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.     

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。     

侵染广西甜椒的西瓜银斑驳病毒分子鉴定

During a survey in 2019, sweet pepper plants (GXTJ) showing symptoms of ring spots and chlorotic on leaves and fruits were collected in Wuming district of Guangxi province. Serological tests by DAS-ELISA demonstrated that the GXTJ was reacted positively against to antisera of watermelon silver mottle virus (WSMoV), but negatively against to antisera of the tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Total RNA was extracted and 2 pairs specific primers were designed to amplify the WSMoV N gene sequence by RT-PCR. Two expected fragments (769 bp and 654 bp) were obtained from GXTJ, The results of sequence analysis showed that the sequence of N gene was 828 nt and sharing 100% identity with WSMoV isolates from Taiwan (X78556, U78734). Base on the phylogenetic analysis, the N gene sequence of WSMoV-GXTJ was grouped in the same clades as WSMoV isolates from Taiwan. These results indicate that the virus isolate from sweet pepper in Guangxi is an isolate of WSMoV.     

广东西瓜上检测到甜瓜黄斑病毒

在广东发现了可能被番茄斑萎病毒属(Tospovirus)病毒侵染的西瓜,采用ELISA和RT-PCR法对该西瓜病样进行了检测,西瓜病叶粗汁液不与番茄斑萎病毒(Tomato spotted wilt virus,TSWV)和西瓜银斑驳病毒(Watermelon silver mottle virus,WSMoV)的血清发生反应;利用引物J13/UHP通过RT-PCR可以扩增出约1400 bp的基因片段,该片段包括一个840 bp的核衣壳蛋白ORF,其推导的氨基酸序列与已报道的Melon yellow spot virus(MYSV)NP基因氨基酸序列的同源率都为99%,进化树分析表明侵染广东西瓜的病毒(命名为MYSV-GZ)属于Tospovirus的MYSV血清组。     

番茄褐色皱果病毒SYBR Green Ⅰ实时荧光RT-PCR检测方法的建立和应用

根据番茄褐色皱果病毒(tomato brown rugose fruit virus,ToBRFV)外壳蛋白(coat protein,CP)的保守基因序列设计l对特异性引物,建立了基于SYBR Green Ⅰ的ToBRFV实时荧光RT-PCR检测方法,并对其进行了特异性、灵敏度检测,对自然感染ToBRFV的番茄、辣椒...     

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.     

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.     

Use of Degenerate Primers in a RT-PCR Assay for the Identification and Analysis of Some Filamentous Viruses, with Special Reference to Clostero- and Vitiviruses of the Grapevine

RT-PCR with degenerate primers was used for the screening of the genome of some members of the Closterovirus, Vitivirus and Trichovirus genera. Two sets of primers, targeted to conserved sequences of the heat shock protein 70 homologue of closteroviruses or to the RNA dependent RNA polymerase genes of tricho- and vitiviruses, amplified the expected fragments from total RNA extracts or double-stranded RNAs of infected plants. Amplified cDNAs were cloned, sequenced and phylogenetically analyzed. Results support the allocation of grapevine viruses A, B, D and heracleum latent virus (HLV) in the genus Vitivirus, whereas, the detection of a HSP70 homologue in grapevine leafroll-associated viruses agrees with their assignment in the genus Closterovirus. The use of degenerate primers for the identification of grapevine viruses belonging to Vitivirus and Closterovirus genera is envisaged.     

广东番茄上检测到Tospovirus病毒

 Some tomato samples possibly infected by tospovirus in Guangdong were detected with indirect ELISA and RT-PCR. The results showed that the virus infected tomato did not react with the antiserum of Tomato spotted wilt virus (TSWV), but about 500 bp fragment of RT-PCR shared 83%-84% nucleotide identities with N gene of those reported tospoviruses. The phylogenetic tree of the N gene fragment compared with those of other tospoviruses indicated that the virus infected tomato was belonged to Tospovirus.     

Comparison of ambisense m RNA of watermelon silver mottle virus with other tospoviruses

ABSTRACT Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5'-AGAGCAAU...-3' at their 5' ends and 5'-...AUUGCUCU-3' at their 3' ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid 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。     

番茄斑萎病毒4种检测方法比较

本研究根据番茄斑萎病毒(TSWV)S RNA上的核衣壳蛋白(N)基因保守序列设计特异性引物,比较了4种检测方法的灵敏度。结果表明,特异性引物可扩增出397bp的片段,序列和已发表的TSWV核苷酸序列同源性高达99%,可用于常规PCR和荧光定量RT-PCR(qRT-PCR)检测。qRT-PCR的灵敏度比快速检测试纸条、双抗体夹心酶联免疫吸附法(DAS-ELISA)和常规PCR分别高出15 625倍、3 125倍和125倍,且能够准确定量;常规PCR灵敏度较高,但不能准确定量;DAS-ELISA方法适用于批量定性测定,但检测时间较长;试纸条法检测速度最快,但灵敏度最低,使用时可根据症状程度和试验条件选择适宜的检测方法。     

Serological Comparison and Molecular Characterization for Verification of Calla lily chlorotic spot virus as a New Tospovirus Species Belonging to Watermelon silver mottle virus Serogroup

ABSTRACT Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A(405) readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5' and 3' ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3'-terminal sequence (5'-AUUGCUCU-3') that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.     

Identification of different isolates of Cassava brown streak virus and development of a diagnostic test

An RT-PCR based detection method for Cassava brown streak virus (CBSV)-infected cassava has been developed. The RT-PCR detection method described includes RNA extraction methods for cassava leaves, a distinct primer set for the virus and RT-PCR conditions. The primers were designed to the virus coat protein gene and generate a virus-specific product of 231 bp from infected cassava. The test can detect the virus in the new growth of cassava sticks before any disease symptoms are visible. This test was used successfully with infected cassava from both Tanzania and Mozambique. Three isolates from Tanzania were found to exhibit different symptoms on the secondary host plants Nicotiana benthamiana and N. tabacum SR1. They have nucleotide sequence variation within the coat protein region of up to 8% and amino acid differences of up to 6%.     

Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup

ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.     

黄瓜花叶病毒NASBA检测技术的建立

 以香蕉花叶病病样为材料,初步建立了黄瓜花叶病毒核酸序列依赖性扩增(Nucleic acid sequence based amplifica-tion,NASBA)的检测技术。通过以香蕉叶片总RNA为模板,在黄瓜花叶病毒(Cucumber mosaic virus,CMV)亚组ⅠRNA 2高保守区设计特异引物,进行NASBA反应,经5%琼脂糖凝胶电泳检测,阳性样品中出现了预期大小为310 bp的条带,而阴性和空白对照中均未出现。并对11份香蕉样品分别进行NASBA反应,并经过斑点杂交验证与RT-PCR检测比较,两者的检测结果一致,灵敏度相当,检出限量可达100 pg。