Complete Coat Protein Sequences Research Articles

Molecular Analysis of the Global Population of Potato Virus S Redefines Its Phylogeny, and Has Crop Biosecurity Implications.

In 2020, 264 samples were collected from potato fields in the Turkish provinces of Bolu, Afyon, Kayseri and Niğde. RT-PCR tests, with primers which amplified its coat protein (CP), detected potato virus S (PVS) in 35 samples. Complete CP sequences were obtained from 14 samples. Phylogenetic analysis using non-recombinant sequences of (i) the 14 CP's, another 8 from Tokat province and 73 others from GenBank; and (ii) 130 complete ORF, RdRp and TGB sequences from GenBank, found that they fitted within phylogroups, PVSI, PVSII or PVSIII. All Turkish CP sequences were in PVSI, clustering within five subclades. Subclades 1 and 4 were in three to four provinces, whereas 2, 3 and 5 were in one province each. All four genome regions were under strong negative selection constraints (ω = 0.0603-0.1825). Considerable genetic variation existed amongst PVSI and PVSII isolates. Three neutrality test methods showed PVSIII remained balanced whilst PVSI and PVSII underwent population expansion. The high fixation index values assigned to all PVSI, PVSII and PVSIII comparisons supported subdivision into three phylogroups. As it spreads more readily by aphid and contact transmission, and may elicit more severe symptoms in potato, PVSII spread constitutes a biosecurity threat for countries still free from it.

First Insight into Genetic Variation and Population Structure of The Emerging Citrus chlorotic dwarf-associated virus (CCDaV, genus Citlodavirus)

Citrus spp. is widely planted in tropical and subtropical regions, including in Turkey and other Mediterranean countries. Due to its widespread vector and climate change, Citrus chlorotic dwarf-associated virus (CCDaV), a member of the newly formed genus Citlodavirus, is one of the emerging viruses that can be a serious constraint to Citrus crops production in the coming years. Therefore, in-silico analysis on all available isolates in NCBI GenBank was performed to provide the first insight into the genetic population and evolution of CCDaV, which may contribute to its control. CCDaV phylogroups based on full genome, complete movement protein, and complete coat protein sequences were found to be not associated with isolate origins or host species, and all isolates also shared a high genetic identity among them. However, neutrality tests indicated that the current populations are expanding, driven by new mutations. Low Fixation index (FST) values (0.00000-0.36207) confirmed no genetic separation among different ORFs of isolates from three countries. The constructed TimeTree suggested that CCDaV emergence was very recent compared to the other three members of the genus Citlodavirus. Therefore, the obtained results of this study could also expand our knowledge on other even more obscure citladovirus and even other plant DNA viruses, which are still less studied than RNA viruses.

First Report of Cucurbit Chlorotic Yellows Virus Infecting Cucumber and Zucchini in Algeria

This work was supported by the Spanish Ministerio de Ciencia, Innovacion y Universidades, cofunded with FEDER funds (project nos. AGL2017-85563-C2-1-R and RTA2017-00061-0O3-03 [INIA]) and the programa para grupos de investigacion de excelencia from the Conselleria d'Educacio, Investigacio, Cultura i Esport, (Generalitat Valenciana) (Prometeo Program 2017/078). A. Kheireddine thanks the Erasmus+ Programme of the European Union for her mobility project (KA107 2018-20). C. Saez is a recipient of a predoctoral fellowship from Generalitat Valenciana, cofunded by the Operational Program of the European Social Fund (FSECV 2014-2020) (grant no. ACIF/2016/188). Plant Dis. 104: 1264, 2020; published online as https://doi.org/10.1094/PDIS-10-19-2091PDN.Accepted for publication 19 December 2019.

Development of RT-PCR assays for the detection and the resultant phylogenetic analysis of four grapevine vitiviruses based on the coat protein sequences

Four sets of primers were designed based on the alignment of the complete coat protein (CP) gene sequences of several isolates of four different vitiviruses, i.e. grapevine virus B (GVB), GVD, GVE and GVF, and tested for their efficiency in RT-PCR assays to detect vitiviruses infections in grapevine. The resultant RT-PCR amplicons were sequenced and analyzed for their genetic variability and phylogenetic studies. The results of the RT-PCR assays showed that these primers were highly efficient in detecting different vitivirus isolates in grapevine material originating from ten different Mediterranean countries. In particular, 76 out of 218 tested samples (ca. 35%) were infected with at least one vitivirus. GVE was the most detected (14.7%), followed by GVF (11.5%), GVB (6.9%), and GVD (2.8%). Nucleotide (nt) sequence analysis of the CP genes from this study and Genbank showed that the sequence identity matrixes among isolates of GVB and GVE were the most variable, with nt identity ranging from 77% to 100%, whereas isolates of GVD and GVF showed more conserved nt identities ranging between 82% to 100% and 86.4% to 99.8%, respectively. The phylogenetic trees constructed based on the CP sequences distinguished two main groups of isolates for each vitivirus species, except for the GVD isolates, which did not show any particular subdivision. In general, the distributions of the isolates in the phylogenetic tree were associated with their geographical origin, thus suggesting limited movement of grapevine materials between the different countries. This study reported for the first time: (i) the development of primers based on the complete CP gene sequences for RT-PCR assays for the universal detection of vitivirus species, (ii) the high genetic variability among Mediterranean isolates of vitiviruses and (iii) the presence of GVD in Jordanian vines, of GVE in grapevines from Hungary, Italy, Jordan, Malta and Palestine, and of GVF in grapevines from Afghanistan, Bulgaria, China, France, Hungary, Italy, Jordan, Lebanon and Malta.

Prevalence and genetic diversity of Grapevine virus A in Tunisia.

Prevalence and genetic diversity of the complete CP gene of Grapevine virus A (GVA) were assessed in isolates from rootstocks, wine and table grape varieties and autochthonous grapevines. Wine grapes were the most infected (63%), followed by table grapes (49%) and rootstocks (44%). Autochthonous grapevines were the less infected (35%). Analyses of the complete coat protein sequences of 20 GVA isolates from the main grapevine growing areas of Tunisia identified three phylogroups, accounting, respectively, for 70% (group I), 25% (group IV) and 5% (group III) of the isolates. No sequences clustered into group II. Phylogenetic analyses indicated that Tunisian GVA isolates are not grouped by the host cultivar or geographic origin.

Biological and molecular characterization of a basal-Brassica/Raphanus Turnip mosaic virus isolate from Eruca sativa

Eruca sativa (rocket salad or arugula) and Raphanus raphanistrum (raphanus) plants with mosaic symptoms were found in the field during 2016 in Sao Paulo State, Brazil. Initially, the plants were submitted to indirect ELISA using a potyvirus antiserum, and then total RNA extraction and RT-PCR were performed using the ELISA-positive samples. The complete coat protein sequence was obtained and the virus was identified as Turnip mosaic virus (TuMV). Biological and Bayesian analysis grouped the TuMV rocket isolate in the Brassica-Raphanus (BR) clade that includes isolates infecting Brassica and Raphanus species. This clade has two sub-clusters, the basal-Brassica/Raphanus (basal-BR) and the Asian-Brassica/Raphanus (Asian-BR), and the rocket isolate was placed in the basal-BR cluster. TuMV from rocket was aphid-transmitted to raphanus and rocket, and sap-transmitted to Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum, N. glutinosa, Datura stramonium, Beta vulgaris subsp. vulgaris, Brassica rapa and to seven rocket cultivars, which were heavily affected by the virus. Cabbage and cauliflower were not infected by the virus. According to the phylogenetic analysis, at least two different introductions of TuMV isolates occurred in Brazil, corresponding to the basal-BR and world-B types, infecting Brassica/Raphanus and Brassica, respectively.

First Report of Youcai mosaic virus in Raphanus raphanistrum subsp. sativus in Korea

HomePlant DiseaseVol. 101, No. 11First Report of Youcai mosaic virus in Raphanus raphanistrum subsp. sativus in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Youcai mosaic virus in Raphanus raphanistrum subsp. sativus in KoreaS.-K. Choi, S.-H. Park, B.-W. Jeon, S.-W. Jang, and J.-Y. YoonS.-K. Choi†Corresponding author: S.-K. Choi, E-mail: E-mail Address: viroid73@gmail.comSearch for more papers by this author, S.-H. ParkSearch for more papers by this author, B.-W. JeonSearch for more papers by this author, S.-W. JangSearch for more papers by this author, and J.-Y. YoonSearch for more papers by this authorAffiliationsAuthors and Affiliations S.-K. Choi † S.-H. Park B.-W. Jeon S.-W. Jang , Department of Vegetable Research, National Institute of Horticultural and Herbal Science, RDA, Wanju, 55365, South Korea J.-Y. Yoon , Virology Unit, Department of Horticultural Environment, National Institute of Horticultural and Herbal Science, RDA, Wanju, 55365, South Korea. Published Online:7 Sep 2017https://doi.org/10.1094/PDIS-05-17-0730-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Radish (Raphanus raphanistrum subsp. sativus), a species of the family Brassicaceae, is widely consumed as Kimchi and various other foods in South Korea. During October 2016, virus-like symptoms, including stunting and mild mosaic of leaves, were observed on a radish plant (cv. Seoho) on a farm located in Wanju, South Korea. The symptomatic leaf sample was also analyzed by DAS-ELISA using four different polyclonal antibodies specific to Cucumber mosaic virus, Turnip mosaic virus, Turnip yellow mosaic virus, and Turnip yellows virus (Loewe Biochemica, Germany). None of the four viruses was detected in the radish leaf sample. The symptomatic leaf sample was also analyzed by transmission electron microscopy (TEM) in leaf dip preparations. Typical tobamovirus-like particles of rigid rod shape and about 300 × 18 nm length/width were observed in the leaf sample of radish plant. To further confirm the obtained TEM result, total RNA was extracted from the symptomatic radish plant and analyzed by reverse transcription (RT)-PCR using degenerate primers specific to genus Tobamovirus (Agdia, U.S.A.). An approximate 400-bp RT-PCR product was amplified from the radish sample and the amplified RT-PCR product was purified and cloned into pCR4-TOPO vector (ThermoFisher Scientific, U.S.A.). Selected cDNA clones were sequenced, and the BLASTN search showed that determined sequences of the amplified RT-PCR product had the highest identity with isolate Br of Youcai mosaic virus (accession no. JN634066). To confirm that YoMV (named YoMV-Rad) was the cause of the disease observed in radish, pathogenicity test was carried out using two Nicotiana species containing the N-gene, resulting in necrotic local lesions 4 days postinoculation (dpi). To fulfill Koch’s postulates, virus-free radish plants inoculated mechanically by sap from local lesions on N. glutinosa, showing identical symptoms observed on the symptomatic radish 30 dpi and YoMV-Rad was reisolated from the inoculated radish plants. Mock-inoculated radish plants remained symptomless and virus-free, suggesting that YoMV is the causal virus of the symptomatic radish plant. To determine the complete coat protein (CP) gene sequence of YoMV-Rad, primers YoMV-CP-For (5′-ATGTCTTACAACATCACGAGCTCGAAT-3′) and YoMV-CP-Rev (5′-CTATGTAGCTGGCGCAGTAGTCCAAGG-3′) were synthesized according to the sequence of YoMV-Br. A 474-bp RT-PCR product amplifying the CP gene was amplified from the symptomatic radish. Sequencing result of selected cDNA clones for YoMV-Rad showed the determined sequences of the amplified RT-PCR products matched with YoMV CP gene. A multiple alignment of the obtained CP sequence of YoMV-Rad (accession no. LC270132) with other YoMV isolates using MEGA6.0 showed 92.0 to 98.0% identity to YoMV strains described previously. All these results demonstrate that YoMV is the cause of the disease in radish. During fall and winter 2016, 300 Korean radish plants showing virus-like symptoms on farms were collected in Wanju and Kochang and subjected to RT-PCR using the above YoMV-CP primers. Incidence of YoMV in radish plants was 3%. To our knowledge, this is the first report of YoMV infecting radish in Korea.DetailsFiguresLiterature CitedRelated Vol. 101, No. 11 November 2017SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 16 Oct 2017Published: 7 Sep 2017First Look: 11 Jul 2017Accepted: 5 Jul 2017 Page: 1959 Information© 2017 The American Phytopathological SocietyCited byYoucai mosaic virusCABI Compendium, Vol. CABI CompendiumRaphanus sativus (Radish)6 June 2020

Molecular characterization of a grapevine leafroll-associated virus 4 from Slovenian vineyards.

During a survey conducted in vineyards in Slovenia, variety of grapevine leafroll disease symptoms were observed. Mixed infection with grapevine leafroll-associated viruses 3 and 4 (GLRaV-3, -4) in two grapevines from a vineyard in south-western part of Slovenia was confirmed by DAS-ELISA in 2010. The 3'final 1769 nucleotides of the Slovenian GLRaV-4 isolate were assembled from amplicons obtained by IC RT-PCR. The complete coat protein (CP) and p23 gene sequences were compared with other GLRaV-4 sequences from GenBank. Results showed that CP and p23 amino acid sequences of Slovenian variant (055-SI) are 88% and 85%, respectively, identical to corresponding genes of reference sequence GLRaV-4 LR106 (GenBank Acc. No. FJ467503). Phylogenetic analyses show that Slovenian variant clusters together with other corresponding strains of GLRaV-4. The sequencing results show great variability of the N-terminal part of the CP sequence indicating that this part of the genome is not suitable for molecular detection of the virus. To our knowledge this is also the first report of GLRaV-4 in Slovenian vineyards.

Survey of viral infections in spontaneous grapevines from natural environments in Sicily

The presence of nine of the most economical important grapevine viruses was surveyed in eight spontaneous grapevine populations from natural areas of Sicily. Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1, 2 and 3 (GLRaV-1, −2 and −3), Grapevine virus A and B (GVA, GVB), Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine fleck virus (GFKV) infections were assessed by molecular and serological methods in 73 vines collected in summer 2013 and winter 2013–2014. Reverse transcription-multiplex PCR detected nine GRSPaV- and one GFLV-infected plants in five and one grapevine populations, respectively, while no ArMV, GLRaV-1, GLRaV-2, GLRaV-3, GVA, GVB, GFKV infections were detected. ELISA tests on dormant wood confirmed the result of the molecular detection. The relationship of Sicilian wild grapevine isolates with isolates from grapevines of different geographic origin was investigated through sequencing of the complete coat protein (CP) region of GRSPaV, and partial CP and homing protein (HP) domains of GFLV. Pairwise comparison among the Sicilian GRSPaV CP sequences showed identity scores ranging from 81.67 % to 99.87 % and from 92.66 % to 100 % at nucleotide (nt) and amino acid level (aa), respectively. For GFLV, the CP sequence showed identity ranges from 84.28 % to 90.48 % (nt) and from 89.39 % to 97.11 % (aa); higher variability was obtained analysing the HP domain with identity scores ranging from 68.42 to 93.28 % (nt) and from 58.02 % to 93.23 % (aa). According to phylogenetic analyses, GRSPaV and GFLV isolates from Sicilian wild grapevines clustered with isolates from cultivated grapevines without any correlation between isolate distribution and their geographic origin.

The European race of Gremmeniella abietina hosts a single species of Gammapartitivirus showing a global distribution and possible recombinant events in its history

The population genetics of the family Partitiviridae was studied within the European race of the conifer pathogen Gremmeniella abietina. One hundred sixty-two isolates were collected from different countries, including Canada, the Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. A unique species of G. abietina RNA virus–MS1 (GaRV-MS1) appears to occur indistinctly in G. abietina biotypes A and B, without a particular geographical distribution pattern. Forty-six isolates were shown to host GaRV-MS1 according to direct specific RT-PCR screening, and the virus was more common in biotype A than B. Phylogenetic analysis based on 46 partial coat protein (CP) cDNA sequences divided the GaRV-MS1 population into two closely related clades, while RNA-dependent RNA polymerase (RdRp) sequences revealed only one clade. The evolution of the virus appears to mainly occur through purifying selection but also through recombination. Recombination events were detected within alignments of the three complete CP and RdRp sequences of GaRV-MS1. This is the first time that recombination events have been directly identified in fungal partitiviruses and in G. abietina in particular. The results suggest that the population dynamics of GaRV-MS1 do not have a direct impact on the genetic structure of its host, G. abietina, though they might have had an innocuous ancestral relationship.

Genetic Diversity ofGrapevine virus Ain Washington and California Vineyards

Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the rugose wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera) cultivars from California and Washington were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from California and Washington into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.

First Report of Cowpea mild mottle virus in Cowpea and French Bean in Taiwan.

In 2009, more than 50% of vine type French beans were found bearing severe viral symptoms in a vegetable garden in Nantou County, Taiwan. Infected plants were stunted and exhibited pronounced mottling symptoms on their leaves. The symptomatic plants were mechanically inoculated on Chenopodium quinoa and local lesions developed 7 to 10 days after inoculation. The virus source established by back isolation the single lesion from C. quinoa on French beans developed symptoms similar to those found in the field. Host range test showed that this isolate could only infect leguminous plants, including soybean, mung bean, pea, peanut, asparagus bean, cowpea, adzuki bean, and lima bean, but not cucurbitaceous and solanaceous plants. Since only Cucumber mosaic virus (CMV) has been reported in Taiwan to induce similar symptoms in French beans, we tested both the field collected and inoculated French beans by CMV antiserum in ELISA but obtained a negative result. Due to subsequent electron microscopy studies that found potyvirus and carlavirus like particles in the leaf dips of infected French beans, we conducted reverse transcription (RT)-PCR using generic degenerate primers for potyviruses (Hrp5/Pot1 (2) and PotZ/Pot1 (3)) and carlaviruses (Decarla-u2 (5'-TGCACTGARTCMGAYTATGARGCYTT-3' and Decarla-d1 (5'-GCACATRTCRTCVCCDGCAAA-3') previously designed in our lab. No amplification was found from the potyvirus primers, while the carlavirus one gave an expected amplicon of 285 bp, which was found sharing 81% nucleotide sequence identity with the replicase gene of Cowpea mild mottle virus (CpMMV) (GenBank Accession No. FJ560903). A primer pair (CpMMV-CPu: 5'-TTTACTCTTAggTWATggAgTC-3' and CpMMV-CPd: 5'-CCTATTAAAACACACAAHTCAAA-3') was thus designed to amplify the complete coat protein (CP) gene based on the reported CP sequences and obtained an expected 867-bp product from our French bean isolate. This 867-bp sequence (JX020701) was confirmed to have 97.6% amino acid sequence identities with the CP gene of a Puerto Rican CpMMV isolate (GU191840). In a separate survey, another isolate from asparagus bean (CpMMV-V) causing mild mottling symptom was obtained. Analyses of the CP gene of CpMMV-V (JX070669) confirmed that it shared 88.8% and 97.8% of nucleotide and amino acid sequence identities with the French bean isolate, respectively. Different from most carlaviruses with aphid transmissibility, CpMMV has been shown to be transmitted non-persistently by whiteflies (1). Both CpMMV isolates from Taiwan were confirmed to be transmitted by silver leaf whiteflies (Bemicia argentifolii Bellows and Perring). This is the first record of whitefly transmissible legume virus in Taiwan. Since whitefly has been a problem in agriculture worldwide, CpMMV can be a new emerging threat for Taiwan's legume crop production.

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C. maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.

Indigenous and introduced potyviruses of legumes and Passiflora spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.

Molecular variability analyses of Apple chlorotic leaf spot virus capsid protein

The complete sequences of the coat protein (CP) gene of 26 isolates of Apple chlorotic leaf spot virus (ACLSV) from India were determined. The isolates were obtained from various pome (apple, pear and quince) and stone (plum, peach, apricot, almond and wild Himalayan cherry) fruit trees. Other previously characterized ACLSV isolates and Trichoviruses were used for comparative analysis. Indian ACLSV isolates among themselves and with isolates from elsewhere in the world shared 91-100% and 70-98% sequence identities at the amino acid and nucleotide levels, respectively. The highest degree of variability was observed in the middle portion with 9 amino acid substitutions in contrast to the N-terminal and C-terminal ends, which were maximally conserved with only 4 amino acid substitutions. In phylogenetic analysis no reasonable correlation between host species and/or geographic origin of the isolates was observed. Alignment with capsid protein genes of other Trichoviruses revealed the TaTao ACLSV peach isolate to be phylogenetically closest to Peach mosaic virus, Apricot pseudo chlorotic leaf spot virus and Cherry mottle leaf virus. Recombination analysis (RDP3 ver.2.6) done for all the available ACLSV complete CP sequences of the world and Indian isolates indicate no significant evidence of recombination. However, one recombination event among Indian ACLSV-CP isolates was detected. To the best of our knowledge, this is the first report of complete CP sequence variability study from India and also the first evidence of homologous recombination in ACLSV.

First Report of Grapevine fleck virus from Washington Vineyards.

Grapevine fleck virus (GFkV) is a positive-sense, single-stranded RNA virus with a genome size of 7,564 nucleotides (3). The virus is present in many grape-growing regions (1,2,4). GFkV is phloem-limited and graft transmissible, but a biological vector is not yet known (4). It causes latent infections in Vitis vinifera cultivars, but induces specific foliar symptoms in the indicator host, V. rupestris. While testing samples from wine grape cultivars, samples from cv. Chardonnay tested positive for GFkV in single-tube one-step reverse transcription (RT)-PCR assay using forward primer GFkV585F (5'-CTCAGCCTCCACCTTGCCCCGT-3') and reverse primer GFkV1117R (5'-CAATTTGGCTGGGCGAGAAGTACA-3'). The forward primer is identical to nt 585 to 606 and the reverse primer is complementary to nt 1094 to 1117 in the GFkV genome (Accession No. AJ309022) and encompass a portion of the RNA-dependent RNA polymerase. The primer pair amplified a 533-nt fragment from 15 of 37 individual grapevines in the Chardonnay block. The amplicons obtained from five grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones per amplicon were sequenced in both orientations. Sequences were edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparisons of these sequences (Accession Nos. GU372367 to GU372371) with a corresponding sequence of a GFkV isolate deposited in the GenBank (Accession No. AJ309022) showed 89 to 97% identity at the nucleotide and 95 to 98% identity at the amino acid level. To further support these results, we amplified a 714-nt fragment specific to the complete coat protein (CP) gene of GFkV from three of the five isolates sequenced above using primers GFkV-6351F (5'-CTCTCCGCCTCGTCTGATGA-3') and GFkV-7064R (5'-TCGGTTCATGACGAGGGAGT-3'). The amplicons were cloned and sequenced as described above. A comparison of these sequences (Accession Nos. GU372372 to GU372374) with the CP sequence of GFkV available in the GenBank (Accession No. AJ309022) showed 94 to 95% and 98 to 100% identity, respectively, at the nucleotide and amino acid level. ELISA with GFkV-specific antibodies (BIOREBA AG, Reinach, Switzerland) further confirmed the presence of the virus in samples that were positive in RT-PCR. ELISA results validated the data described above and confirmed the presence of GFkV in Chardonnay samples that tested positive by RT-PCR assay. Previously, GFkV was documented in grapevines in California and Missouri (2), Australia (4) and Europe (1). To our knowledge, this is the first confirmed report of the occurrence of GFkV in Washington vineyards. The results expand our current knowledge on the distribution of GFkV and help to prevent its dissemination through the supply of grapevine cuttings by 'clean' plant programs.

Genetically distinct strains of Cassava brown streak virus in the Lake Victoria basin and the Indian Ocean coastal area of East Africa

Six isolates of Cassava brown streak virus (CBSV, genus Ipomovirus; Potyviridae) from the Lake Victoria basin in Uganda and Tanzania were characterized. Virus particles were 650 nm long. The complete coat protein (CP)-encoding sequences (1,101 nucleotides, nt) were 90.7-99.5 and 93.7-99.5% identical at the nt and amino acid (aa) levels, respectively. The 3' untranslated region was 225, 226 or 227 nt long. These eight isolates were only 75.8-77.5% (nt) and 87.0-89.9% (aa) identical when compared to the partial CP sequences (714 nt) of six CBSV isolates characterized previously from the costal lowlands of Tanzania and Mozambique. Hence, two genetically different and geographically separated populations of CSBV exist in East Africa.

Rapid determination of the nucleotide sequences of potyviral coat protein genes using semi-nested RT-PCR with universal primers

A rapid method (<20 h) for determining the nucleotide sequences of potyviral coat protein (CP) genes was developed. The procedure involves reverse transcription using total RNA from a potyvirus-infected plant, followed by polymerase chain reaction (PCR) with the universal primers Sprimer/M4. The expected fragment, purified from an agarose gel, is used in semi-nested PCR with three degenerate primer sets: Sprimer/D1000, Sprimer/RYAFDFY-C and U341/M4. The three purified fragments are directly sequenced using the respective primers D1000, RYAFDFY-C and U341, then the complete CP sequence is assembled. All 11 potyviruses tested were efficiently sequenced by this method.

Molecular Characterization of Tuberose mild mosaic virus and Preparation of Its Antiserum to the Coat Protein Expressed in Bacteria

A 2-kb DNA product was amplified from purified Tuberose mild mosaic virus (TMMV) virions as well as from infected tissues of tuberose by the use of degenerate primers for potyvirus. The PCR product was subsequently cloned and its sequence analyzed. It was found comprised of 1947 nucleotides (nts) corresponding to the 3′-terminal region of potyviruses. The deduced amino acid sequence contained 598 residues encoding part of the 3′-terminal region of NIb gene (319 residues) and the complete sequence of coat protein (CP) gene (279 residues). A 136 nts of non-coding region (NCR1 was found located at the 3′-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 19-2 1 residues from the N-terminus of CP gene. Compared to the known sequences of potyviruses, the percent of nucleotide identities of the CP gene and the NCR were less than 62% and 39%, respectively. Similarly, percent identities of TMMV’s CP amino acid sequence to those of other known potyviruses were all below 58%, confirming our previous finding that TMMV is a new species of Polyvirus. Using directional cloning technology, a 39-kDa fusion protein containing a complete CP sequence of TMMV and a partial sequence encoded by the expression vector plasmid (pET-30b, Novagene) was highly expressed and purified from E. coli cell cultures. The antigenicity of the fusion protein was determined to be indistinguishable from the viral CP. Antiserum prepared against this fusion protein showed comparable reactivities in the serological detection of TMMV with the conventional antibodies against purified virus particles.