, single-strand positive sense RNA genome of PRSV-P Mex-VrPO was 10320 nt in length (excluding the poly(A) tail) and contained a single large predicted ORF with 3344 aa. The comparative analysis of our PRSV isolates and five others reported before, showed the most variable proteins were P1, P3, 6 K and CP with 13–33%, 5–7%, 6–9% and 5–9% divergence respectively. The most conserved ones were CI, NIb and HC-Pro (2–3%, 3–5% and 4–5%). The phylogenetic analysis showed a close relation between the Mexican (Mex-VrPO) and Hawaiian (PRSV-P HA) isolates. This work provided the first opportunity to establish the foundation for (1) understanding whole genome and polyprotein variability between Asian and American PRSV isolates, and (2) elucidating major trends in the relative evolution of viral cistrons as deduced from in silico recombination analyses. An erratum to this article can be found at     

Cowpea aphid borne mosaic virus-Morocco and South AfricanPassiflora virus are strains of the same potyvirus

Summary High performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South AfricanPassiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South AfricanPassiflora strain of CABMV.     

The complete sequence of the genomic RNAs of spinach latent virus

Summary.  We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus – CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C₂H₂ type “zinc finger”-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus. Received October 21, 1996 Accepted December 3, 1996     

The genomic sequence and biological properties of Pennisetum mosaic virus, a novel monocot-infecting potyvirus

The complete nucleotide sequences of two isolates of Pennisetum mosaic virus (PenMV) were determined. The viral genome comprised 9,611 nucleotides (nt) excluding the 3′-terminal poly(A) sequence, with the capacity of encoding a single polyprotein of 3,065 amino acids. The large open reading frame is flanked by a 172-nt 5′-untranslated region (UTR) and a 244-nt 3′-UTR. Sequence comparisons and phylogenetic analyses of the complete genome and polyproteins suggest that PenMV is closely related to other monocot potyviruses such as Maize dwarf mosaic virus, Sorghum mosaic virus and Sugarcane mosaic virus (SCMV), and thus represents a distinct potyvirus within the SCMV subgroup. The host range of PenMV is limited to Gramineae, and the virus naturally infects maize, sorghum and some wild grasses, causing mosaic symptoms on the leaves. This virus could be transmitted by both mechanical inoculation and by at least four species of aphids.     

Complete sequence of the genomic RNA of the prevalent strain of a potyvirus infecting maize in China

Summary.  The complete nucleotide sequence of the prevalent strain of a potyvirus isolated from maize in Beijing, China was determined and compared with other closely related potyviruses. The viral genome comprises 9595 nucleotides, excluding the poly (A) tail, and encodes a putative polyprotein of 3063 amino acid residues. Sequence comparison of the coat proteins showed that this isolate was most closely related to most other potyviral isolates infecting maize across China with identities of about 99% and thus represented the prevalent strain. It was also closely related to most isolates of Sugarcane mosaic virus (SCMV) infecting maize in Europe with maximum identity of about 95% at the amino acid level. The polyprotein sequence of the Beijing isolate shares identities of 98% with those of two other Chinese maize isolates and shares identity of 69% with Maize dwarf mosaic virus-Bulgarian isolate, respectively. Phylogenetic analyses of the sequences indicated that the Beijing isolate can be tentatively referred to as a prevalent strain of SCMV. Received November 15, 2001; accepted December 4, 2002     

Recombination between satellite and genomic RNAs of turnip crinkle virus.

New recombinant molecules formed from satellite and genomic RNAs of turnip crinkle virus (TCV) have been characterized. Known collectively as sat-RNA CX, these molecules are composed of a nearly full-length segment of a previously characterized TCV satellite RNA (sat-RNA D) at the 5' end joined to variable lengths of TCV genomic RNA 3' terminal sequence. Sat-RNA CX molecules fall into two classes: molecules of 420 to 435 bases and larger species of 501 to 506 bases. The TCV sequence at the junction of the larger molecules is purine-rich and is similar to a motif found at the 5' ends of the TCV satellite RNAs and at the junctions of some TCV defective interfering RNAs. The TCV sequence at the junction of the smaller sat-RNA CX molecules is pyrimidine-rich and is similar to the sequence at the right side of a junction of one TCV defective interfering RNA as well as sequence immediately downstream of the internal initiation site of the 1.45-kb TCV subgenomic RNA. We propose that the latter motif is another putative signal recognized by the viral replicase during the generation of defective interfering and recombinant RNAs in the TCV system.