Molecular analysis of Greek isolates of cucumber mosaic virus from vegetables shows a low prevalence of satellite RNAs and suggests the presence of host-associated virus strains.

Cucumber mosaic virus (CMV) is a generalist pathogen that infects many economically important crops in Greece. The present study was designed to evaluate the genetic variability of Greek CMV isolates in combination with their satellite RNAs (satRNAs). To achieve this goal, 77 CMV isolates were collected from symptomatic Greek vegetables, mainly tomatoes and cucurbits, alongside their neighboring crops, during a four-year period from 2015 to 2018. Phylogenetic analysis of a partial coat protein (CP) gene segment revealed that all of the isolates belong to CMV subgroups IA and IB and that they are closely related to previously reported Greek isolates. It should be noted, however, that the latter mainly included tomato isolates. Network analysis of the evolutionary relationships among the CP sequences of the Greek isolates in comparison to the corresponding sequences obtained from the GenBank database indicated two predominant common ancestors and at least three differentiated peripherals, and possibly host-associated (tomatoes, legumes, cucurbits) haplogroups (strain groups). More specifically, host-adaptive evolution can be postulated regarding the tomato isolates in subgroup IB. Necrogenic or non-necrogenic satRNAs were detected in four samples from tomato and melon, and this is the first report of non-necrogenic satRNAs in CMV in Greece.

Evolutionary timescale and geographical movement of cucumber mosaic virus, with focus on Iranian strains.

Cucumber mosaic virus (CMV) is a geographically widespread plant virus with a very broad host range. The virus has been detected in diverse crops all over Iran. In this study, we estimated the timescale of the evolution of CMV subgroup I and the geographical movement of the virus with a focus on Iranian strains. Analyses using the MP and CP genes and their concatenation revealed that the CMV population within subgroup I had a single ancestor dating back to about 450-550 years ago. The Iranian strains formed three clusters in a maximum-clade-credibility phylogenetic tree. It was found that the most recent common ancestor of the Iranian strains within each cluster dates back to less than 100 years ago. Our results also suggest that both short- and long-distance migration of Iranian CMV strains has occurred in the last 100 years.

[Molecular identification of cucumber mosaic virus in woad (Isatis tinctoria) with mosaic disease in Beijing].

To detect possible pathogenic virus(es) in woad (Isatis tinctoria) cultivated at Institute of Medicinal Plant Development in Beijing, reverse transcription(RT)-PCR was performed using total RNA of symptomatic woad leaves with primers for poty-, polero-, tobamovirus, broad bean wilt virus 2(BBWV2) and cucumber mosaic virus (CMV). A 657 bp fragment was amplified from symptomatic woad using CMV primers. Sequencing and BLAST analysis indicated that this fragment shared 99% nucleotide identity and 100% amino acid identity with CMV-Vi isolate. The isolate was named CMV-Isatis tinctorial (CMV-It). Phylogenetic analysis based on nucleotide sequences of CP genes showed that CMV-It clustered with CMV-K and belonged to subgroup I. To our knowledge, this is first identification of CMV in woad by RT-PCR and the CP gene was analyzed. This work provided data for research and control of woad mosaic disease.

Virome analysis of lily plants reveals a new potyvirus.

Lily plants exhibiting virus-like symptoms of leaf yellowing, twisting and brownish necrotic spots were collected, and next-generation sequencing of small RNAs was conducted to identify the associated viruses. Cucumber mosaic virus, lily symptomless virus and a hitherto unrecorded potyvirus, tentatively named "lily yellow mosaic virus" (LYMV), were detected. The genomic RNA of LYMV was 9811 nt in length, encoding a large polyprotein of 3,124 amino acids with a predicted Mr of 353.3 kDa. BLAST analysis showed that LYMV shared a high degree of amino acid sequence identity with Thunberg fritillary mosaic virus (55%), bean yellow mosaic virus (52%), clover yellow vein virus (51%), leek yellow stripe virus (51%), and lily mottle virus (52%), and these viruses clustered together in a phylogenetic tree.

Genetic diversity, distant phylogenetic relationships and the occurrence of recombination events among cucumber mosaic virus isolates from zucchini in Poland.

In recent years, the occurrence of cucumber mosaic virus (CMV) has been noted in zucchini crops in Poland. Beside characteristic isolates, which displayed mosaics and chlorosis on infected plants, new necrotic isolates have also been identified. Here, we analysed the molecular variability of 27 isolates of CMV collected from zucchini in various regions of the country. Sequence and phylogenetic analysis based on the genes encoding the coat (CP) and movement (MP) proteins revealed that the Polish isolates belong to two subgroups: IA and II, with the prevalence of subgroup II. New recombinant variants with an IA-MP/II-CP pattern for RNA3 were also detected.

Development of a molecular assay for the detection of Cucumber mosaic virus and the discrimination of its subgroups I and II.

A nucleic acid based test for the detection of the economically important plant virus Cucumber mosaic virus (CMV) based on the Luminex xTAG technology was developed. This technology has the advantage of allowing the simultaneous detection of various targets. Applying this method, we prove the presence of CMV in general and differentiate between its two subgroups I and II for which significant differences concerning severity of symptoms and virulence have been reported. For the development of the test procedure the coat protein gene sequences of 29 CMV isolates were cloned, sequenced and classified into subgroups. Sequences from GenBank were used to design primers. Additionally, a subgroup specific ELISA was conducted for comparison. This work is part of a project which aims to develop a test for the simultaneous detection of various plant pathogens (viral, bacterial and fungal) in plant material.

A genetically novel, narrow-host-range isolate of cucumber mosaic virus (CMV) from rosemary.

An isolate of cucumber mosaic virus (CMV), designated CMV-Rom, was isolated from rosemary (Rosmarinus officinalis) plants in several locations near Avignon, France. Laboratory studies showed that, unlike typical CMV isolates, CMV-Rom has a particularly narrow host range. It could be transmitted by aphids Aphis gossypii and Myzus persicae, but with low efficacy compared to a typical CMV isolate. Phylogenetic analysis of the nucleotide sequences of the CMV-Rom genomic RNAs shows that this isolate does not belong to any of the previously described CMV subgroups, IA, IB, II or III.

Sequence analysis and genetic diversity of five new Indian isolates of cucumber mosaic virus.

Cucumber mosaic virus (CMV) is an important virus since it causes severe losses to many economically important crops worldwide. Five new isolates of CMV were isolated from naturally infected Hippeastrum hybridum, Dahlia pinnata, Hemerocallis fulva, Acorus calamus and Typhonium trilobatum plants, all exhibiting severe leaf mosaic symptoms. For molecular identification and sequence analyses, the complete coat protein (CP) gene of these isolates was amplified by RT-PCR. The resulting amplicons were cloned and sequenced and isolates were designated as HH (KP698590), DP (JF682239), HF (KP698589), AC (KP698588) and TT (JX570732). For study of genetic diversity among these isolates, the sequence data were analysed by BLASTn, multiple alignment and generating phylogenetic trees along with the respective sequences of other CMV isolates available in GenBank Database were done. The isolates under study showed 82-99% sequence diversity among them at nucleotide and amino acid levels; however they showed close relationships with CMV isolates of subgroup IB. In alignment analysis of amino acid sequences of HH and AC isolates, we have found fifteen and twelve unique substitutions, compared to HF, DP and TT isolates, suggesting the cause of high genetic diversity.

Genotyping of Cucumber mosaic virus isolates in western New York State during epidemic years: Characterization of an emergent plant virus population.

In the early 2000s an epidemic of cucumber mosaic virus (CMV) spread within the Midwestern and Eastern US affecting snap and dry bean (Phaseolus vulgaris L.) cultivation. Fifty one CMV isolates from this period were partially characterized from varied hosts by sequencing a section from each of the three genomic RNAs. Aside from one subgroup II strain from pepper, all isolates, including those from snap bean, fell within the IA subgroup. The nucleotide sequence diversity of virus populations sampled at multiple sites and at different years was significantly higher than that of a population from single site in a single year, although in general the number of polymorphisms was low (<11%). Complementary DNA (cDNA) clones of Bn57, a representative isolate from snap bean, were engineered for the production of infectious in vitro RNA transcripts initiated from a T7 promoter. Infections from these cDNAs resulted in symptoms consistent with those of the original field isolate, indicating that a satellite RNA is not involved in symptom expression in snap bean. These infectious clones were used to assess symptom determinants and the effects of virus infection on plant growth. Inoculations with pseudorecombinants derived from Bn57 and the non-bean infecting strain Fny confirmed RNA2 as a specific determinant for snap bean infection. Bn57, along with almost all isolates identified in this study contained the Y631 locus in the 2a protein, a determinant for systemic infection in bean. The presence of this locus extended to all non-bean hosts except two pepper infecting isolates. Infection by Bn57 in snap bean had a significant effect on pod number and mass with a 55 and 41 percent reduction in greenhouse assays, respectively. To our knowledge Bn57 is the first CMV strain isolated from P. vulgaris to be fully sequenced and cloned, providing a useful tool for analyses of CMV-host interactions.

Molecular characterization of cucumber mosaic virus isolates infecting tomato in Hamedan and Tehran provinces of Iran.

Here we identified four isolates, MS, 3H, 50A, and 2K of cucumber mosaic virus (CMV) infecting tomato, on the basis of their non-coding intergenic region and a part of the coat protein (CP) sequence in the CMV genomic RNA3. The sequences from the four isolates were compared with other previously characterized isolates of CMV isolated from different plant species across the globe. Sequence comparisons revealed that the two CMV isolates from Hamedan province (MS and 3H) had the highest sequence identity with CMV-G10 (98%), which was previously reported as a severe Hellenic tomato isolate of CMV, while the CMV isolates from Tehran province, including CMV-2K (isolated from Karaj region) and CMV-50A (isolated from Varamin region), had the highest sequence identity with that of CMV-ALF (99%). Phylogenetic analysis of the nucleotide sequences showed that CMV-MS and CMV-3H belong to group IB, while CMV-2K and CMV-50A belong to group IA. This is the first report on the molecular characterization of novel isolates of CMV infecting tomato plants in Iran.

Fixation of emerging interviral recombinants in cucumber mosaic virus populations.

Interstrain recombinants were observed in the progenies of the Cucumber mosaic virus (CMV) reassortant L(1)L(2)F(3) containing RNAs 1 and 2 from LS-CMV and RNA 3 from Fny-CMV. We characterized these recombinants, and we found that their fixation was controlled by the nature of the replicating RNAs 1 and 2. We demonstrate that the 2b gene partially affects this fixation process, but only in the context of homologous RNAs 1 and 2.

Cucumber mosaic virus.

Cucumber mosaic virus (CMV) is an important virus because of its agricultural impact in the Mediterranean Basin and worldwide, and also as a model for understanding plant-virus interactions. This review focuses on those areas where most progress has been made over the past decade in our understanding of CMV. Clearly, a deep understanding of the role of the recently described CMV 2b gene in suppression of host RNA silencing and viral virulence is the most important discovery. These findings have had an impact well beyond the virus itself, as the 2b gene is an important tool in the studies of eukaryotic gene regulation. Protein 2b was shown to be involved in most of the steps of the virus cycle and to interfere with several basal host defenses. Progress has also been made concerning the mechanisms of virus replication and movement. However, only a few host proteins that interact with viral proteins have been identified, making this an area of research where major efforts are still needed. Another area where major advances have been made is CMV population genetics, where contrasting results were obtained. On the one hand, CMV was shown to be prone to recombination and to show high genetic diversity based on sequence data of different isolates. On the other hand, populations did not exhibit high genetic variability either within plants, or even in a field and the nearby wild plants. The situation was partially clarified with the finding that severe bottlenecks occur during both virus movement within a plant and transmission between plants. Finally, novel studies were undertaken to elucidate mechanisms leading to selection in virus population, according to the host or its environment, opening a new research area in plant-virus coevolution.

Population genetics of cucumber mosaic virus infecting medicinal, aromatic and ornamental plants from northern Italy.

The genetic variation and evolution of cucumber mosaic virus (CMV) from aromatic, medicinal and ornamental plants in northern Italy was studied by sequence analysis of the movement protein gene and comparison with equivalent sequences of isolates from other countries. Comparison of nonsynonymous and synonymous substitutions suggested that 30% of amino acid sites were under negative selection and only one was under positive selection. Phylogenetic, nucleotide diversity and genetic differentiation analyses suggested that long-distance migration plays a role in the evolution and determination of the genetic structure and diversity of CMV in northern Italy and other areas.

Cucumber mosaic virus: viral genes as virulence determinants.

TAXONOMIC RELATIONSHIPS: Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus in the family Bromoviridae, which also encompasses the Peanut stunt virus (PSV) and the Tomato aspermy virus (TAV). Nucleotide sequence similarity among these three cucumoviruses is 60%-65%. CMV strains are divided into three subgroups, IA, IB and II, based on the sequence of the 5' untranslated region of the genomic RNA 3. Overall nucleotide sequence similarity among CMV strains is approximately 70%-98%. GEOGRAPHICAL DISTRIBUTION, HOST RANGE AND SYMPTOMATOLOGY: CMV is distributed worldwide, primarily in temperate to tropical climate zones. CMV infects more than 1200 species of 100 plant families, including monocot and dicot plants. Symptoms caused by CMV infection vary with the host species and/or CMV strain, and include mosaic, stunt, chlorosis, dwarfing, leaf malformation and systemic necrosis. CMV disease is spread primarily by aphid transmission in a nonpersistent manner. PHYSICAL PROPERTIES: In tobacco sap, the thermal inactivation point of the viral infectivity is approximately 70 °C (10 min), the dilution end-point is approximately 10(-4) and viral infectivity is lost after a few days of exposure to 20 °C. Viral infectivity can be retained in freeze-dried tissues and in the form of virions purified using 5 mm sodium borate, 0.5 mm ethylenediaminetetraacetic acid and 50% glycerol (pH 9.0) at -20 °C. CMV particles are isometric, approximately 28-30 nm in diameter and are composed of 180 capsid subunits arranged in pentamer-hexamer clusters with T= 3 symmetry. The sedimentation coefficient (s(20) ,(w) ) is c. 98 S and the particle weight is (5.8-6.7) × 10(6) Da. The virions contain 18% RNA. The RNA-protein interactions that stabilize the CMV virions are readily disrupted by sodium dodecylsulphate or neutral chloride salts. GENOMIC PROPERTIES: The genomic RNAs are single-stranded messenger sense RNAs with 5' cap and 3' tRNA-like structures containing at least five open reading frames. The viral RNA consists of three genomic RNAs, RNA 1 (c. 3.3 kb), RNA 2 (c. 3.0 kb) and RNA 3 (c. 2.2 kb), and two subgenomic RNAs, RNA 4 (c. 1.0 kb) and RNA 4A (c. 0.7 kb). The 3' untranslated regions are conserved across all viral RNAs. CMV is often accompanied by satellite, noncoding, small, linear RNA that is nonhomologous to the helper CMV.

Multiplex RT-PCR detection of Cucumber mosaic virus subgroups and Tobamoviruses infecting Tomato using 18S rRNA as an internal control.

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), using 18S rRNA as an internal control. Species- and subgroups-specific primers designed to differentiate CMV subgroups I and II, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum). Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parameters were optimized and a multiplex RT-PCR procedure was established. Six fragments of 704, 593, 512, 421, 385, 255 bp, specific to CMV subgroup II, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were simultaneously amplified. The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). This method was successfully used for field detection. Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found. The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.

Molecular variability of five Cucumber mosaic virus isolates from China.

Cucumber mosaic virus (CMV) isolates are currently divided into two main groups, I and II according to their genomic sequences. The group I is further divided into two subgroups IA and IB. We performed a phylogenetic analysis of the genome regions containing 1a, 2a, 2b, coat protein (CP), and movement protein (MP) genes of 5 CMV isolates from China and other 28 CMV isolates available in the GenBank. The results indicated that CMV isolates could be genetically divided into three groups I, II, and III according to the genes encoding MP, CP, 1a, and 2a proteins and to the 2 groups according to the gene 2b. Group I could be further divided into two subgroups (IA and IB) according to the genes encoding CP, MP, 2a, and 2b proteins and to the three subgroups (IA, IB, and IC) according to the gene encoding 1a protein. Four of 5 examined Chinese CMV isolates belonged to the subgroup IB, while the remaining isolate was a natural inter-subgroup reassortant. We found that the 2b gene of CMV was under positive selection, while the other genes were under negative selection. No evidence of the selection associated with a host adaptation or geographic distribution was found.

The 3' untranslated region of cucumber mosaic virus (CMV) subgroup II RNA3 arose by interspecific recombination between CMV and tomato aspermy virus.

Recombination in single-stranded RNA viruses is one of the principal mechanisms responsible for their evolution. Here we show, using a variety of different methods, that the 3' untranslated region (3'UTR) of subgroup II strains of cucumber mosaic virus [CMV(II)] is related more closely to that of tomato aspermy virus (TAV) than to those of CMV(I) strains. These results suggest that the CMV(II) 3'UTR arose by interspecific CMV/TAV recombination. The putative crossover is close to the 5' end of the 3'UTR, at a recombination hot spot previously observed in short time-frame experiments. The CMV(II) strains show divergence from TAV at specific points along the 3'UTR that most probably indicate adaptive changes due to natural selection. Thus, for the large majority of CMV(II) strains examined, the 3'UTR has two discrete regions, W (nt 1902-1971) and Y (nt 2126-2184), that are more similar to the corresponding regions of TAV than to those of CMV(I) strains.

Complete nucleotide sequence of a Polish strain of Peanut stunt virus (PSV-P) that is related to but not a typical member of subgroup I.

Peanut stunt virus (PSV) is a common legume pathogen present worldwide. It is also infectious for many other plants including peanut and some vegetables. Viruses of this species are classified at present into three subgroups based on their serology and nucleotide homology. Some of them may also carry an additional subviral element - satellite RNA. Analysis of the full genome sequence of a Polish strain - PSV-P - associated with satRNA was performed and showed that it may be classified as a derivative of the subgroup I sharing 83.9-87.9% nucleotide homology with other members of this subgroup. A comparative study of sequenced PSV strains indicates that PSV-P shows the highest identity level with PSV-ER or PSV-J depending on the region used for analysis. Phylogenetic analyses, on the other hand, have revealed that PSV-P is related to representatives of the subgroup I to the same degree, with the exception of the coat protein coding sequence where PSV-P is clustered together with PSV-ER.

A simple technique for separation of Cowpea chlorotic mottle virus from Cucumber mosaic virus in natural mixed infections.

A simple technique was developed to separate Cowpea chlorotic mottle virus (CCMV) from Cucumber mosaic virus (CMV) in natural mixed infections. Sap from cowpea leaves infected naturally with a mixture of CCMV and CMV was inoculated mechanically on the first tri-foliolate leaf of cowpea seedlings. Both inoculated and non-inoculated upper leaves were sampled 3 or 8 days post-inoculation and tested by reverse transcription polymerase chain reaction (RT-PCR) using primers specific to CCMV and CMV. RT-PCR analysis showed the presence of only CCMV in the inoculated leaf and both viruses in the non-inoculated systemically infected upper leaves. Total RNA from the inoculated leaves positive to CCMV only was further confirmed upon re-inoculation to cowpea seedlings. Typical CCMV symptoms were produced within 1 week and RT-PCR analysis showed only the presence of CCMV in both inoculated and non-inoculated systemically infected upper leaves. Systemically infected upper leaves of the same plants were used for CCMV purification. RT-PCR analysis of the purified virion and RNA extracted from the virion further confirmed the absence of CMV contamination. To our knowledge, this is the first report of a method separating CCMV directly from mixed infections with CMV in cowpea.

Nucleotide sequence analysis of peanut stunt virus Rp strain suggests the role of homologous recombination in cucumovirus evolution.

The complete nucleotide (nt) sequence of peanut stunt virus Robinia strain (PSV-Rp) was determined and compared to other PSV strains and to representatives of the genus Cucumovirus. Nt sequence comparison showed 74.1-84.6% identity with the known PSV strains. Phylogenetic analysis revealed the different origin of the two genes encoded by RNA3. While the 3a gene clustered with PSV-W, the coat protein gene clustered with PSV-Mi. Recombination breakpoint analysis revealed two recombination points on RNA3. Based on these results, the establishment of a fourth PSV subgroup is proposed. This work revealed that homologous recombination occurred during the evolution of PSV.