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.     

Identification and characterization of Lisianthus necrotic ringspot virus,a novel distinct tospovirus species causing necrotic disease of lisianthus (Eustoma grandiflorum)

In 2007, lisianthus (Eustoma grandiflorum) plants with necrotic ringspots on the leaves were found in Kochi Prefecture, Japan. Tospovirus-like spherical enveloped particles with ca. 160 nm in diameter were observed with electron microscopy. The complete nucleotide sequence of the S RNA segment of the virus was determined, and phylogenetic analysis using deduced amino acid sequences of the nucleocapsid protein and the nonstructural S protein indicated that the virus is phylogenetically distinct from any known tospovirus species. The results suggest that the virus is a new member of the genus Tospovirus, in the family Bunyaviridae. The virus is the fifth distinct tospovirus occurring naturally in lisianthus in Japan. The necrotic symptoms were reproduced on lisianthus seedlings after mechanical inoculation. The host range of the virus isolate on several test plants was also examined.     

A new tomato-infecting tospovirus from iran

ABSTRACT A new tospovirus species serologically distinct from all other established tospoviruses was found in tomato in Iran. Typical disease symptoms observed include necrotic lesions on the leaves and yellow ring spots on the fruits, hence the name Tomato yellow ring virus (TYRV) was proposed. The S RNA of this virus was cloned and its 3,061 nucleotide long sequence showed features characteristic for tospoviral S RNA segments. The nucleocapsid (N) protein with a predicted Mr of 30.0 kDa showed closest relationship to the N protein of Iris yellow spot virus (74% sequence identity).     

Increase of tospoviral diversity in Brazil with the identification of two new tospovirus species, one from chrysanthemum and one from zucchini

ABSTRACT During a survey conducted in several different regions of Brazil, two unique tospoviruses were isolated and characterized, one from chrysanthemum and the other from zucchini. The chrysanthemum virus displayed a broad host range, whereas the virus from zucchini was restricted mainly to the family Cucurbitaceae. Double-antibody sandwich-enzyme-linked immunosorbent assay and western immunoblot analyses demonstrated that both viruses were serologically distinct from all reported tospovirus species including the recently proposed peanut yellow spot virus and iris yellow spot virus (IYSV) species. The nucleotide sequences of the nucleocapsid (N) genes of both viruses contain 780 nucleotides encoding for deduced proteins of 260 amino acids. The N proteins of these two viruses displayed amino acid sequence similarities with the previously described tospovirus species ranging from 20 to 75%, but they were more closely related to each other (80%). Based on the biological and molecular features, these viruses are proposed as two new tospovirus species, designated chrysanthemum stem necrosis virus (CSNV) and zucchini lethal chlorosis virus (ZLCV). With the identification of CSNV and ZLCV, in addition to tomato spotted wilt virus, groundnut ring spot virus, tomato chlorotic spot virus, and IYSV, Brazil harbors the broadest spectrum of tospovirus species reported.     

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     

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。     

Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

侵染云南烟草的番茄环纹斑点病毒N基因的分子变异分析

 应用电镜观察和ELISA检测从云南11个县（市）烟草种植区采集的烟草上检测到番茄环纹斑点病毒（Tomato zonate spot virus, TZSV）。根据TZSV N基因设计引物扩增得到了31个TZSV分离物N基因全序列。测序分析表明,31个TZSV N基因核苷酸序列与GenBank中报道的序列相似性在94.9%~98%之间,其推导的氨基酸序列同源性为98.2%~99.3%。构建系统进化树发现云南不同地区的TZSV分离物存在一定差异。氨基酸序列比较发现,文山分离物和西畴分离物具有2个该地区独有的氨基酸位点。     

Molecular and biological characterization of <Emphasis Type="Italic">Chrysanthemum stem necrosis virus</Emphasis> isolates from distinct regions in Japan

Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.     

Generation of polyclonal antibodies and serological analyses of nucleocapsid protein of Soybean vein necrosis-associated virus: A distinct soybean infecting tospovirus serotype

Soybean vein necrosis-associated virus (SVNaV) is a newly isolated tospovirus from field-grown soybeans in the United States. Polyclonal antibodies generated against the recombinant Escherichia coli-expressed nucleocapsid protein (NP) of the virus, reacted specifically with SVNaV and exhibited low, if any, cross-reactivity with other species within the genus Tospovirus. The serological results are in agreement with low sequence homology of the SVNaV-NP gene when compared with those of other tospovirus species, some of which are capable of infecting soybean naturally. Phylogenetic analysis utilizing NP sequences from several SVNaV isolates collected from different geographical regions and various soybean genotypes over 4?years showed close relationships, but distinct from the representatives of all the established serogroups or distinct serotypes within the genus of Tospovirus. All SVNaV isolates examined reacted strongly with the generated polyclonal antibodies. Collectively, our serological analyses suggest that SVNaV represents a new and distinct soybean-infecting tospovirus serotype. Furthermore, our data demonstrate that antiserum against NP has the potential to serve as a reliable probe for SVNaV detection in field-grown soybeans.     

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.     

Molecular Variability of the Capsid Protein of the Prune Dwarf Virus

Sequences of the capsid protein gene and the preceding intergenic region of eleven isolates of prune dwarf virus from central Europe were determined. The isolates were obtained from plum, cherry and peach trees. Comparison of all sequenced isolates (including two sequences published previously) revealed high (88%) conservation of the capsid protein gene. The highest degree of identity was observed in the C-terminal half, where only 13 amino acid substitutions could be observed in contrast to the N-terminal half with 22 substitutions. No reasonable correlation between amino acid substitutions and host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other ilarviruses revealed apple mosaic virus, elm mottle virus, lilac ring mottle virus and prunus necrotic ringspot virus as the most related to prune dwarf virus. Unlike the isolates of related prunus necrotic ringspot virus all the isolates of prune dwarf virus shared extensive conservation of the intergenic region. Portions of RNA3 were selected for design of universal primers for PCR detection.     

小麦黄色花叶病毒不同分离物外壳蛋白(CP)基因序列的比较分析

 核苷酸序列分析结果表明,小麦黄色花叶病毒(W YMV)不同分离物的外壳蛋白基因存在一定的差异。邓州分离物CP基因在其31~33nt处均缺失了3个核苷酸,其余分离物与潢川分离物及日本分离物长度一致,均为882nt。不同分离物CP基因核苷酸序列同源性为97.3%~98.9%,由此推导的氨基酸序列同源性为97.6%~99.3%,外壳蛋白N末端的110个氨基酸和C末端的55个氨基酸在各个分离物间是高度保守的。潢川分离物有5个氨基酸与其它5个分离物明显不同。WYMV不同分离物外壳蛋白序列分析结果进一步确认了WYMV与WSSMV为Bymovirus属的2种不同病毒。     

Molecular characterization of the CP gene and 3'UTR of Chilli veinal mottle virus from South and Southeast Asia

Twenty-four isolates of Chilli veinal mottle virus (ChiVMV) from China, India, Indonesia, Taiwan and Thailand were analysed to determine their genetic relatedness. Pathogenicity of virus isolates was confirmed by induction of systemic mosaic and/or necrotic ringspot symptoms on Capsicum annuum after mechanical inoculation. The 3' terminal sequences of the viral genomic RNA were determined. The coat protein (CP) coding regions ranged from 858 to 864 nucleotides and the 3' untranslated regions (3'UTR) from 275 to 289 nucleotides in length. All isolates had the inverted repeat sequence GUGGNNNCCAC in the 3'UTR. The DAG motif, conserved in aphid-transmitted potyviruses, was observed in all isolates. All 24 isolates were considered as belonging to ChiVMV because of their high CP amino acid and nucleotide identity (more than 94·8 and 89·5%, respectively) with the reported ChiVMV isolates including the pepper vein banding virus (PVBV), the chilli vein-banding mottle virus (CVbMV) and the CVbMV Chiengmai isolate (CVbMV-CM1). Based on phylogenetic analysis, ChiVMV isolates including all 24 isolates tested, PVBV, CVbMV and CVbMV-CM1 can be classified into three groups. In addition, a conserved region of 204 amino acids with more than 90·2% identity was identified in the C terminal of the CP gene of ChiVMV and Pepper veinal mottle virus (PVMV), and may explain the serological cross reaction between these two viruses. The conserved region may also provide useful information for developing transgenic resistance to both ChiVMV and PVMV.     

First report of <Emphasis Type="Italic">Impatiens necrotic spot virus</Emphasis> infecting chrysanthemum (<Emphasis Type="Italic">Chrysanthemum morifolium</Emphasis>) in Japan

In 2009, chlorotic mottle and necrosis were observed on chrysanthemums (cv. Jimba) in Aomori Prefecture, Japan. A virus was isolated from the chrysanthemum plants by serial local-lesion transfer. The symptoms exhibited by the test plants, the particle morphology, the features of the protein and the potential for transmission by thrips were similar to those for Impatiens necrotic spot virus (INSV). The partial nucleotide sequences of the nucleocapsid protein gene and the 3′-untranslated sequence of the S RNA shared 99% identity with that of an INSV isolate. This report is the first of INSV infection of chrysanthemums in Japan.     

Nucleotide Sequence of the 3′ -terminal Region of Carnation vein mottle virus RNA

The 2326 nucleotides of the 3′-terminal region of Carnation vein mottle virus (CVMV) RNA, which included part of the nuclear inclusion b gene, the complete coat protein (CP) gene and the entire 3′-noncoding region (3′-NCR) were determined. The region encoding the CP gene is 843 nucleotides long and the deduced protein consists of 280 amino acids. A search of the EMBL and PIR databases showed that the amino acid sequence of CVMV CP most resembled that of Plum pox virus with a similarity of 67.9%. The 3′-NCR of CVMV RNA is 541 nucleotides long, second longest in the genus Potyvirus. These results indicate that CVMV is closely related to Plum pox virus but is a distinct species in the genus Potyvirus. Received 8 October 1999/ Accepted in revised form 9 January 2000     

Melon yellow spot virus: A Distinct Species of the Genus Tospovirus Isolated from Melon

ABSTRACT A tospovirus-like virus recovered from netted melon was transmitted by Thrips palmi in a persistent manner but had different cytopathological features from tospoviruses previously reported. Viral nucleocapsid (N) was purified with two protective reagents, 2-mercaptoethanol and L-ascorbic acid, and RNA extracted from the viral nucleocapsid was used for genomic analysis. The virus had a genome consisting of three single-stranded RNA molecules. The open reading frame on the viral complementary strand, located at the 3' end of the viral S RNA, encoded the N protein. The 3' terminus of this RNA also contained an eight-nucleotide sequence similar to the conserved sequence at the 3' end of genomic RNA molecules of tospoviruses. These features of the viral genome are identical to those of tospoviruses; therefore, this virus is considered to belong to the genus Tospovirus. Its N protein comprised 279 amino acids and had a molecular mass of 31.0 kDa. Comparisons of its amino acid sequence with those of known tospoviruses revealed less than 60% identity. This melon virus is concluded to be a distinct species in the genus Tospovirus, and the name Melon yellow spot virus is proposed.