Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates

Tomato spotted wilt virus (TSWV) infects numerous host plants and has three genome segments, called L, M and S. Here, we report the complete genome sequences of three Korean TSWV isolates (TSWV-1 to -3) infecting tomato and pepper plants. Although the nucleotide sequence of TSWV-1 genome isolated from tomato is very different from those of TSWV-2 and TSWV-3 isolated from pepper, the deduced amino acid sequences of the five TSWV genes are highly conserved among all three TSWV isolates. In phylogenetic analysis, deduced RdRp protein sequences of TSWV-2 and TSWV-3 were clustered together with two previously reported isolates from Japan and Korea, while TSWV-1 grouped together with a Hawaiian isolate. A phylogenetic tree based on N protein sequences, however, revealed four distinct groups of TSWV isolates, and all three Korean isolates belonged to group II, together with many other isolates, mostly from Europe and Asia. Interestingly, most American isolates grouped together as group I. Together, these results suggested that these newly identified TSWV isolates might have originated from an Asian ancestor and undergone divergence upon infecting different host plants.     

Protein composition of tomato spotted wilt virus

Analysis of the protein composition of tomato spotted wilt virus (TSWV), purified by an improved procedure, by polacrylamide gel electrophoresis, revealed three major structural proteins (of MW 84,000, 50,000, and 29,000d) and a minor one of MW 220,000d. The three major proteins constitute about 98% of the total viral protein and all three were shown to be glycoproteins. One of the major proteins (MW 29,000d) and the minor protein were shown to be associated with subviral particles isolated by treatment of virus with the nonionic detergent Nonidet P-40. Only traces of the other two proteins were detected in the subviral particles.Synthesis of virus-induced proteins in TSWV-infected tobacco leaves was studied by labeling infected and healthy tissue with [³H]and [¹⁴C]valine, respectively. The labeled tissues were then fractionated into crude subcellular fractions and protein patterns of healthy and infected tissues were compared by coelectrophoresis on polyacrylamide gels. Only one virus-specific protein (of MW 49,000d) was detected in the virus-enriched fractions; this corresponded with the viral structural protein of MW 50,000d.     

Purification and properties of tomato spotted wilt virus

Tomato spotted wilt virus was purified by chromatography on columns of calcium phosphate, precipitation with polyethylene glycol, density gradient centrifugation, and ascending zone electrophoresis in a sucrose density gradient. Examination of the virus by electron microscopy revealed particles of different shapes in different suspending media. Particles fixed with glutaraldehyde were spherical and somewhat flattened and had a corrected diameter of 85 nm.Antisera with a titer of $\text{1}\text{128}$ against the virus were prepared which did not react with plant antigens.The virus had a sedimentation coefficient s_20,w of 530 S and, when fixed in 0.5% glutaraldehyde, it had an electrophoretic mobility at pH 7 of ?19.8 × 10^-5 cm² V^?1 sec^?1.     

The first complete genome sequences of clinical isolates of human coronavirus 229E

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 3??UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.     

Status of tobacco viruses in Serbia and molecular characterization of tomato spotted wilt virus isolates

In a four-year survey to determine the presence and distribution of viruses in tobacco crops at 17 localities of the Vojvodina Province and Central Serbia, 380 samples were collected and analyzed by DAS-ELISA. Out of the seven viruses tested, tomato spotted wilt virus (TSWV), potato virus Y (PVY), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV) were detected in 37.9, 33.4, 28.7, 23.9, and 15.5% of the total tested samples, respectively. TSWV was the most frequently found virus at the localities of Central Serbia, while PVY and CMV were the most frequent viruses in the Vojvodina Province. Single infections were prevalent in years 2005-2007 and the most frequent were those of PVY. A triple combination of those viruses was most frequent mixed infection type in 2008. The presence of all five detected viruses was confirmed in selected ELISA-positive samples by RT-PCR and sequencing. The comparisons of obtained virus isolate sequences with those available in NCBI, confirmed the authenticity of serologically detected viruses. Phylogenetic analysis based on partial nucleocapsid gene sequences revealed a joint clustering of Serbian, Bulgarian and Montenegrin TSWV isolates into one geographic subpopulation, which was distinct from the other subpopulation of TSWV isolates from the rest of the European countries. The high incidence of viruses in Serbian tobacco crops highlights the importance of enhancing farmers knowledge towards better implementation of control strategies for preventing serious losses.     

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.     

Complete genome sequence of a tomato spotted wilt virus isolate from China and comparison to other TSWV isolates of different geographic origin

Tomato spotted wilt virus (TSWV) is well established in most countries worldwide, while it is rarely reported in China. In this report, we have determined the complete nucleotide sequence of a TSWV isolate named TSWV-YN infecting tomato in Yunnan province in southwestern China. The tripartite genome of TSWV-YN was found to consist of L, M and S RNAs of 8910, 4773 and 2970 nt, respectively. The complete genome sequence and the sequence of each genomic region of TSWV-YN from China were compared to those of four other TSWV isolates from Brazil and Korea. The phylogenetic relationship of the Chinese TSWV-YN isolate to other TSWV isolates of different geographic origin, based on the nucleotide sequences of the glycoprotein (GP) and nucleocapsid (N) genes, was also analyzed in this study.     

Comparison of complete nucleotide sequences and genome organization of six distinct cherry leaf roll virus isolates from New Zealand

The complete genomic sequences of six phenotypically distinct cherry leaf roll virus (CLRV) isolates from New Zealand were determined and compared. RNA1 and RNA2 are 7,919-7,921 and 6,361-6,363 nucleotides in length, respectively, excluding the poly(A) tails. Both genome segments contain a single open reading frame and encode one polyprotein of 2,113 amino acids for RNA1 and 1,590 aa for RNA2, with corresponding molecular masses of 235.5-236.1 kDa and 174.2-174.8 kDa, respectively. The six RNA1 sequences share 92.0-99.7 % nt and 97.0-99.5 % aa similarity, and the RNA2 sequences share 91.4-99.8 % nt and 94.3-99.7 % aa similarity. Phylogenetic analysis established close associations between the six New Zealand CLRV isolates and members of the subgroup C nepoviruses.     

Effect of Virazole (Ribavirin) on tomato spotted wilt virus in two systemic hosts,tomato and tobacco

Summary Virazole (1, beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide = Ribavirin), a synthetic nucleoside analogue, has been successfully used against tomato spotted wilt virus (TSWV) in tomato and tobacco plants. In tomato, the most efficient concentration to control TSWV was 500 mg/l while in tobacco, concentrations of 100 mg/l were sufficient to control systemic infection. When the plants did not show a systemic reaction virus could not be recovered from inoculated and treated tomato and tobacco plants by means of the local lesion assay. The results point out the response of a new plant virus to Virazole and indicate a possibility of controlling TSWV systemic infection.     

Electron microscopy of developmental stages of tomato spotted wilt virus in plant cells

Shortly after infection with TSWV, leaf parenchyma cells of Tropaeolum majus, Nicotiana tabacum cv. Samsun NN, and Cucumis sativus show typical, dark, diffuse masses containing locally denser striated spots. The striation is about 5 nm. The dark, diffuse masses intimately surrounded by ribosomes are freely scattered in the cytoplasm. They occur alone or in combination with characteristic virus particles in clusters within the cisternae of the endoplasmic reticulum system. Therefore it is assumed that they represent an early phase in the assembly process of the virus particles. The results of pronase incubation of sections suggest a proteinaceous nature of the denser striated spots.     

The complete nucleotide sequence and genome organization of tomato chlorosis virus

Summary. The crinivirus tomato chlorosis virus (ToCV) was discovered initially in diseased tomato and has since been identified as a serious problem for tomato production in many parts of the world, particularly in the United States, Europe and Southeast Asia. The complete nucleotide sequence of ToCV was determined and compared with related crinivirus species. RNA 1 is organized into four open reading frames (ORFs), and encodes proteins involved in replication, based on homology to other viral replication factors. RNA 2 is composed of nine ORFs including genes that encode a HSP70 homolog and two proteins involved in encapsidation of viral RNA, referred to as the coat protein and minor coat protein. Sequence homology between ToCV and other criniviruses varies throughout the viral genome. The minor coat protein (CPm) of ToCV, which forms part of the “rattlesnake tail” of virions and may be involved in determining the unique, broad vector transmissibility of ToCV, is larger than the CPm of lettuce infectious yellows virus (LIYV) by 217 amino acids. Among sequenced criniviruses, considerable variability exists in the size of some viral proteins. Analysis of these differences with respect to biological function may provide insight into the role crinivirus proteins play in virus infection and transmission.     

Phylogenetic analysis of New Zealand tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution

Tomato spotted wilt virus (TSWV) is an internationally significant pathogen with a wide host range, vectored by thrips. We have studied the sequence variation and evolutionary mechanisms at play in parts of the L, M and S subgenomes of 23 New Zealand TSWV isolates collected between 1992 and 2009, aiming to identify the possible geographic origins of isolates. Maximum-likelihood-based phylogenetic analyses of New Zealand and overseas TSWV isolates placed the L and M subgenome sequences of two isolates (MAF04 and PFR04) in distinct clades composed primarily of Korean, Japanese and Chinese isolates, in contrast to the remaining 21 isolates, which clustered with a cosmopolitan group of isolates. The nucleocapsid (N) gene sequences of MAF04 and PFR04 plus MAF02 clustered with Japanese isolates. Consequently, we postulate that these isolates may represent a distinct incursion into New Zealand, but we do not have enough evidence to indicate an incursion pathway. Alternately, these isolates may have arrived with an incursion that included a mixture of TSWV isolates of diverse international origins. The sequences of four of the TSWV isolates contained a number of sites with a mixture of nucleotides, suggesting that these isolates either consisted of several sequence variants or were from plants with mixed infections. One isolate (MAF02) was shown to be a either a reassortant or an S subgenome recombinant. Large amounts of low-level polymorphism were detected with low amino acid change fixation rates (purifying selection). Negative selection was indicated at four amino acid sites in the New Zealand TSWV N gene sequences.