Zucchini tigré mosaic virus is a distinct potyvirus in the papaya ringspot virus cluster: molecular and biological insights

In recent years, three new potyviruses have been described in the papaya ringspot virus (PRSV) cluster. In addition, two types of PRSV are recognized, type W, infecting cucurbit plants, and type P, infecting papaya and also cucurbits. A third type, PRSV-T, was also partially described in Guadeloupe. Complete genome sequencing of four PRSV-T isolates showed that this virus is a related virus that is distinct from PRSV, and the name zucchini tigré mosaic virus (ZTMV) is proposed, in reference to the typical symptoms observed in zucchini squash. Eleven other viral isolates from different geographic origins were confirmed as ZTMV isolates using the complete sequence of the cylindrical inclusion (CI) coding region, whereas pairwise sequence similarities in the coat protein (CP) coding region did not unambiguously distinguish ZTMV isolates from PRSV isolates. The use of the CI coding region for species demarcation appears more suitable than the CP coding region for closely related viruses. Principal coordinates analysis based on the biological behavior of the viral isolates studied clustered PRSV-P, PRSV-W and ZTMV isolates into three different groups. Therefore, ZTMV is different from PRSV in its molecular and biological properties.     

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     

Sequence characterization of a Peruvian isolate of Sweet potato chlorotic stunt virus: further variability and a model for p22 acquisition

Sweet potato chlorotic stunt virus (SPCSV) is probably the most important virus infecting sweetpotato worldwide, causing severe synergistic disease complexes with several co-infecting viruses. To date only one isolate (Ug), corresponding to the EA strain has been completely sequenced. It was later shown to be unusual in that, in contrast to most isolates, it encoded an additional p22 protein at the 3' end of RNA1. We report the complete sequence and genome organization of a Peruvian isolate of SPCSV (m2-47) as determined by siRNA deep sequencing. We confirm that the ORF encoding p22 is lacking from m2-47 and all tested Peruvian and South American isolates, whereas additional isolates containing p22 were identified from Uganda. Other potentially important genomic differences such as two small ORFs encoding putative small hydrophobic proteins instead of one, upstream the hsp70h gene and a more divergent sequence at its RNA1 3'-UTR in contrast to SPCSV isolates that contain p22 are discussed and a model for recent acquisition of p22 in Uganda is proposed. A role for p22 as a pathogenicity enhancer of SPCSV is also provided by complementary expression of p22 in transgenic sweetpotato plants.     

Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus

Summary. The complete nucleotide sequence of the genome of a Taiwan isolate of papaya ringspot potyvirus (PRSV YK) was determined from three overlapping cDNA clones and by direct RNA sequencing. Comparison was made with the reported Hawaii isolate of PRSV HA. Both genomes are 10 326 nucleotides long, excluding the poly(A)-tail. They encode a polyprotein of 3 344 amino acids with a 5′ leader of 85 nucleotides and a 3′ non-translated region of 209 nucleotides. The two genomes share an overall nucleotide identity of 83.4% and an amino acid identity of 90.6%. The 3′ non-translated regions show 92.3% identity. The first 23 nucleotides of the leaders are identical, while the remaining parts of the leaders only show 51.6% identity. The P1 protein genes of the two isolates are very different, with 70.9% nucleotide identity and 66.7% encoded amino acids identity. However, the other viral proteins of the two virus isolates are similar, with a 82.5–89.8% nucleotide identity of their genes and 91.2–97.6% amino acid identity, indicating that they are strains of the same potyvirus. Analysis of the ratios of nucleotide differences to the actual amino acid changes revealed that there are only 2.63 nucleotide changes for each amino acid change in the P1 protein, whereas for the other proteins 4.0–16.4 nucleotide changes are required for each amino acid replacement. The P1 protein has 58% of all the differences of polyprotein. The unusual variation in the leader sequences and the P1 proteins suggests that the two PRSV strains were derived from different evolutionary pathways in different geographic areas. Received February 5, 1996 Accepted September 17, 1996     

Phylogeography and molecular epidemiology of Papaya ringspot virus

Papaya ringspot virus (PRSV) is the most important virus affecting papaya and cucurbit plants in tropical and subtropical areas. PRSV isolates are divided into biotypes P and W: both the P and W types naturally infect plants in the family Cucurbitaceae, whereas the P type naturally infects papaya (Carica papaya). Understanding the origin and nature of the PRSV genetic diversity and evolution is critical for the implementation of control strategies based on cross-protection and the deployment of transgenic plants that show resistance to virus isolates highly similar to the transgene. The molecular epidemiology of PRSV was evaluated by analyzing the nucleotide sequence of the capsid protein (CP) and helper component-proteinase (HC-Pro) genes of isolates from around the world, including newly characterized ones from Colombia and Venezuela, using a relaxed molecular clock-based approach and a phylogeographic study. Our results confirm previous estimates on the origin of PRSV around 400 years ago and suggest distinct dispersion events from the Indian Peninsula to the rest of Asia, via Thailand, and subsequently to the Americas. A historical reconstruction of the P- and W-type characters in the phylogenetic study supports the need to revise the hypothesis that PRSV-P derives from PRSV-W since our results suggest that the ancestral state could be either of the two biotypes. Moreover, estimates of epidemic growth predict an increasing genetic diversity of the virus over time that has direct implications for control strategies of PRSV based on cross-protection and the use of transgenic plants.     

Ambient temperature perception in papaya for <Emphasis Type="Italic">papaya ringspot virus</Emphasis> interaction

Temperature dramatically affects the host–virus interaction. Outbreaks of viral diseases are frequently associated with the ambient temperature required for host development. Using papaya as a host and Papaya ringspot virus (PRSV) as a pathogen, we studied the effect of temperature on the intensity of disease symptoms and virus accumulation. The phenotypic expression of symptoms and viral accumulation were found to be maximum at ambient temperature (26–31°C) of papaya cultivation. However, there was a drastic difference, 10°C above and below the ambient temperature. The underlying mechanism of these well-known observations are not yet understood completely; however, these observations might help find answers in RNA silencing mechanism of plants. Since viral-derived silencing suppressor proteins play a significant role in RNA silencing mechanism, here we show that PRSV-derived Helper component proteinase (HC-Pro) protein has an affinity for small RNAs in a temperature-dependent manner. This suggested the probable role of HC-Pro in the temperature-regulated host–virus relationship.     

Sequence and phylogenetic analysis of the RNA1 and RNA2 segments of Korean <Emphasis Type="Italic">Rice stripe virus</Emphasis> isolates and comparison with those of China and Japan

Rice stripe virus (RSV) is one of the most destructive pathogens of rice plants in East Asia. The RSV genome consists of four single-stranded RNA segments. We have determined and compared the complete nucleotide sequences of the RNA1 and RNA2 segments and the deduced amino acid sequence of each ORF of the 13 Korean RSV isolates and established their relationships with reported RSV sequences from China and Japan. Our results showed that the average percent nucleotide divergence based on the full-length genome is higher in RNA2 (2.2%) than in RNA1 (2.0%). The average percent amino acid variation of the RNA-dependent RNA polymerase (RdRp), glycoprotein and NS2 genes encoded by viral complementary (vc) RNA1, viral RNA2 and vcRNA2, showed 2.8, 2.5 and 6.46%, respectively. On the other hand, the average percent nucleotide variation in the intergenic region (IGR) of RNA2 among the 13 Korean-RSV isolates was 3.5%. Phylogenetic analysis of the 13 Korean, 1 Japanese and 5 Chinese isolates based on their complete nucleotide sequences revealed two distinct types of RNA1 and three distinct types of RNA2. Most Chinese isolates grouped with one of the RNA1 types, but they were distributed among the three types when grouped by RNA2. Japanese isolate T was grouped with Korean isolates into one of the RNA1 and RNA2 genotypes. Taken together, our results suggest that the RSV population in Korea consists of mixtures of RNA1–RNA4 genome segments originating from distinctive ancestors, most likely due to either reassortment or recombination events among isolates.     

Phylogenetic analyses of an isolate obtained from potato in 1985 revealed potato virus X was introduced to China via multiple events

Potato virus X (PVX) is one of the most common plant viruses that cause great economic losses to solanaceous plants. We have previously reported the complete genomic sequence of the 2006 Chinese potato isolate FX21 and demonstrated that PVX isolates cluster into two groups: Eurasia and America. Here, we present the complete genomic sequence of one PVX isolate collected from potato in 1985 (PVX-1985). The genome of PVX-1985 is identical to that of FX21 in length and has the same genomic structure. PVX-1985, which like FX21 fell within the Eurasia group, clustered together with isolates from Europe, whereas FX21 clustered together with isolates of primarily Asian origin. Phylogenetic analyses of the complete genomic sequences and of CP gene sequences showed that Chinese PVX isolates have different origins and were introduced via multiple events. Though all the open reading frames of PVX are under negative/purifying selection, the central region of RNA-dependent RNA polymerase is under positive/diversifying selection.     

N-terminal of <Emphasis Type="Italic">Papaya ringspot virus</Emphasis> type-W (PRSV-W) helper component proteinase (HC-Pro) is essential for PRSV systemic infection in zucchini

The Papaya ringspot virus (PRSV) is one of the limiting factors affecting papaya and cucurbits production worldwide. PRSV belongs to the potyvirus genus which consists of 30% of known plant viruses. Two serological closely related strains, namely type-P and -W, have been reported. PRSV type-P infects both papaya and cucurbits, while type-W infects only cucurbits. The genome of PRSV Thailand isolate consists of a (+) RNA molecule of 10323 nucleotides, which is first translated into a single polypeptide and further cleaved by three viral encoded proteases into ten gene products. Helper-component proteinase (HC-Pro), which is encoded by the 2nd cistron of the potyviral genome, has been implicated in aphid transmission, viral movement, viral replication and suppression of host viral defense system. Studies of the Tobacco etch virus (TEV), Lettuce mosaic virus (LMV), Onion yellow dwarf virus (OYDV) and Wheat streak mosaic virus (WSMV) indicate that the N-terminal of HC-Pro is dispensable for systemic infection in their respective hosts. However, deletion analysis of the Tobacco vein mottling virus (TVMV) indicates otherwise. In this study, we examined whether HC-Pro is essential for PRSV systemic infection in cucurbits and the role of its N-terminal in systemic infection. Our results indicated that HC-Pro is indispensable for PRSV infection in zucchini. Deletion analysis of PRSV HC-Pro showed that deletion of as few as 54 amino acids at the N-terminal of HC-Pro completely abolished the infectivity of the corresponding cDNA clone. Therefore, it is proposed that the N-terminal of HC-Pro is involved in systemic infection of PRSV, in addition to its conserved function in aphid transmission.

The genome structure of turnip crinkle virus

The nucleotide sequence of turnip crinkle virus (TCV) genomic RNA has been determined from cDNA clones representing most of the genome. Segments were confirmed using dideoxynucleotide sequencing directly from viral RNA, and the 3' terminal sequence was confirmed by chemical sequencing of end-labeled genomic RNA. Three open reading frames (ORFs) have been identified by examination of the deduced amino acid sequences and by comparison with the ORFs found in the genome of carnation mottle virus. ORF 1 initiates near the 5' terminus of the genome and is punctuated by an amber termination codon. Translation of ORF 1 would yield a 28-kDa protein and an 88-kDa read-through product. The read-through domain possesses amino acid sequence similarities with putative viral RNA polymerases. ORFs 2 and 3 encode products of 38 (coat protein) and 8 kDa, respectively, which are expressed from subgenomic mRNAs. The organization of the TCV genome suggests that TCV is closely related to carnation mottle virus and distinct from members classified in other small RNA virus groups, such as the tombus- and sobemoviruses.     

A set of host proteins interacting with papaya ringspot virus NIa-Pro protein identified in a yeast two-hybrid system

The protein-protein interactions between viral and host proteins play an essential role in plant virus infection and host defense. The potyviral nuclear inclusion protein a?protease (NIa-Pro) is involved in various steps of viral infection. In this study, the host proteins interacting with papaya ringspot virus (PRSV) NIa-Pro were screened in a?Carica papaya L. plant cDNA library using a?Sos recruitment two-hybrid system (SRS). We con?rmed that the full-length EIF3G, FBPA1, FK506BP, GTPBP, MSRB1, and MTL from?papaya can interact speci?cally with PRSV NIa-Pro in yeast, respectively. These proteins fufill important functions in plant protein translation, biotic and abiotic stress, energy metabolism and signal transduction. In this paper, we discuss possible functions of interactions between these host proteins and NIa-Pro in PRSV infection and their role in host defense. Keywords: Sos recruitment two-hybrid system; papaya ringspot virus; NIa-Pro; protein-protein interaction.     

Nucleotide sequence of a Thai isolate of Papaya ringspot virus type W

The complete nucleotide sequence of a Thai isolate of Papaya ringspot virus (PRSV) type W (PRSV-W) was determined. The viral genome is 10,323 nucleotides (nts) long and contains an ORF encoding polyprotein 3,343 amino acids (aa) long, flanked with 5'- and 3'-non-coding regions (NCRs) of 85 and 206 nts, respectively. Out of the ten putative proteins P1 is the most variable (73.9% similarity) as compared to the PRSV type P (PRSV-P) sequences, while the CI protein is most conserved (99.1% similarity). The sequence similarity among the type W and P isolates also suggests that the P type arose from the W type. However, no significant difference between types P and W that would account for the host specificity was disclosed.     

Identification and molecular characterization of a new potyvirus from Panax notoginseng

A potyvirus causing distortion and mosaic symptoms in the herbal plant Sanqi (Panax notoginseng) was isolated from Yunnan province, China, and the complete nucleotide sequence of one isolate and the partial sequences of two other isolates were determined. The viral RNA genome comprised 9,750 nt excluding the 3′-terminal poly(A) tail, with the capacity to encode a single polyprotein of 3,089 amino acids. Phylogenetic analysis with other completely sequenced potyviruses revealed that the virus in this study was most closely related to plum pox virus, with 56.3% nt identity in the genomic RNA sequence and 53.3% aa identity in the polyprotein. However, the most closely related 3′-terminal sequences were from four partially sequenced potyviruses infecting plants of the family Apiaceae (67.7–75.3% nt identity and 73.8–76.7% aa identity in their coat protein cistrons), especially Angelica virus Y. These results suggest that this virus isolate should be designated a member of a new species in the genus Potyvirus, which is tentatively named Panax virus Y (PanVY).     

Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing

RNA interference is a natural defense against viruses in plants. To date, the only viral siRNAs characterized have been those for positive-sense RNA viruses with one or two genome components. Here, we characterized siRNAs derived from rice stripe virus (RSV), a member of the genus Tenuivirus with four genomic RNAs and an ambisense coding strategy. Deep sequencing of small RNAs from infected rice leaves showed that siRNAs were derived almost equally from virion and complementary RNA strands and were mostly 20–22 nucleotides long. Most viral siRNAs were produced within the coding sequences and 5′ termini of the RSV genome. RSV siRNAs had a higher G and lower C content than the viral genome but a strong A/U bias at the first nucleotide and a U bias at the final one, suggesting preferential targeting of such sequences by rice Dicer-like proteins.     

Identification of a satellite double-stranded RNA in the parasitic protozoan Trichomonas vaginalis infected with T. vaginalis virus T1

Co-infection by a 0.5-kb small double-stranded (ds) RNA together with Trichomonas vaginalis virus (TVV) genomic 4.6-kb dsRNA is commonly observed in a number of T. vaginalis isolates. By molecular cloning and primer extension experiments, the 497-bp cDNA sequence of a 0.5-kb dsRNA co-infecting with TVV-T1 in T vagina/is T1 isolate was elucidated. Consistent with the replication cycle of a typical dsRNA virus, a plus-strand viral RNA beginning at +1 of the 0.5-kb dsRNA was identified in infected T. vaginalis T1 cells by primer extension and Northern hybridization studies. The 0.5-kb dsRNA was separately encased in TVV capsids from the viral genomic dsRNA, as shown by protein analysis and electron microscopic examination of viral particles purified by multiple rounds of CsCl gradient centrifugation. The riboprobes transcribed from a cloned cDNA of the 0.5-kb dsRNA exhibited strong hybridization to a small dsRNA in a T vaginalis T9 isolate, which harbors a TVV-T9 distantly related to TVV-T1, but the same probes showed very little hybridization to the viral genomic dsRNA of both TVV-T1 and TVV-T9. Very little sequence homology between the 0.5-kb dsRNA and the 4.6-kb dsRNA in TVV-T1 was found by computer-assisted analysis, suggesting that the small dsRNA in T. vaginalis T1 is not derived from the genome of TVV-T1 or other distantly related T. vaginalis viruses. These results suggest that the small dsRNAs in T vaginalis are satellite RNAs of T. vaginalis virus.     

Specific binding of Bluetongue virus NS2 to different viral plus-strand RNAs

The Reoviridae have double-stranded RNA genomes of 10-12 segments, each in a single copy in the mature virion. The basis of genome segment sorting during virus assembly that ensures each virus particle contains the complete viral genome is unresolved. Bluetongue virus (BTV) NS2 is a single-stranded RNA-binding protein that forms inclusion bodies in infected cells. Here, we demonstrate that the specific interaction between NS2 and a stem-loop structure present in BTV S10 RNA, and phylogenetically conserved in other BTV serotypes, is abolished by mutations predicted to disrupt the structure. Subsequently, we mapped RNA regions in three other genomic segments of BTV that are bound preferentially by NS2. However, structure probing of these RNAs did not reveal secondary structure motifs that obviously resembled the stem-loop implicated in the NS2-S10 interaction. In addition, the specific binding by NS2 to two different viral RNAs was found to occur independently. Together, these data support the hypothesis that the recognition by NS2 of different RNA structures may be the basis for discrimination between viral RNAs during virus assembly.     

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.     

Complete genomic characterization of a potato mop-top virus isolate from the United States

Potato mop-top virus (PMTV; family Virgaviridae) was reported recently in the Pacific Northwestern USA. To better understand the genetic diversity of this virus, the complete genome of an isolate from Washington State (WA), USA, was characterized. Sequence comparisons of the WA isolate with other known sequences revealed that the RNA-Rep-encoded RdRp protein and the RNA-CP-encoded coat protein displayed >99 % amino acid sequence identity to those of two Nordic (RdRp) and several European and North American isolates (CP), respectively. The RNA-TGB-encoded TGB 1 and TGB 3 protein sequences had >99 % amino acid sequence identity to the corresponding proteins of Czech and Danish isolates, whereas the TGB 2 protein is identical to those of Colombian isolates. Phylogenetic analysis of the viral genes of the WA isolate reflected the close relationship between WA and European isolates. RFLP analysis of corresponding DNA of RNA TGB and RNA CP revealed that the WA isolate has the RNA TGB-II and RNA CP-B types, which are prevalent in Europe and other parts of world. This is the first report of the complete genome characterization of PMTV from the Americas.