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     

Effect of temperature on RNA silencing of a negative‐stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative‐stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft‐inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV‐derived small RNAs (CPsV‐sRNAs) slightly increased at 32/26°C, with the ratio of CPsV‐sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.     

Molecular Characterization of <Emphasis Type="Italic">Bean yellow mosaic virus </Emphasis>from Vegetatively Propagated Dwarf <Emphasis Type="Italic">Gentiana </Emphasis>Plants

The causative virus (isolate No. 4) of gentian (Gentiana spp.) mosaic, which had been identified previously as Clover yellow vein virus (C1YVV) on the basis of host range and serological reactions, was re-identified as Bean yellow mosaic virus (BYMV) on the basis of the nucleotide sequences of the gene for the coat protein (CP) and the 3′-noncoding region, as well as the predicted amino acid sequence of CP. Received 16 April 2002/ Accepted in revised form 19 June 2002     

Complete Nucleotide Sequence of Ryegrass Mottle Virus : A New Species of the Genus Sobemovirus

The genome of Ryegrass mottle virus (RGMoV) comprises 4210 nucleotides. The genomic RNA contains four open reading frames (ORFs). The largest ORF 2 encodes a polyprotein of 947 amino acids (103.6 kDa), which codes for a serine protease and an RNA-dependent RNA polymerase. The viral coat protein is encoded on ORF 4 present at the 3′-proximal region. Other ORFs 1 and 3 encode the predicted 14.6 kDa and 19.8 kDa proteins of unknown function. The consensus signal for frameshifting, heptanucleotide UUUAAAC and a stem-loop structure just downstream is in front of the AUG codon of ORF 3. Analysis of the in vitro translation products of RGMoV RNA suggests that the 68 kDa protein may represent a fusion protein of ORF 2-ORF 3 produced by frameshifting. The protease region of the polyprotein and coat protein have a low similarity with that of the sobemoviruses (approximately 25% amino acid identity), while the RNA-dependent RNA polymerase region has particularly strong similarity (54 to 60% of more than 350 amino acid residues). The sequence similarities of RGMoV to the sobemoviruses, together with the characteristic genome organization indicate that RGMoV is a new species of the genus Sobemovirus. Received 28 June 2000/ Accepted in revised form 14 November 2000     

The Complete Nucleotide Sequence of a Japanese Isolate of White clover mosaic virus Strain RC

The complete nucleotide sequence was determined for genomic RNA of White clover mosaic virus (WClMV-RC) isolated from red clover (Trifolium pratense) in Japan, It is 5843 nucleotides in length, excluding the poly(A) tail at the 3' terminus. Similar to other potexviruses, it contains five open reading frames (ORFs 1 through 5), which putatively encode an RNA-dependent RNA polymerase (RdRp) (147 kDa), a triple gene block (TGB) (26 kDa/13 kDa/7 kDa), and a coat protein (CP) (22 kDa), respectively. The deduced amino acid sequence of the WClMV-RC CP was identical to that of WClMV-O, one of two New Zealand isolates, but only 85% identical to that of WClMV-M, the other New Zealand isolate, because of heterogeneity in the C-termini of CP amino acid sequences. The implication of this CP heterogeneity is discussed. Received 30 August 2001/ Accepted in revised form 11 January 2002     

Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2

Citrus psorosis virus (CPsV), the type species of genus Ophiovirus, is the presumed causal agent of a bark scaling disease in citrus plants. CPsV virions are kinked filaments composed of three negative‐strand RNA molecules and a ～48‐kDa coat protein. The virus induces two different syndromes: psorosis A (PsA), characterized by limited bark scaling lesions in the trunk and main limbs, and a more aggressive form of the disease called psorosis B (PsB) with rampant bark lesions affecting even thin branches and chlorotic blotches in old leaves. In the greenhouse, the PsA and PsB syndromes can be induced by graft inoculating healthy citrus seedlings with non‐lesion or with lesion bark inoculum from PsA‐affected field trees. PsA‐ and PsB‐inducing CPsV sub‐isolates obtained by this procedure from the same tree showed identical single‐strand conformation polymorphism (SSCP) profiles in homologous segments of the RNAs 1 and 3, whereas segments of the RNA 2 enabled discrimination between PsA‐ and PsB‐associated sequence variants. SSCP analysis of the RNA 2 population present in different tissues of psorosis‐infected plants showed that: (i) PsA‐inducing isolates contain PsB‐associated sequence variants at low frequency, (ii) the PsB‐associated sequence variant is predominant in blistered twigs and gummy pustules affecting old leaves, characteristic of PsB isolates, and (iii) the PsB‐associated sequence variant accumulates preferentially in bark lesions of the trunk and limbs. SSCP analysis of the RNA 2 population also enabled monitoring of interference between PsA‐ and PsB‐associated variants in plants co‐inoculated with both psorosis types.     

<Emphasis Type="Italic">Amaranthus leaf mottle virus</Emphasis>: 3′-end RNA sequence proves classification as distinct virus and reveals affinities within the genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Amaranthus leaf mottle virus (AmLMV) was classified as a member of the genus Potyvirus on the basis of its particle morphology, serology, and biological properties (Casetta et al., 1986). Based on these properties, an Amaranthus viridis-infecting virus isolated in Spain, causing mottle and leaf blistering as well as reduced growth has been identified as AmLMV. The 3′ terminal genomic region of this and a reference isolate from Italy has been sequenced and reveals a 95% nucleotide identity between the two isolates. The sequenced part comprises the coat protein with 281 amino acids and 315 nucleotides of the 3′ untranslated region (UTR) preceding a polyadenylated tail. Pairwise comparisons and phylogenetic analysis of the nucleotide and deduced amino acid sequences of the CP and 3′ UTR of the cloned cDNAs with those of other potyviruses shows that AmLMV is a distinct potyvirus closely related to Potato virus Y.     

Occurrence of Citrus psorosis virus in Campania, southern Italy

Citrus psorosis virus (CPsV), genus Ophiovirus, is associated with a severe disease of citrus worldwide. Double antibody sandwich (DAS) ELISA using a polyclonal antiserum, and triple antibody sandwich (TAS) ELISAs, employing the IgG monoclonal antibody (mab) 13C5, and the IgM mab 2A3, were used to detect CPsV in orchards of different citrus varieties in Campania, southern Italy. TAS ELISA with 13C5 detected all the infections detected by DAS ELISA. Overall, 14% of trees younger than 15 years were positive, but only 1% of older trees, suggesting that infected propagating material has been increasingly used in recent years, in the absence of certification. Highest infection rates were in younger trees of sweet orange (22.8%) and clementine (18.6%). CPsV could easily be detected at all seasons of the year tested (June–January); these and earlier results indicate that TAS ELISA using 13C5 is a sensitive, broad-spectrum and reliable diagnostic method useful for routine tests and certification programmes. Of 44 field isolates responding strongly to DAS ELISA and 13C5-TAS ELISA, mab 2A3 gave similar results with 29 isolates, but gave low values with the others, thus providing a degree of differentiation among isolates. To confirm that the ELISA tests were indeed detecting CPsV, samples of 42 ELISA-positive plants were analysed by ISEM in a blind test, and in 38 of these, characteristic virus particles were clearly seen. Although CPsV was frequently and consistently detected in the area sampled, bark scaling symptoms were not seen: possible reasons for this are discussed.     

Identification of divergent variants of Grapevine virus A

Eight isolates of Grapevine virus A (GVA), which induced different symptoms in leaves of Nicotiana benthamiana, were recovered from various grapevines. The dsRNA patterns of two isolates, which consistently induced mild vein clearing (referred here as mild isolates of GVA) were similar, but different from those of other isolates of GVA. Analysis based on overall nucleotide (nt) sequence identity in the 3 terminal part of the GVA genome, comprising part of ORF3 (putative movement protein, MP), entire ORF4 (capsid protein, CP), entire ORF5 and part of 3 UTR, revealed that GVA isolates separate into three groups (I, II, III), sharing 91.0–99.8% nt sequence identity within groups and 78.0–89.3% nt sequence identity between groups. Mild isolates of the virus were group III and shared only 78.0–79.6% nt sequence identity with the other isolates. The comparison of predicted amino acid sequences for MP and CP revealed many amino acid alterations, revealing distinct local net charges of these proteins for mild isolates of the virus. Based on both conserved and divergent nt regions in the CP and ORF5, oligonucleotide primers were designed for the simultaneous RT-PCR detection of all GVA isolates and for the specific detection of the most divergent virus variants represented here by mild isolates of the virus.     

Molecular Identification of Oat Mosaic Virus as a Bymovirus

A partial sequence of Oat mosaic virus (OMV) has been obtained for four isolates of the virus from four European countries. This represents the first available sequence data for this important disease of winter-sown oats. The longest clone of 1699 nucleotides was obtained from infected English oats using a degenerate primer, designed to members of the Potyviridae family. Alignment of the predicted amino acid sequence with members of the Potyviridae showed closest identity with viruses of the Bymovirus genus. The predicted amino acid sequence has one open reading frame corresponding to part of the NIb and capsid protein, with a 3 untranslated region of 351 nucleotides, followed by a poly(A) tail. PCR primers were designed to the coat protein and NIb gene of members of the Bymovirus genus and used to obtain partial sequences of 1441 nucleotides at the 3 end of infected oats from both Wales and France. A specific primer set designed to the English isolate was used to generate a product of 701 nucleotides from OMV-infected oat leaves from Ireland. All four isolates are highly conserved at the amino acid level.The first two authors contributed equally to the work     

Complete nucleotide sequence and latency of a novel blueberry-infecting closterovirus

A novel latent closterovirus was detected from highbush blueberry in Japan and provisionally named blueberry virus A (BVA). The BVA genome (17,798 nucleotides) contains 10 open reading frames, but no minor coat protein could be identified in the virus genome. The BVA RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h), and major coat protein (CP) shared the highest amino acid sequence identities with those of viruses in the genus Closterovirus (61.2, 27.6, and 20.9 %, respectively). In a phylogenetic analysis of the RdRp, HSP70h, and CP, BVA did not cluster with any genus in the family Closteroviridae.     

New potyvirus isolated from <Emphasis Type="Italic">Cryptotaenia japonica</Emphasis>

 A potyvirus, for which the name Japanese hornwort mosaic virus (JHMV) is proposed, was isolated from Japanese hornwort plants (Cryptotaenia japonica) with mosaic disease symptoms. The virus was used to inoculate mechanically 34 plants belonging to 33 species of 10 families. Of these species seven from two families were infected. Faint chlorotic spots appeared on the inoculated leaves of Chenopodium quinoa and C. amaranticolor, but no systemic infection occurred in these plants. JHMV systemically infected only Umbelliferae plants; they did not infect 26 other species in eight families. JHMV was transmitted in a nonpersistent manner by aphids (Myzus persicae). The virus was a flexuous rod-shaped particle about 750 nm in length. Sequencing the nucleotides in the 3′ terminal region of JHMV revealed that the coat protein contains 280 amino acids with a molecular mass of 32.2 kDa. The nucleotide sequence of the coat protein of JHMV had the highest similarity with that of Zantedeschia mosaic virus (83.3%) compared to those of other potyviruses (57.0%–64.9%). An antiserum against JHMV reacted strongly with JHMV and weakly with Potato virus Y. These results indicate that JHMV is a new potyvirus. Received: September 9, 2002 / Accepted: November 7, 2002 RID="*" ID="*" The nucleotide sequence determined in this work appears in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB081518     

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

A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.     

Sequence analysis within the RNA 3 of seven Spanish tomato isolates of Parietaria mottle virus (PMoV-T) reveals important structural differences with the parietaria isolates (PMoV)

The genomic regions encoding the putative movement protein (MP), coat protein (CP) and intergenic region (IGR) of seven Spanish isolates of the Parietaria mottle virus which infects tomato plants (PMoV-T) were sequenced. Values for the genetic diversity of the PMoV-T isolates were 0.056, 0.047 and 0.013 for the CP, MP genes and IGR, respectively. Nucleotide and amino acid sequence comparison of the seven PMoV-T isolates with those of PMoV revealed significant differences. All of them had a cytosine deletion at position 1366, also confirmed in an Italian tomato isolate, which involves a start codon for the CP gene different from that for the PMoV sequence, resulting in a CP 16 amino acids shorter than the PMoV CP. The certainty of a cytosine deletion only associated to the tomato isolates or the possibility of a mistake in the PMoV published sequence are the two hypotheses that could explain this difference. Structural motifs highly conserved in Ilarviruses were identified in PMoV-T MP and CP. A stable hairpin structure is proposed for IGR, by the initiation site for subgenomic RNA 4 synthesis. Phylogenetic analysis of CP and MP amino acid sequences showed that Spanish PMoV-T isolates form a separate group from PMoV and other members of the Ilarvirus genus. Comparative analysis with different PMoV isolates including tomato isolates from other regions and isolates from different hosts are necessary to confirm this differentiation.     

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     

甘蔗黄叶病毒复制酶基因的克隆及序列分析

 The gene of RNA-dependent RNA polymerase (RdRP) was cloned by RT-PCR from Sugarcane yellow leaf virus-Fuzhou isolate (CHN-FJ1) and then cloned into pMD18-T vector. The sequence showed that the fragment comprised 1 212 nucleotides including part gene of ORF1 and ORF2. The ORF2 was involved the RdRP gene consisted of 995 nucleotides and encoded putative protein of 331 amino acids. Compared the nucleo-tide sequence and encoded putative protein of CHN-FJ1 with the other isolates from different countries, they shared the homology above 92.0%. Phylogenetic tree suggested that the sixteen isolates were classified into four types according to the amino acid sequence of the RdRP. One of the groups contained CHN-FJ1 and the other isolates from China, American, Brazil, Australia and Colombia;however, there was the closest relation between CHN-FJ1 and BRA-YL1 isolate from Brazil.     

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。     

D-Alanine-D-Alanine Ligase Gene (ddl) of Erwinia chrysanthemi Strain EC16 I. Isolation and Gene Dosage Effect on Pectate Lyase Synthesis

New regulatory gene for pectate lyase (Pel) production of Erwinia chrysanthemi strain EC16 was searched by observing the gene dosage effect of each cosmid library in Pel production. From this survey, a cosmid clone, p5A, had reduced in Pel production under both inducing and non-inducing conditions and caused less tissue maceration of potato tubers. The 2.64-kb HindIII fragment from p5A was found to be responsible for this phenotype and to contain one major ORF consisting of 1107 nucleotides, which had homology with ddlA (49.6%) and ddlB (49.6%) of Escherichia coli and with ddlA (44.6%) of Salmonella typhimurium. These genes had been shown to encode D-alanine-D-alanine ligase (Ddl), an enzyme which is involved in the synthesis of peptidoglycan. This ORF encodes 368 amino acid residues and its molecular weight is estimated to be 43 kDa. This ORF of EC16 could complement ddl deficient mutant of Escherichia coli. Received 15 May 2002/ Accepted in revised form 20 June 2002     

棉铃虫类肌钙蛋白基因的克隆、原核表达纯化及其时空表达谱分析

为探索棉铃虫Helicoverpa armigera(Hübner)类肌钙蛋白基因HaCal的生理功能及其所编码蛋白的生物特性,采用PCR方法克隆了HaCal的开放阅读框(open reading frame,ORF)序列,借助在线软件对其进行生物信息学分析,通过原核表达技术诱导表达和纯化其编码的蛋白,并通过实时荧光定量PCR技术分析其在棉铃虫不同发育龄期和组织中的表达量。结果显示,HaCal的ORF序列长度为564 bp,编码187个氨基酸,理论分子量为20.52 kD,理论等电点为7.64。其保守结构域与Calponin家族一致,但不含跨膜区和信号肽,系亲水性蛋白。原核表达结果显示,融合蛋白大小与理论值一致,纯化获得了纯度较高的目的蛋白。HaCal在棉铃虫幼虫不同发育阶段和不同组织中均有表达,在6龄幼虫体内表达量最高,是1龄幼虫的2.34倍;在5龄幼虫中肠中表达量最高,是头部的257.14倍。表明棉铃虫类肌钙蛋白基因HaCal可能与棉铃虫的生长发育过程密切相关。