Serological and Molecular Characterization of a High Temperature-recovered Virus Belonging to Tospovirus Serogroup IV

A serologically and cytologically distinct gloxinia tospovirus (HT-1) previously isolated from a gloxinia plant infected with Impatiens necrotic spot virus (INSV) when propagated in a high-temperature environment was characterized. Rabbit antisera produced for INSV and Tomato spotted wilt virus (TSWV) nucleocapsids (N) failed to react with HT-1 proteins in western blot analysis. The HT-1 antibodies reacted strongly with homologous antigen but failed to react with INSV and TSWV. However, the HT-1 antiserum reacted in ELISA with Watermelon silver mottle virus (WSMV) from Taiwan and in western blot analysis with the WSMV N protein. A reciprocal test showed that the antiserum prepared against the N protein of WSMV also reacted with the HT-1 N protein in both ELISA and western blot analysis. DNA probes derived from the N gene of HT-1 or WSMV hybridized to RNAs prepared from plants infected with either virus. Stronger signals were obtained with homologous than with heterologous reactions. Neither probe detected INSV or TSWV. The M and S RNAs of HT-1 were sequenced. The M RNA contains two open reading frames (ORF) ; one in the sense orientation encoding a nonstructural (NSm) protein of 308-amino-acids (aa) and the other in the ambisense orientation, a 1122-aa precursor of Gl and G2 glycoproteins. The S RNA also contains two ORFs ; one in the sense orientation encoding a nonstructural (NSs) protein of 439 aa and the other in the ambisense orientation, an N protein of 277 aa. HT-1 is distantly related to INSV and TSWV as shown by low nucleotide (40–52%) and amino acid (28–48%) similarities in the four ORF sequences. The HT-1 virus shares high nucleotide (76–81%) and amino acid (85–92%) similarities with WSMV and peanut bud necrosis virus (PBNV). Based on the serological properties and sequence data, we propose that HT-1 is a distinct species of serogroup IV in the genus Tospovirus. This is the first time that a tospovirus similar to those found in the Far East and in Southeast Asia has been identified in the US. Received 16 October 1999/ Accepted in revised form 20 December 1999     

Serological and Molecular Characterization of Peanut chlorotic fan-spot virus, a New Species of the Genus Tospovirus

ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.     

Hypovirulence-Associated Double-Stranded RNA from Sclerotinia homoeocarpa Is Conspecific with Ophiostoma novo-ulmi Mitovirus 3a-Ld

ABSTRACT The nucleotide sequence of the hypovirulence-associated double-stranded RNA (dsRNA) in hypovirulent isolate Sh12B of Sclerotinia homoeocarpa, the causal agent of dollar spot of turf grass, was determined. This large dsRNA (L-dsRNA) is 2,632 bp long and is A and U rich (61.0% A+U residues). One strand of this dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 720 amino acids. This ORF contains 12 UGA codons, predicted to encode tryptophan in ascomycete mitochondria, and has a codon bias typical of mitochondrial genes, which is consistent with a mitochondrial localization of this dsRNA. The amino acid sequence contains conserved motifs typical of RNA-dependent RNA polymerases (RdRps). Sequence analyses of the nucleotide and RdRp-like protein revealed that the L-dsRNA is homologous with previously characterized mitochondrial viruses and dsRNAs from other phytopathogenic fungi, and shares 92.4% nucleotide and 95.1% amino acid sequence identities with the Ophiostoma novo-ulmi mitovirus 3a-Ld from Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The results indicate that these two dsRNAs are conspecific. This is the first report that a hypovirulence-associated dsRNA virus naturally occurs in two taxonomically distinct fungi, and indicates that horizontal transmission of this dsRNA virus may have occurred between these fungi.     

Characterization of two Tospoviruses in Italy: tomato spotted wilt and impatiens necrotic spot

Tospovirus serogroups I and III have recently been designated as species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), while the species status of serogroup II isolates remains undefined. Fifteen Tospovirus isolates from ornamental and vegetable crops in Liguria, Italy, were found to belong either to TSWV (seven isolates) or to INSV (eight isolates) on the basis of test-plant reactions, serological techniques using DAS ELISA kits raised against the nucleoproteins of the type members of the two species, and cytopathology. None of them could be assigned to serogroup II using DAS ELISA kits raised against nucleoproteins of this serogroup. Italian isolates representative of the two species reacted in indirect ELISA using a polyclonal antiserum against the entire particle of a TSWV isolate, but with higher intensity for our TSWV isolates than for the INSV isolates. Western blots and dot immunobinding assays confirmed that the nucleoproteins of the two species are unrelated whereas the glycoproteins are related. The cytopathology was similar for two isolates representative of TSWV and INSV, except that the type of filaments encountered was different, and appeared to be characteristic of the species.     

Identification and characterization of a tospovirus causing chlorotic ringspots on <Emphasis Type="Italic">Phalaenopsis</Emphasis> orchids

A putative virus-induced disease showing chlorotic ringspots on leaves of Phalaenopsis orchids has been observed in Taiwan for several years. A virus culture, 91-orchid-1, isolated from a Phalaenopsis orchid bearing chlorotic ringspot symptoms was established in Chenopodium quinoa and Nicotiana benthamiana, and characterized serologically and biologically. The virus reacted slightly with the antiserum of Watermelon silver mottle virus (WSMoV) but not with those of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Groundnut ringspot virus (GRSV). Isometric particles measuring about 70–100 nm were observed. Inoculation with isolated virus was conducted to confirm that 91-orchid-1 is the causal agent of chlorotic ringspot disease of Phalaenopsis orchids. To determine the taxonomic relationships of the virus, the conserved region of L RNA and the complete nucleocapsid gene (N gene) were cloned and sequenced. The sequence of conserved region of L RNA shares 83.8, 82.5, 64.4 and 64.9% nucleotide identities and 96.5, 97.7, 67.3 and 67.6% amino acid identities with those of Peanut bud necrosis virus (PBNV), WSMoV, TSWV and INSV, respectively, indicating that 91-orchid-1 is a tospovirus related to WSMoV. The complete nucleotide sequence of the N gene determined from a cDNA clone was found to be 828 nucleotides long encoding 275 amino acids. Sequence analyses of the N gene showed that 91-orchid-1 is an isolate of Capsicum chlorosis virus (CaCV) which has been reported to infect tomato and capsicum plants in Australia and Thailand. 91-orchid-1 is therefore designated as CaCV-Ph. To our knowledge, this is the first formal report of a tospovirus infecting Phalaenopsis orchids.     

云南西瓜银灰斑驳病毒病害的鉴定

 应用DAS-ELISA和RT-PCR方法从褪绿和银色斑驳的西瓜叶片中检测到病毒分离物（WSMoV-YN）,感病样品能与WSMoV/GBNV复合抗血清（Agdia）呈阳性反应。获得WSMoV N蛋白的多克隆抗体,抗体能与WSMoV血清组成员CaCV和TZSV反应,但不能与INSV、TSWV、HCRV和GYSV反应。为明确引起该病害的病毒种类,采用Tospovirus通用引物对样品的总RNA进行RT-PCR扩增,获得长度为3 554 nt的S RNA全序列,经Blastn比对分析与WSMoV中国台湾分离物同源性最高,为95.8%,其N和NSs蛋白氨基酸序列同源性分别为99%和97.6%。构建系统进化树发现,西瓜银灰斑驳病毒云南分离物（WSMoV-YN）与其他WSMoV聚为一支。确定引起云南西瓜病害的病毒为WSMoV。     

Phylogenetic relationships within the family potyviridae: wheat streak mosaic virus and brome streak mosaic virus are not members of the genus rymovirus

ABSTRACT The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5' leader and 149-nt 3'-untranslated region and is polyadenylated at the 3' end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5'-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3'). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is the type species. Phylogenetic analyses were conducted with complete polyprotein or NIb protein sequences of 11 members of the family Potyviridae, including viruses of monocots or dicots and viruses transmitted by aphids, whiteflies, and mites. WSMV and the monocot-infecting, mite-transmitted brome streak mosaic virus (BrSMV) are sister taxa and share a most recent common ancestor with the whitefly-transmitted sweet potato mild mottle virus, the type species of the proposed genus "Ipomovirus." In contrast, RGMV shares a most recent common ancestor with aphid-transmitted species of the genus Potyvirus. These results indicate that WSMV and BrSMV should be classified within a new genus of the family Potyviridae and should not be considered species of the genus Rymovirus.     

Purification and serology of virions of impatiens necrotic spot tospovirus

Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.     

Characterization of a vector-non-transmissible isolate of Tomato spotted wilt virus

The characteristics of a thrips‐non‐transmissible isolate of Tomato spotted wilt virus (TSWV), designated TSWV‐M, were compared with those of a thrips‐transmissible isolate, designated TSWV‐T. TSWV‐M showed a narrower host range than TSWV‐T. Adult thrips failed to transmit TSWV‐M, although the vector acquired the virus during the larval stages. TSWV‐M was detected by RT‐PCR in adult thrips bodies, but not in thrips heads, suggesting that loss of thrips transmissibility was the result of the absence of virus in adult thrips salivary glands. Whereas N (nucleoprotein), NSs (non‐structural protein) and G_C (the C‐terminal portion of the glycoprotein precursor protein) were present in similar amounts in leaf tissue from TSWV‐M‐ or TSWV‐T‐infected plants, G_N (the N‐terminal portion of the glycoprotein precursor protein) was present at much lower amounts in TSWV‐M‐ than in TSWV‐T‐infected plants. SDS‐PAGE and immunoblotting analysis of TSWV‐M and TSWV‐T virion preparations with G_N‐ and G_C‐specific antibodies revealed similar amounts of the G_N and G_C glycoproteins in TSWV‐T virions, but lower amounts of G_N than G_C in TSWV‐M virions. This resulted in a statistically significant reduction in the G_N/G_C ratio in TSWV‐M virions. In affinoblots, the G_C and G_N glycoproteins of TSWV‐M exhibited weak binding with lectins showing affinity for N‐linked oligosaccharide structures. Sequence analysis of M RNA (medium segment of the TSMV genome) revealed no deletions or frameshift mutations in the G_N/G_C precursor of TSWV‐M. However, five amino acid changes were detected in the G_N/G_C precursor. A single, relatively conservative amino acid substitution (V→I) was observed in the NSm protein. Sequence analysis of S RNA (small portion of the TSMV genome) revealed a large intergenic region with no changes in the N protein and with three amino acid changes in the NSs protein.     

北京地区侵染茄子的番茄斑萎病毒分子鉴定与分析

调查发现北京地区一温室栽培茄子Solanum melongena L.出现严重病毒病。利用基于小RNA的高通量测序技术和RT-PCR方法,明确了引起茄子病害的病毒种类为番茄斑萎病毒,将其命名为TSWV-eggplant分离物。进一步克隆了该病毒的基因组全长(S RNA、M RNA、L RNA),并构建其系统发育树。结果表明,该分离物的S RNA与美国分离物亲缘关系较近,M RNA与中国分离物亲缘关系较近,而L RNA与韩国分离物亲缘关系较近。因此,本研究发现的TSWV分离物与国内已发生报道的分离物不同,该分离物是否存在不同分离物之间基因组的重组需要进一步研究。     

深度测序技术分析番茄斑萎病毒病害寄主及介体中病毒种类

 正番茄斑萎病毒属病毒(orthotospoviruses)是严重危害云南蔬菜等重要农业经济作物的病毒病原之一。采用血清学检测、小RNA深度测序以及RT-PCR验证相结合的方法,从云南省昆明市晋宁区的主要作物寄主(番茄、辣椒、油麦菜)、重要中间寄主(鬼针草)和传毒介体(蓟马)中鉴定到TSWV、TZSV、PCSV和INSV 4种病毒,其中TSWV为该地区的主要优势病毒,而PCSV则是首次报道侵染鬼针草。通过对云南番茄斑萎病毒病害重病区作物寄主、中间寄主及蓟马三者进行病毒种类分析研究,明确TSWV为引起云南省昆明市晋宁区作物的主要病毒,TZSV、PCSV和INSV零星发生于不同寄主中。     

番茄斑萎病毒核衣壳蛋白基因的克隆与分析

从云南表现褪绿黄化症状的番茄上分离到番茄斑萎病毒(Tomato spotted wilt virus)YN-1株系,利用RT-PCR方法克隆了该株系的核衣壳蛋白基因,并测定了该基因的序列,全长为777 bp,编码258个氨基酸。对该基因的氨基酸序列分析结果表明,YN-1 N基因与韩国3个株系的氨基酸序列相似性均高达97%。     

Effects of Temperature and Host on the Generation of Tomato Spotted Wilt Virus Defective Interfering RNAs

ABSTRACT The generation of defective interfering (DI) RNA molecules of tomato spotted wilt tospovirus (TSWV) was studied by serially passaging in-ocula from plant to plant under different controlled conditions. DI RNAs were generated at higher rates in plants at 16 degrees C than in plants incubated at higher temperatures. Another factor promoting the TSWV DI RNA generation was the use of high virus concentrations in the inocula. The solanaceous species Capsicum annuum, Datura stramonium, Lycopersicon esculentum, Nicotiana benthamiana, and N. rustica supported the generation of DI RNAs, whereas the virus recovered from the inoculated composite species, Emilia sonchifolia, remained free of any DI RNA under all conditions tested. This study resulted in a strategy to maintain DI RNA-free TSWV isolates, as well as in an efficient way to produce a large population of different DI RNA species. A single DI RNA species usually became dominant in an isolate after a few rounds of serial inoculations. The possible mechanisms involved in TSWV DI RNA generation under different inoculation circumstances are discussed.     

Ornamental plants and thrips populations associated with tomato spotted wilt virus in Greece

A survey was conducted in order to record the ornamental plants that are hosts of tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) in Greece. Polyclonal antibodies prepared against the N protein of a Greek isolate of TSWV fromGerbera jamesonii (GR-34) were used. Leaf samples were taken from plants showing typical symptoms of tospovirus infection such as chlorotic and necrotic rings on the leaves and malformation and necrosis of the flowers. The samples were tested by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antibodies to the N proteins of TSWV and INSV (NL-07). ELIS A-positive samples were mechanically transmitted to plants ofPetunia hybrida, Nicotiana rustica andN. benthamiana to confirm infection. Although none of the samples was found infected with INSV, TSWV presence was recorded in 42 botanical species that belong to 40 genera in 27 families. Among them the speciesBeloperone guttata, Coleus barbatus, Impatiens petersiana andLilium auratum are reported for the first time as hosts of TSWV, whereasBegonia sp.,Catharanthus roseus Celosia cristata, Dianthus chinensis, Fuchsia hybrida andStephanotis floribunda are found as new hosts of the virus in Greece. Thrips collected from TSWV-infected plants were in most cases identified asFrankliniella occidentalis, except from plants ofDendranthema sp. andDianthus caryophyllus whereThrips tabaci individuals were also identified. Different percentages of transmitters were noticed when the thrips populations collected from TSWV-infected ornamental hosts were tested for transmission of TSWV.     

Tomato Spotted Wilt Tospovirus Adapts to the TSWV N Gene-Derived Resistance by Genome Reassortment

ABSTRACT Pathogen- and host-derived resistance have been shown to suppress infection by many plant viruses. Tomato spotted wilt tospovirus (TSWV) is among these systems; however, it has easily overcome nearly all host resistance genes and has recently been shown to overcome resistance mediated by the TSWV N gene. To better understand the resistance-breaking mechanisms, we have chosen TSWV N gene-derived resistance (TNDR) as a model to study how plant viruses defeat resistance genes. A defined viral population of isolates TSWV-D and TSWV-10, both suppressed by TNDR, was subjected to TNDR selection by serial passage in an N-gene transgenic plant. The genotype analysis demonstrated that the mixed viral population was driven to form a specific reassortant, L(10)M(10)S(D), in the presence of TNDR selection, but remained as a heterogeneous mixture in the absence of the selection. A genotype assay of 120 local lesion isolates from the first, fourth, and seventh transfers confirmed the shift of genomic composition. Further analysis demonstrated that the individual L(10), M(10), and S(D) RNA segments were each selected independently in response to TNDR selection rather than to a mutation or recombination event. Following the seventh transfer on the N-gene transgenic plants, TSWV S RNA remained essentially identical to the S RNA from TSWV-D, indicating that no intermolecular recombination occurred between the two S RNAs from TSWV-10 and TSWV-D nor with the transferred N gene. These results support the hypothesis that TSWV utilizes genome reassortment to adapt to new host genotypes rapidly and that elements from two or more segments of the genome are involved in suppression of the resistance reaction.     

A novel putative member of the family Benyviridae is associated with soilborne wheat mosaic disease in Brazil

Soilborne wheat mosaic disease (SBWMD), originally attributed to infections by Soilborne wheat mosaic virus (SBWMV) and Wheat spindle streak mosaic virus (WSSMV), is one of the most frequent virus diseases and causes economic losses in wheat in southern Brazil. This study aimed to characterize molecularly the viral species associated with wheat plants showing mosaic symptoms in Brazil. Wheat leaves and stems displaying mosaic symptoms were collected from different wheat cultivars in Passo Fundo municipality, Rio Grande do Sul State, southern Brazil. Double-stranded RNA was extracted and submitted to cDNA library synthesis and next-generation sequencing. No sequences of SBWMV and WSSMV were detected but the complete genome sequence of a putative new member of the family Benyviridae was determined, for which the name wheat stripe mosaic virus (WhSMV) is proposed. WhSMV has a bipartite genome with RNA 1 and RNA 2 organization similar to that of viruses belonging to Benyviridae. WhSMV RNA 1 has a single open reading frame (ORF) encoding a polyprotein with putative viral replicase function. WhSMV RNA 2 has six ORFs encoding the coat protein, the major protein (read-through), triple gene block movement proteins (TGB 1, 2 and 3) and ORF 6 (hypothetical protein). In addition to the genomic organization and nucleotide and amino acid sequence identities, phylogenetic analyses also corroborated that WhSMV is a virus species of the Benyviridae. However, isolates of WhSMV formed a clade distinct from members of the genus Benyvirus. It was also demonstrated that the plasmodiophorid Polymyxa graminis is associated with wheat roots showing SBWMD symptoms and infected by WhSMV.     

Double-stranded RNA comprising the putative avocado virus 1 has a high degree of sequence homology to the avocado genome

The hypothesis that the double-stranded (ds) RNA isolated from avocado is indicative of infection by RNA plant viruses has never been proven. However, it has not been disproven and the different dsRNAs are still known as putative avocado viruses (AV) 1, 2 and 3. We have probed restriction fragments of avocado cellular DNA with cloned ³²P-labelled AV1-specific cDNA and discovered that the dsRNA nucleotide sequences are also present on plant DNA. Our evidence contradicts the hypothesis that viruses are responsible for the formation of the dsRNA species in avocado.