Coat Protein Cistron Research Articles

Coat protein genealogy and complete genome characterization of field isolates of rice yellow mottle virus from Zambia

AbstractRice yellow mottle virus (RYMV) is widespread in mainland Africa and adjoining islands but to date its occurrence in Zambia is unknown. In March 2022, field surveys were conducted in Luapula, Northern, Western and Eastern provinces of Zambia to determine the occurrence of RYMV and its genetic relationship with global isolates of the virus. Thirty‐three paddy rice fields were visited and 108 leaf tissue samples were collected for analysis. The incidence of yellow mottle symptoms ranged from 25% to 43% in 10 (30.3%) of 33 fields and RYMV was detected in 35 (32.4%) of 108 samples by RT‐PCR with virus‐specific primers. A subset of 27 RYMV‐positive samples was constituted from which full‐length (720 bp) coat protein (CP) regions were amplified followed by bidirectional Sanger sequencing. Phylogenetic analysis of the CP cistron revealed distinct clustering of the isolates from Zambia in a monophyletic clade as subtype of the RYMV strain S4. Three isolates were randomly selected and used to obtain complete RYMV genomes of 4448–4449 nucleotides (nt). Comparative analysis of both the CP and the complete RYMV genomes from Zambia with their corresponding sequences in GenBank revealed that they shared 92%–93.2% nt identities with strain S4 isolates. These results confirm for the first time occurrence of RYMV strain S4 in Zambia and further reinforce the need for phytosanitary vigilance to safeguard rice production in Zambia.

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

First Report of Tobacco Mosaic Virus Infecting Papaya in China.

Papaya (Carica papaya. L) is widely cultivated in tropical and subtropical regions of China and has high nutritional and medicinal values. More than 11 species of papaya viruses have been recorded in the world, but the most destructive one for papaya production in China is papaya ringspot virus (PRSV) (Li, 2019). In order to control PRSV, a transgenic papaya cultivar, designated as 'Huanong No.1', carrying the nuclear inclusion b (Nib) cistron of PRSV Ys isolate, was successfully commercialized in 2006, and has shown a wide range of resistance to PRSV in China (Li et al. 2007). However, more than 10% of 'Huanong No.1' plants developed different virus-like symptoms on leaves, including mosaic, yellow mottle, and deformation in some plantations of Guangdong Province, China in 2020 (Suppl Figure 1a, b, and c). Based on observation of the symptomatic phenotypes, the field surveys indicated that the disease incidence ranged from 10% to 40%, resulting in significant loss of papaya fruit. The virus particles were purified from symptomatic papaya plants following Gooding and Hebert (1967) and rigid filamentous particles resembling tobacco mosaic virus (TMV) were observed by transmission electron microscopy. Purified virus samples were further utilized to mechanically inoculate healthy seedlings of papaya, Nicotiana glutinosa and N. tabacum K326. At 15 days after inoculation, the obvious symptoms of virus infection on different plants were observed. The diseased plants showed systemic mottling and mosaic in the papaya leaves (Suppl Figure 1d), necrotic spots on the leaves of N. glutinosa (Suppl Figure 1e), mosaic and mottling spots on leaves of N. tabacum K326 (Suppl Figure 1f). These symptoms produced on the hosts were exactly the same caused by TMV. In order to reconfirm the species of the infected virus, the total RNA was extracted from the single leaf of 30 diseased papaya plants using RNAiso Plus kit (Takara, Japan) and reverse transcription--polymerase chain reaction was performed using TMV coat protein cistron specific primers (TMV-CP-R: 5'-TCAAGTTGCAGGACCAGA-3' and TMV-CP-F 5'- ATGTCTTACAGTATCACTAC-3') as described previously (Srivastava et al. 2015). An expected 480-bp fragment was amplified from all of the samples. Sequence analysis revealed that the diseased papaya was infected with TMV, designated as Cpa-TMV. In order to understand the difference among TMV isolates on papaya and other host plants, the whole genomic sequence of TMV from papaya was obtained and analyzed. The total length of the genome of Cpa-TMV was 6395 bp, and the sequence was submitted to the NCBI database (GenBank no. OK149218). A neighbor-joining phylogenetic tree of 19 TMV isolates was constructed using MEGA X software. Phylogenetic analysis indicated that the 19 TMV isolates were divided into Clade I, II and III (Suppl Figure 2). Interestingly, Clade I was composed of 12 Chinese mainland isolates, which further was grouped into IA (Northern China) and IB (Southern China), while 6 isolates from other countries and 1 isolate (pet-TMV) from China Taiwan belong to Clade II and III. It is inferred that the TMV isolates from Chinese mainland are quite different from other countries and China Taiwan. This suggests that geographical differences between Northern and Southern China may lead to the gradual differentiation of TMV isolates and eventually induce those isolates to evolve into two subclades. To our knowledge, this is the first report of TMV infection on papaya under natural conditions. It is necessary to find effective methods to control TMV in transgenic papaya.

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019–2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96–99.5% nucleotide and 94.40–99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li’s F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.

First Report of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 in Watermelon (Citrullus lanatus) Plants Coinfected with Cucurbit chlorotic yellows virus in Florida.

Watermelon (Citrullus lanatus L.) and other cucurbits are major crops in Florida. During the 2020 and 2021 seasons, watermelon plants with foliar virus-like symptoms of yellow mottling and chlorosis, mild leaf wrinkling and thickened leaves were observed in commercial fields (40 to 150 ha) in five counties (Desoto, Glades, Osceola, Seminole, and Charlotte) at >50% field incidence. Initial screening of 13 field-collected samples (2 to 4/County) for potyviruses with the Agdia POTY Immunostrip (Agdia, Inc. Elkhart, IN) were negative. Total nucleic acid extracts from each sample (RNeasy Plant Mini Kit, Qiagen, Germantown, MD) were used in one-step RT-PCR (Qiagen OneStep RT-PCR kit) with species-specific primer targeting squash vein yellowing virus (SqVYV), papaya ringspot virus-W (PRSV-W) (Adkins et al., 2008), cucurbit yellow stunting disorder virus (CYSDV) (Polston et al. 2008), cucurbit chlorotic yellows virus (CCYV) (Hernandez et al., 2021a), watermelon crinkle leaf-associated virus 1 (WCLaV-1), and WCLaV-2 (Hernandez et al., 2021b). The samples were also tested for cucurbit leaf crumple virus (CuLCrV) as per Hagen et al. (2008). All 13 samples were negative for SqVYV, PRSV-W, CuLCrV, and CYSDV, but 7 samples (53.8%) from 4 counties tested positive for CCYV, 12 (92.3%) from 5 counties were positive for WCLaV-1, and WCLaV-2 was detected in 8 samples (61.5%) from 5 counties. Three samples were singly infected with WCLaV-1 while the remaining 10 were mixed infected with different combination of 2 or 3 viruses. Notably, symptoms on all 13 plants were visually indistinguishable. To verify the results, two randomly chosen gene-specific fragments per virus, obtained with primers CCYV-v1330/c2369, CCYV-v4881/c5736, WCLaV-1vRP/1cRP, WCLaV-1vMP/1cMP, WCLaV-2vRP/2cRP, and WLaV 2vMP/2cMP, were excised from the gel, cloned, and Sanger-sequenced as described (Hernandez et al., 2021a, 2021b). In pairwise comparisons, the ~1 kb partial ORF1a (GenBank accession nos. MZ325846 to MZ325847) and 753 bp complete coat protein cistron (MZ325848 to MZ325849) of CCYV from Florida shared 98.9-99.5%/98.4-99.6% nucleotide (nt)/amino acid (aa) and 99.4-99.8%/99.6-100% nt/aa identities, respectively with the corresponding sequences of global CCYV isolates. The partial RNA1 (MZ325850 to MZ325851) and RNA2 (MZ325852 to MZ325853) sequences of WCLaV-1 from Florida shared 99.2-99.8%/100% nt/aa and 98.9-100%/99.3-100% nt/aa identities, respectively with the corresponding global sequences of WCLaV-1 isolates. Lastly, the partial RNA1 (MZ325854 to MZ325857) and RNA2 (MZ325858 to MZ325861) sequences of WCLaV-2 from Florida shared 96.4-99.8%/97-100% nt/aa and 96.5-100%/95.9-100% nt/aa identities, respectively with the corresponding global sequences of WCLaV-2 isolates. This is the first report of WCLaV-1 and WCLaV-2 from Florida and the first documentation of the occurrence of CCYV in South Florida. CCYV has been reported previously from California (Wintermantel et al. 2019), Georgia (Kavalappara et al. 2021), and recently from North Florida (M. Paret, pers. comm) but WCLaV-1 and WCLaV-2 have only been reported from Texas (Hernandez et al., 2021b), after their discovery in China (Xin et al. 2017). The results indicate further expansion of the geographical range of these cucurbit-infecting viruses, although their longer but undetected presence in Florida is plausible due to the resemblance of their associated symptoms with those attributed to known viruses. References Adkins, et al., 2008. Plant Dis. 92:1119-1123. https://apsjournals.apsnet.org/doi/10.1094/PDIS-92-7-1119. Hagen, et al. 2008. Plant Dis. 92:781-793. https://apsjournals.apsnet.org/doi/pdfplus/10.1094/PDIS-92-5-0781. Hernandez, et al. 2021a. Plant Dis. https://doi.org/10.1094/PDIS-02-21-0378-PDN. Hernandez, et al., 2021b. Plant Dis. https://doi.org/10.1094/PDIS-02-21-0249-PDN Kavalappara, et al., 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-20-2429-PDN. Polston, et al. 2008. Plant Dis. 92(8):1251. https://apsjournals.apsnet.org/doi/10.1094/PDIS-92-8-1251B. Wintermantel, et al., 2019. Plant Dis. 103(4):778. https://doi.org/10.1094/PDIS-08-18-1390-PDN. Xin, et al., 2017. Front. Microbiol. 8:1514, doi: 10.3389/fmicb.2017.01514.

English

Cucumber mosaic virus (CMV) is a destructive and menace to a wide range of plant species including eggplant crop worldwide. This study was carried out to define the current status of CMV infecting eggplant crop in Pothwar region of Pakistan and characterization of CMV eggplant isolates as well as to recognize the sources of resistance in eggplant genotypes. A total of 660 leaf samples with the virus and virus-like symptoms were collected from different sites of Pothwar region and subjected to CMV specific DAS-ELISA using monoclonal antibodies. ELISA results indicated that approximately 56% of samples were positive for CMV infection and the disease incidence was highest (62.50%) in Jhelum district followed by Rawalpindi (58.33%), Chakwal (58.33%) and Attock (55.55%) districts with the lowest disease incidence (46.15%) was recorded in Islamabad district. Two ELISA positive samples from each district were further confirmed by RT-PCR and subsequently sequenced based on their coat protein cistron. All isolates were identical to each other so the nucleotide sequence of one eggplant isolate (CMV-Pak) was deposited in GenBank with accession number MH119063. BLAST and phylogenetic analysis revealed that CMV-Pak eggplant isolate shared 90-94% nucleotide and 80-100% amino acid similarities with other isolates available in GenBank. Moreover, 10 eggplant genotypes were tested against CMV-Pak eggplant isolate, ELISA results categorized three genotypes (Singh Nath 666, Neelum, and Brinjal Jamak) as moderately resistant, two genotypes (Round Black, and Global Brinjal PPL) as susceptible and rest of the five genotypes (Bemissal, Brinjal Shilpa, Short Purple, Brinjal PPL, and Namyal Ratchburi) as moderately susceptible. Therefore, the three moderately resistant genotypes could be recommended to farmers for farming and plant breeders for developing new eggplant resistant genotypes to CMV. To the best of our knowledge, this study is the first time provided evidence that CMV infects eggplant in Pakistan.

First report of Cucurbit chlorotic yellows virus infecting cantaloupe (Cucumis melo L.) in Texas.

Texas is a major producer of cucurbits such as cantaloupe (Cucumis melo L.), but outbreaks of virus-like diseases often adversely affect yields. Little is known about the identity of the causal or associated viruses. During studies conducted in fall 2020 to explore the virome of cucurbit fields in Texas, a commercial cantaloupe field (~4.1 ha) in Cameron County was observed with virus-like symptoms of interveinal chlorotic mottle and foliar chlorosis and disease incidence was estimated at 100%. Virus-like symptoms including mosaic and leaf curl were also observed in six additional fields across five south and central Texas counties of Atascosa, Hidalgo, Fort Bend, Frio, and Wharton. Forty-six plants, which included 32 symptomatic and 14 non-symptomatic, were sampled from these fields for virus diagnosis and each sample was subjected to total nucleic acid extraction according to Dellaporta et. al. (1983). Initially, equal amounts of nucleic acids from 14 symptomatic plants (two/field) were pooled into one composite sample for preliminary diagnosis by high throughput sequencing (HTS). The cDNA library obtained from the composite sample with a TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina) was sequenced on the Illumina NextSeq 500 platform, generating ~26.3 M single-end HTS reads (75 nucleotides [nt] each). Analyses of the reads according to Al Rwahnih et al. (2018) revealed several virus-like contigs; among them 23 contigs (206 to 741 nt) shared 98 to 100% nt identities to isolates of cucurbit chlorotic yellows virus (CCYV), genus Crinivirus, family Closteroviridae. Three pairs of CCYV-specific primers were designed from the HTS contigs with primers CCYV-v1330: 5'-AGTCCCTTACCCTGAGATGAA/CCYV-c2369: 5'-CGGAGCATTCGACAACTGAATA targeting ~1 kb fragment of the ORF1a (RNA1), primers CCYV-v4881: 5'-ATAAGGCGGCGACCTAATC/CCYV-c5736: 5'-GATCACTTGACCATCTCCTTCT targeting a ~0.9 kb fragment encompassing the coat protein (CP) cistron of CCYV (RNA2), and primers CCYV-v6362: 5'-CACCTCTTCCAGAACCAGTTAAA/CCYV-c7423: 5'-TGGGAACAACTTATTTCTCCTAGC targeting ~1 kb spanning partial minor coat protein (CPm) and p26 sequences (RNA2). Total nucleic acid extracts of each of the 46 samples from the seven fields were tested by two-step reverse transcription polymerase chain reaction using all three CCYV-specific primer pairs and they yielded amplicons of expected sizes from all five symptomatic cantaloupe samples from the Cameron County field and one additional symptomatic butternut squash sample from a field in Hidalgo County. The DNA bands from three randomly chosen cantaloupe samples were cloned and sequenced as previously described (Oke et al. 2020). In pairwise comparisons, the obtained 1,040 nt ORF1a (MW584332-334), 753 nt complete CP (MW584335-337), and 1,062 nt CPm/p26 (MW584338-340) gene specific sequences from the three samples shared 100% nt identity with each other, and 99-100% nt identities with corresponding RNA1 (AB523788) and RNA2 (AB523788) sequences of the exemplar isolate of CCYV. This is the first report of CCYV in Texas, thus expanding the current geographical range of the virus in the U.S. that includes California (Wintermantel et al. 2019) and Georgia (Kavalappara et al. 2021). The abundance of whiteflies of the Bemisia tabaci species complex in south Texas and other major U.S. cucurbit production areas presents additional challenges to virus disease management.

FIRST REPORT OF CHILLI RINGSPOT VIRUS ON CHILLI PEPPER IN PAKISTAN

Viral diseases hamper successful production of solanaceous crops in Pakistan (Ahmad and Ashfaq, 2017; Ashfaq and Ahmad, 2017). In August 2014, 25 chilli pepper leaf samples with symptoms of mottling, interveinal chlorosis and dark green vein-banding were collected from three sites in the Multan district. All diseased samples and two healthy samples were screened against potyvirus infection by indirect PTA-ELISA using “potyvirus group test” kit (Bioreba AG, Switzerland). Only 16 samples were positive for potyvirus infection and were further tested for the presence of specific potyviruses by RT-PCR. Total RNAs extracted using TRIzol reagent (Life Technologies) were subsequently subjected to RT-PCR using primers Poty3 and Oligo(dT)18 (Tsai et al., 2008). All 16 RT-PCR amplicons (ca. 0.8 kb) were purified using QIAquick® PCR purification kit (Qiagen) and sequenced. Sequencing results confirmed the presence of chilli veinal mottle virus (ChiVMV), potato virus Y (PVY) and chilli ringspot virus (ChiRSV) in 11, three and two (Capsicum annuum var. sanam) samples, respectively. ChiRSV and PVY presence were confirmed in chilli fields in Basti Malook and Basti Islampur, respectively, while ChiVMV presence was recorded in all three sites of Multan district. Aphids were also observed (2-4 aphids per plant) on a few plants in the fields from where ChiRSV positive samples were collected. ChiRSV was further confirmed using ChiRSV-specific forward primer (5’- AAGAAGCTGTACACAGGAGAGGA-3’, designed from GenBank Accession No. JQ234922) and Oligo(dT)18. A total of 997 nucleotides (nt) were obtained from each amplicon including the full coat protein cistron and 181 nt of the 3’ nontranslated region, and the sequences deposited in GenBank (KX816566, KX816567); these two sequences shared >98% nt identity. BLASTn revealed 92-98% sequence identities with chilli (C. annuum) isolates of ChiRSV from China (KX258620, KX379001, JN008909) and Vietnam (DQ925438, DQ925439). This virus, first reported in 2008 in Vietnam and China in 2009 from chilli pepper, has not been found elsewhere in the world (Wang et al., 2012). To the best of our knowledge, this is the first confirmed report of ChiRSV on chilli pepper in Pakistan.

Occurrence and Evolutionary Analysis of Coat Protein Gene Sequences of Iranian Isolates of Sugarcane mosaic virus.

Sugarcane mosaic virus (SCMV) is one of the most damaging viruses infecting sugarcane, maize and some other graminaceous species around the world. To investigate the genetic diversity of SCMV in Iran, the coat protein (CP) gene sequences of 23 SCMV isolates from different hosts were determined. The nucleotide sequence identity among Iranian isolates was more than 96%. They shared nucleotide identities of 75.5–99.9% with those of other SCMV isolates available in GenBank, the highest with the Egyptian isolate EGY7-1 (97.5–99.9%). The results of phylogenetic analysis suggested five divergent evolutionary lineages that did not completely reflect the geographical origin or host plant of the isolates. Population genetic analysis revealed greater between-group than within-group evolutionary divergence values, further supporting the results of the phylogenetic analysis. Our results indicated that natural selection might have contributed to the evolution of isolates belonging to the five identified SCMV groups, with infrequent genetic exchanges occurring between them. Phylogenetic analyses and the estimation of genetic distance indicated that Iranian isolates have low genetic diversity. No recombination was found in the CP cistron of Iranian isolates and the CP gene was under negative selection. These findings provide a comprehensive analysis of the population structure and driving forces for the evolution of SCMV with implications for global exchange of sugarcane germplasm. Gene flow, selection and somehow homologous recombination were found to be the important evolutionary factors shaping the genetic structure of SCMV populations.

Triticum mosaic virus exhibits limited population variation yet shows evidence of parallel evolution after replicated serial passage in wheat

An infectious cDNA clone of Triticum mosaic virus (TriMV) (genus Poacevirus; family Potyviridae) was used to establish three independent lineages in wheat to examine intra-host population diversity levels within protein 1 (P1) and coat protein (CP) cistrons over time. Genetic variation was assessed at passages 9, 18 and 24 by single-strand conformation polymorphism, followed by nucleotide sequencing. The founding P1 region genotype was retained at high frequencies in most lineage/passage populations, while the founding CP genotype disappeared after passage 18 in two lineages. We found that rare TriMV genotypes were present only transiently and lineages followed independent evolutionary trajectories, suggesting that genetic drift dominates TriMV evolution. These results further suggest that experimental populations of TriMV exhibit lower mutant frequencies than that of Wheat streak mosaic virus (genus Tritimovirus; family Potyviridae) in wheat. Nevertheless, there was evidence for parallel evolution at a synonymous site in the TriMV CP cistron.

BrazilianPotato virus Yisolates identified as members of a new clade facilitate the reconstruction of evolutionary traits within this species

Potato virus Y (PVY) is a plant virus distributed worldwide that causes damage to several species of the Solanaceae family. It was established long ago that groups of PVY isolates defined by phylogenetic analyses correlate strongly with those demarcated by differential biological properties. Consequently, life-history traits of this viral species can be inferred by phylogenetic analysis. In this study, characteristics of PVY isolates sampled in different tobacco fields in Brazil were analysed and most of the tested Brazilian PVY isolates were assigned to the recently described unconventional serogroup Y-U. The analysis of molecular diversity of the coat protein (CP) cistron from some Y-U isolates made it possible (i) to identify specific amino acid residues in the N-terminal of the CP protein and (ii) to assign some Y-U isolates to a new PVY clade. The symptoms caused by isolates belonging to this new PVY Brazilian' clade and their ability to infect selected susceptible hosts led to the conclusion that neither veinal necrosis symptoms expressed on infected tobacco plants nor adaptation to potato or pepper hosts are ancestral characteristics of PVY. These observations suggest that PVY has gained a remarkable new biological property and broadened its host range over time.

Wheat streak mosaic virus Infects Systemically despite Extensive Coat Protein Deletions: Identification of Virion Assembly and Cell-to-Cell Movement Determinants

Viral coat proteins function in virion assembly and virus biology in a tightly coordinated manner with a role for virtually every amino acid. In this study, we demonstrated that the coat protein (CP) of Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) is unusually tolerant of extensive deletions, with continued virion assembly and/or systemic infection found after extensive deletions are made. A series of deletion and point mutations was created in the CP cistron of wild-type and/or green fluorescent protein-tagged WSMV, and the effects of these mutations on cell-to-cell and systemic transport and virion assembly of WSMV were examined. Mutants with overlapping deletions comprising N-terminal amino acids 6 to 27, 36 to 84, 85 to 100, 48 to 100, and 36 to 100 or the C-terminal 14 or 17 amino acids systemically infected wheat with different efficiencies. However, mutation of conserved amino acids in the core domain, which may be involved in a salt bridge, abolished virion assembly and cell-to-cell movement. N-terminal amino acids 6 to 27 and 85 to 100 are required for efficient virion assembly and cell-to-cell movement, while the C-terminal 65 amino acids are dispensable for virion assembly but are required for cell-to-cell movement, suggesting that the C terminus of CP functions as a dedicated cell-to-cell movement determinant. In contrast, amino acids 36 to 84 are expendable, with their deletion causing no obvious effects on systemic infection or virion assembly. In total, 152 amino acids (amino acids 6 to 27 and 36 to 100 and the 65 amino acids at the C-terminal end) of 349 amino acids of CP are dispensable for systemic infection and/or virion assembly, which is rare for multifunctional viral CPs.

Survey of Apple mosaic virus in apple-growing provinces of East Anatolia (Malatya and Van) by RNA probe hybridization assay and RT-PCR

Two main apple growing provinces (Van and Malatya) of East Anatolia were surveyed for the presence of the Apple mosaic virus (ApMV). Dot-blot hybridization and RT-PCR tests were implemented to investigate the incidence of ApMV, testing a total of 481 samples collected from commercial apple orchards. Digoxigenin-labeled RNA probes of the ApMV were synthesized from the cloned PCR products and applied in dot-blot hybridization to detect the virus in RNA extracts isolated from fresh leaf tissues. A validated RNA probe (dot-blot) hybridization method was adopted to investigate the presence of ApMV infections in the apple orchards of East Anatolia. In order to determine the most suitable mass extraction methods and to ascertain the presence of virus, 3 RNA preparation procedures (QIAGEN RNA extraction kit, silica capture, and citric buffer) were tested. Among the tested extraction methods, silica capture was determined as the most suitable extraction method for dot-blot hybridization assays. ApMV was found in the surveyed locations with an average incidence of 0.8%. The infected trees showed apparent disease symptoms on the leaves of apple trees. The coat protein (CP) genes of 2 viral isolates selected from Malatya (ApMV-G and ApMV-M) were cloned and their complete nucleotide sequences and deduced amino acids were determined (GenBank acc. nos. JX155668 and JX155669). The CP cistron of the ApMV-G and M isolates contained 224 amino acid residues. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor-joining and the unweighted pair-group mean arithmetic methods with 100 bootstrap replicates. The CP sequence of ApMV-G and ApMV-M varied among the 21 isolates, with overall identity ranging from 88% to 99% and ranging from 91% to 99% at the nucleotide level.

Cloning and sequencing of coat protein gene of Zucchini yellow mosaic virus isolated from squash and muskmelon in Turkey

The coat protein (CP) genes of the genomic RNA of 2 severe Turkish isolates of Zucchini yellow mosaic virus (ZYMV) from squash and muskmelon [ZYMV-Adana (Ad) and ZYMV-Ahlat (Ah), respectively] were cloned, and their complete nucleotide sequences and deduced amino acids were determined. The analysis revealed that both Turkish ZYMV-CP genes contained 837 nucleotides encoded for a CP of about 31.2 kDa. Phylogenetic trees based on nucleic acid sequences were constructed by the neighbor joining and unweighted pair group mean arithmetic (UPGMA) methods with 100 bootstrap replicates. A high degree of homology was detected between the 2 Turkish isolates on the nucleotide sequences (97%). The CP sequence of ZYMV-Ad and ZYMV-Ah varied among the 23 isolates with overall identity of 93%-98% and 94%-99%, respectively, at the nucleotide level. Comparison of the nucleotide sequences of 23 isolates from different geographical regions worldwide showed the ZYMV-Ad isolate clustered with isolates from Middle Eastern countries (Israel, Jordan, and Syria); ZYMV-Ah isolate was clustered with isolates from Far Eastern countries (Korea and Taiwan). The N-terminal of the Turkish ZYMV-Ah CP contained a distinctive sequence at nucleotide positions 9324-9328, which distinguished the Turkish ZYMV-Ah isolate from all previously reported ZYMV isolates. The CP cistron of the ZYMV-Ad and Ah isolates contained 279 amino acid residues. Pairwise nucleotide sequence comparison revealed sequence similarities of 58%-70% between ZYMV Turkish isolates and 22 other potyviruses. Mechanical inoculations showed that ZYMV-Ah produced faster systemic symptom induction than ZYMV-Ad on squash, suggesting that ZYMV-Ah was a more severe isolate than ZYMV-Ad (GenBank accession JF317296-JF317297).

Complete sequence of Fig fleck-associated virus, a novel member of the family Tymoviridae

The complete nucleotide sequence and the genome organization were determined of a novel virus, tentatively named Fig fleck-associated virus (FFkaV). The viral genome is a positive-sense, single-stranded RNA 7046 nucleotides in size excluding the 3′-terminal poly(A) tract, and comprising two open reading frames. ORF1 encodes a polypeptide of 2161 amino acids (p240), which contains the signatures of replication-associated proteins and the coat protein cistron (p24) at its 3′ end. ORF2 codes for a 461 amino acid protein (p50) identified as a putative movement proteins (MP). In phylogenetic trees constructed with sequences of the putative polymerase and CP proteins FFkaV consistently groups with members of the genus Maculavirus, family Tymoviridae. However, the genome organization diverges from that of the two completely sequenced maculaviruses, Grapevine fleck virus (GFkV) and Bombix mori Macula-like virus (BmMLV), as it exhibits a structure resembling that of Maize rayado fino virus (MRFV), the type species of the genus Marafivirus and of Olive latent virus 3 (OLV-3), an unclassified virus in the family Tymoviridae. FFkaV was found in field-grown figs from six Mediterranean countries with an incidence ranging from 15% to 25%.

Biological, Physical, and Molecular Properties of a Papaya lethal yellowing virus Isolate.

Papaya (Carica papaya) is an important tropical fruit crop in northeastern Brazil. Lethal yellowing is a disease caused by Papaya lethal yellowing virus (PLYV) that occurs only in northeastern Brazil. The symptoms are characterized by progressive leaf yellowing and greenish circular spots on the fruits. The virus has isometric particles (30 nm), genomic single-stranded (ss)RNA (1.6 × 106 Da), and a coat protein with a single component (35 kDa). Although no biological vector has been confirmed, the virus is spreading every year. In the present study, an isolate of the virus was characterized biologically, serologically, physically, and molecularly, and its survival in dried tissue was evaluated. Approximately 309.5 mg of virus was purified per kilogram of infected papaya leaves, and a high-titer polyclonal antiserum (1:1,024,000) was obtained from an immunized rabbit. The virus was detected in dried roots and leaves maintained up to 120 days. Its physical properties were thermal inactivation point (80°C), longevity in vitro (60 days), and dilution end point (10-6). The virus was inactivated in leaves and roots eradicated from infected plants when they were submitted to a solarization period of 12 days, but maintained its infectivity when leaves and roots were maintained over the soil at natural conditions for 32 days. Among 58 plant species from 13 botanical families, the PLYV isolate infected only C. papaya, Jacaratia heterophylla, J. spinosa, Vasconcella cauliflora, V. quercifolia, and V. monoica, all from the family Caricaceae. A fragment of 1,098 nt of the virus isolate, corresponding to the 3' replicase (RdRp) and 5' coat protein (CP) cistron, was cloned and sequenced. Phylogenetic analysis of the RdRp nucleotide sequences indicated some similarity of PLYV with members of the genus Sobemovirus.

Identification and molecular characterization of a new potyvirus from Panax notoginseng

A potyvirus causing distortion and mosaic symptoms in the herbal plant Sanqi (Panax notoginseng) was isolated from Yunnan province, China, and the complete nucleotide sequence of one isolate and the partial sequences of two other isolates were determined. The viral RNA genome comprised 9,750 nt excluding the 3'-terminal poly(A) tail, with the capacity to encode a single polyprotein of 3,089 amino acids. Phylogenetic analysis with other completely sequenced potyviruses revealed that the virus in this study was most closely related to plum pox virus, with 56.3% nt identity in the genomic RNA sequence and 53.3% aa identity in the polyprotein. However, the most closely related 3'-terminal sequences were from four partially sequenced potyviruses infecting plants of the family Apiaceae (67.7-75.3% nt identity and 73.8-76.7% aa identity in their coat protein cistrons), especially Angelica virus Y. These results suggest that this virus isolate should be designated a member of a new species in the genus Potyvirus, which is tentatively named Panax virus Y (PanVY).

Potato Virus YNTN: A Coat and P1 Protein Sequences Analysis of a Brazilian Isolate

Potato tubers (Solanum tuberosum L. cv. Monalisa) showing superficial necrotic rings typical of Potato virus Y NTN (PVYNTN) infection were collected from commercial potato fields in the State of Sao Paulo (Brazil). An isolate named IAC-PVYNTN was characterized by sequencing 822 and 836 nucleotides from the coat protein and P1 protein coding genes, respectively. The IAC-PVYNTN was observed as a European/North American recombinant isolate closely related to three Eu-PVYNTN isolates that share an alternative recombination site in the coat protein cistron near position 144. Therefore, the IAC-PVYNTN isolate may represent a novel recombinant variant between PVYN N-605 Swiss (European) and PVY O-139 Canadian (North American). Cluster analysis of the P1 sequences found IAC-PVYNTN in the sub-cluster of Eu-PVYN/NTN/N:O. This study supports previous evidence of a common origin for PVYN/NTN in Europe and Brazil.

Identification and characterization of a novel Tritimovirus species isolated from wild Trisetum flavescens L., family Poaceae

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3' end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).

First Report of Leek yellow stripe virus in Garlic in the State of Guanajuato, Mexico.

Garlic (Allium sativum L.) can be affected by a virus complex (1) consisting of two potyviruses, Onion yellow dwarf virus (OYDV) and Leek yellow stripe virus (LYSV), and two carlaviruses, Garlic common latent virus (GCLV) and Shallot latent virus (SLV) (1). To identify the components of the virus complex that could be present in garlic plants in Guanajuato State, which is the second largest garlic producer in the country and where presumptive viral symptoms were initially observed in December 2004, a survey was carried out in six locations: San Miguel de Allende and San Luis de la Paz in northern Guanajuato; Irapuato and Villagrán in the central region; and Salamanca and Valle de Santiago in the southern part of the state. Enzyme-linked immunosorbent assay (ELISA) was carried out to detect LYSV, OYDV, GCLV, and SLV in 195 garlic leaf samples collected during January 2005 from plants with leaf yellow stripe, mosaic, enation, deformation, or dwarfism symptoms. A set of primers, previously reported and specific to the coat protein cistron of LYSV (1), were synthesized and used in a reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified product (1,020 nucleotides) was cloned into plasmid pGEM T-Easy (Promega, Madison, WI) and sequenced (Gen-bank Accession No. DQ841554). Sequence analysis showed that the cloned DNA fragment shared 97% similarity with the coat protein cistron of LYSV isolate no. 3 from Okinawa (GenBank Accession No. AB194632). The fragment was then radioactively labelled and used as a probe in the RNA blot analysis of all samples to confirm the ELISA results of LYSV. Of the 195 samples, 64 tested positive by RNA blot analysis. Forty-one of these were also positive by ELISA for LYSV. Preliminary, positive ELISA results were also obtained for OYDV, GCLV and SLV. To our knowledge, this is the first report of LYSV in the State of Guanajuato and in Mexico. The correct identification of viruses present in garlic will help to use the appropriate strategies to reduce viral incidence in this garlic-producing region. Reference: (1) T. V. M. Fajardo et al. Fitopatol. Bras. 26:619, 2001.