Family Closteroviridae Research Articles

Selective pressure, putative recombination events and evolutionary relationships among members of the family Closteroviridae: A proposal for a new classification

Molecular evolution of viruses is driven by both positive selection and recombination. In an effort to determine putative recombination events in the entire genome of members of the family Closteroviridae comprising 54 accessions retrieved from the international databases, a detail study was carried out by using RDP algorithm version 3.31β. Only isolates of Citrus tristeza virus and Grapevine leafroll-associated virus 3 were potential recombinants. None of members of Crinivirus genus was possible recombinant, but in turn, they were under positive selection that might be correlated with vector and/or host interactions. In contrast, Citrus tristeza virus isolates were under purifying selection as well as one-third of isolates of Grapevine leafroll-associated virus 3. The evolutionary history of all members of the Closteroviridae showed that they split into three distinct clusters corresponding to the three genera constituting this family. Moreover, the inferred phylogeny reshuffled the existing classification actually adopted by the International Committee on Taxonomy of Viruses. In this regard, each genus should be constituted by two distinct subgroups.

A putative new ampelovirus associated with grapevine leafroll disease

A putative new ampelovirus was detected in Vitis vinifera cv. Carnelian showing mild leafroll symptoms and molecularly characterized. The complete genome consisted of 13,625 nt and had a structure similar to that of members of subgroup I in the genus Ampelovirus (fam. Closteroviridae). In-depth analyses showed that the virus from cv. Carnelian is the most distinct member of the “GLRaV-4 lineage” of ampeloviruses, which comprises GLRaV-4, -5, -6, -9, and the recently characterized GLRaV-Pr, and GLRaV-De. This virus appears to be a new member of the family Closteroviridae, for which the provisional name grapevine leafroll-associated Carnelian virus is proposed.

First Report on the Occurrence of Grapevine leafroll-associated virus 5 in Chilean Grapevines

Grapevine leafroll is one of the most widespread and economically damaging viral diseases of grapevines. At least eight distinct Grapevine leafroll-associated viruses (GLRaVs), all members of the Closteroviridae family, have been associated with this disease (4). GLRaV-5 was recently reported in vineyards from Argentina (2). To determine if GLRaV-5 was present in Chilean grapevines, in addition to the previously reported GLRaV-1, -2, -3, -4, -7, and -9 (1), 45 dormant cane samples from 12 different cultivars were collected from different geographic regions of Chile and screened by reverse transcription-PCR. Two of the forty-five samples (cvs. Sauvignon Blanc and Superior) collected from the III (700 km north of Santiago) and VI (150 km south of Santiago) regions of Chile, respectively, were found to be infected with GLRaV-5 using two different pairs of virus-specific primers. The first pair of primers, LR5-1F: 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R: 5'-CAGACTTCACCTCCTGTTAC-3' (3), was used to amplify a 690-bp fragment corresponding to a partial region of the coat protein gene. The sequences obtained from the two positive samples (GenBank Accession Nos. HM214148 and HM214149) shared 97 and 94% of nucleotide identities, respectively, with the corresponding fragment of a reference GLRaV-5 isolate (GenBank Accession No. EU815935). Both samples shared 99% of amino acid identity with the same reference isolate. A second pair of primers, LR5upF: 5'-CTCTGCTTTTCTGCTGGCA-3' and LR5doR: 5'-TATCTTTTATCTCCCGATAAACGAG-3' (4) that amplified a 160-bp fragment of the HSP70h gene was also used. The positive Chilean samples (GenBank Accession Nos. HM214150 and HM214151) shared in both cases 98% nucleotide and 98% amino acid identities with the corresponding fragment of a reference GLRaV-5 isolate (Accession No. AF039552). The two GLRaV-5-positive plants were additionally infected with other viruses previously reported in Chile (1). The cv. Sauvignon Blanc sample was also infected with GLRaV-2, Grapevine fleck virus, and Grapevine rupestris stem pitting-associated virus. The cv. Superior sample was also infected with GLRaV-3, GLRaV-4, and Grapevine virus A.

First report of Pineapple mealybug wilt associated virus‐3 infecting pineapple in Cuba

Pineapple (Ananas comosus) is a common crop in tropical and subtropical areas of the world. Crop yields are seriously affected by mealybug wilt of pineapple (MWP), a viral disease with mealybugs (Dysmicoccus spp.) as vectors (Sether et al., 2005). Viruses associated with MWP are members of the genus Ampelovirus, family Closteroviridae. In Hawaii, Pineapple mealybug wilt-associated virus- (PMWaV-1) infection has been correlated with 5 to 15% of ratoon crop yield reduction and losses of up to 30% of production due to premature or asynchronous fruit ripeness. However, in that region, the most widespread virus species is PMWaV-2, which causes up to 100% fruit loss (Sether & Hu, 2002). Conversely, PMWaV-2 is uncommon in Australia, a country where MWP also causes major reduction of pineapple fruit yield. In Australia, MWP symptoms are strongly correlated with infections by PMWaV-3 alone or by both PMWaV-1 and -3 (Gambley et al., 2008). Due to the high nucleotide identity and the conserved genome organization between PMWaV-1 and PMWaV-3, it is considered that these two viruses have similar deleterious effects on either growth rate or pineapple fruit yield in Hawaii (Sether et al., 2005, 2009). Currently, there are 5,310 ha of pineapple orchards in Cuba, with annual fruit production that reached 28,908 tonnes in 2009. MWP disease is an economic problem for pineapple production in the island, causing up to 40% crop losses (Anonymous, 1989). PMWaV-2 was first detected in a diseased pineapple plant from Ciego de Avila in 1998, with molecular characterisation further provided by Borroto-Fernandez et al., (2007). During a survey for PMWaVs in 2009, thirty pineapple plants showing typical symptoms of MWP (foliar reddening, leaves with tips curved down and dieback) were collected in the Island of Youth, western region of the country (Fig. 1). Total RNA was extracted using the TRIzol LS Reagent kit (Invitrogen, Scotland, UK). RT-PCR assays for PMWaV-1, PMWaV-2 and PMWaV-3 detection were performed using the 225/226, 223/224 and 263/264 primer pairs, respectively; these amplify fragments corresponding to the HSP70h protein ORF of each viral species (Sether et al., 2005). Fragments of expected size for PMWaV-2 (c. 610 bp) and PMWaV-3 (c. 490 bp) were simultaneously amplified from seventeen plants with appropriate symptoms. Amplicons corresponding to PMWaV-1 were not obtained. DNA bands were purified, ligated to pGEM®-T Easy vector (Promega, Madison, USA) and two individual clones derived from each virus per infected plant were sequenced. PMWaV-3 derived amplicons shared ≥ 98% sequence identity with each other, and a representative sequence was deposited in GenBank (PMWaV-3 Cu, Accession No. GU563497). Sequence comparisons showed the closest nucleotide identity (98%) to PMWaV-3 from Hawaii (DQ399259). On the other hand, a representative sequence of PMWaV-2 Cu amplicons (FN825676) showed the closest nucleotide identity (99%) to PMWaV-2 from Taiwan (EU769115.1) and Thailand (EU016675.1). Phylogenetic analysis based on the PMWaV-3 amplicon sequence of Cuban and reference closteroviruses, grouped PMWaV-3 Cu and PMWaV-3 isolates from Hawaii and Thailand within the same phylogenetic cluster (Fig. 2). The phylogenetic tree also supported the sequence divergence of the cluster composed by PMWaV-3 and PMWaV-1 from that of PMWaV-2Hw, GLRaV-1 and GLRaV-3 (type member of the genus Ampelovirus) as previously observed (Sether et al., 2009). Gathering all these data, this is the first report of the presence of the PMWaV-3 in Cuban pineapple fields and in the Caribbean basin. Noteworthy is that pineapple plants affected by MWP were infected by both PMWaV-3 and PMWaV-2 which suggests that a complex of ampeloviruses may be widespread in Cuban pineapple fields. Results support the need to implement certification procedures for pineapple propagation materials to reduce the economic impact of MWP disease on pineapple crops in Cuba.

Resistance to Tomato chlorosis virus in Wild Tomato Species that Impair Virus Accumulation and Disease Symptom Expression

Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) is an emerging threat to tomato crops worldwide. Although symptoms on fruits are not obvious, yield losses occur through decreased fruit size and number. Control of ToCV epidemics is difficult because the virus is transmitted by several whitefly vector species and its relatively wide host range facilitates establishment in local wild reservoirs. Therefore, breeding for ToCV resistance offers the best control alternative. However, no sources for resistance are available thus far. Here, a screen of tomatoes and wild species relatives was performed in search of ToCV resistance. Two sources of resistance to ToCV were identified in this work, lines '802-11-1' and '821-13-1', each derived by two self-pollinations from ToCV asymptomatic plants of the population 'IAC CN RT' (derived from an interspecific hybrid Solanum lycopersicum x S. peruvianum accession LA0444) and accession LA1028 (S. chmielewskii), respectively. The resistance was expressed by impairing virus accumulation and disease symptom expression, both under natural infection and after challenging with ToCV in controlled inoculations. Genetic control of resistance to ToCV infection in '821-13-1' was conferred by a major locus with mainly additive effects but also partial dominance for higher susceptibility. Also, an additive x dominance epistatic interaction with at least one additional gene was evident.

Host Range and Complete Genome Sequence of Cucurbit chlorotic yellows virus, a New Member of the Genus Crinivirus

Cucurbit chlorotic yellows virus (CCYV) causes chlorotic yellows on cucumber (Cucumis sativus) and melon (Cucumis melo) and is transmitted by Bemisia tabaci biotype B and Q whiteflies. To characterize the host range of CCYV, 21 cucurbitaceous and 12 other plant species were inoculated using whitefly vectors. All tested Cucumis spp. except Cucumis anguria and Cucumis zeyheri were systemically infected with CCYV, although infection rates varied among species. Citrullus lanatus, Cucurbita pepo, and Luffa cylindrica were susceptible to CCYV; however, the infection rates were low and symptoms were unclear. In addition to the cucurbitaceous plants, Beta vulgaris, Chenopodium amaranticolor, Chenopodium quinoa, Spinacia oleracea, Lactuca sativa, Datura stramonium, and Nicotiana benthamiana were also systemically infected by CCYV. Complete RNA1 and RNA2 were reverse-transcribed, cloned, and sequenced. CCYV RNA1 was found to be 8,607 nucleotides (nt) long and contained four open reading frames (ORFs). The first ORF spanned methyltransferase and RNA helicase domains followed by an RNA-dependent RNA polymerase domain, presumably translated by a +1 ribosomal frameshift. CCYV RNA2 was found to be 8,041 nt long and contained eight ORFs, including the hallmark gene array of the family Closteroviridae. Phylogenetic analysis demonstrated that CCYV was genetically close to Lettuce chlorosis virus, Bean yellow disorder virus, and Cucurbit yellow stunting disorder virus. Amino acid sequence similarities of representative proteins with these viruses indicated that CCYV should be classified as a distinct crinivirus species.

First Report on the Susceptibility of Sweet Pepper Crops to Tomato chlorosis virus in Brazil.

In June of 2009, sweet pepper (Capsicum annuum cvs. Elisa and Prador) plants exhibiting interveinal chlorosis, some necrosis, and mild upward leaf curling on the intermediate leaves were found in three protected crops in the municipality of São Miguel Arcanjo, São Paulo state, Brazil. Incidence of symptomatic plants varied from 70 to 100%. Abundant whitefly adults were seen in all crops. Initially, total DNA was separately extracted from seven symptomatic plants and submitted to a PCR reaction using the universal primer pairs PAL1v1978/PAR1c496 and PBL1v2040/PCRc1 for begomovirus (3). The results were negative. The same samples were also analyzed for infection with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). Total RNA was extracted separately from leaves of each symptomatic plant and used for one-step reverse transcription (RT)-PCR using the HS-11/HS-12 primer pair, which amplifies a fragment of 587 bp from the highly conserved region of the heat shock protein (HSP-70) homolog gene reported for TICV and ToCV (1). The RT-PCR product was subsequently tested by nested-PCR for single detection of TICV and ToCV using primer pairs TIC-3/TIC-4 and ToC-5/ToC-6, respectively (1). Only one fragment of approximately 463 bp was amplified from the five plants with the primer pair specific for ToCV. No amplification was obtained with the primers specific for TICV. Four purified amplicons of 463 bp were directly sequenced in both directions. Sequence comparisons of the 419-bp consensus sequence, encompassing nucleotides 750 and 1,167 of the HSP-70 homolog gene, revealed 98% identity with the reported sequences of tomato infecting isolates of ToCV from Brazil (GenBank Accession No. EU868927) and the United States (GenBank Accession No. AY903448). Virus-free adults of Bemisia tabaci biotype B were confined on symptomatic pepper leaves for a 48-h acquisition access period. Twenty adults were transferred to one plant of sweet pepper cv. Magda for a 24-h inoculation access period. The sweet pepper plant exhibited the original symptoms on the leaves 67 days after inoculation under greenhouse conditions. Infection by ToCV was confirmed by RT-PCR. The susceptibility of sweet pepper plants to ToCV was previously reported in Spain (2), whereas in the United States, this species was experimentally found as nonhost for this virus (4). Further studies are needed to better understand the variable susceptibility of sweet pepper to ToCV.

Detection of three closteroviruses in stone fruit trees by multiplex assays.

Simultaneous detection of Plum bark necrosis stem pitting-associated virus (PBNSPaV), Little cherry virus 1 (LChV-1) and Little cherry virus 2 (LChV-2) was achieved with a quadruplex one-step RT-PCR, and a hybridization test with a multi-riboprobe, apparently representing the first successful attempt of concomitant identification of viruses of the family Closteroviridae in stone fruit trees. Quadruplex RT-PCR detected double (natural and artificial) and triple (artificial) infections in all samples tested, amplifying also a plant mRNA as an internal control. Standard and quadruplex RT-PCR were comparable in terms of detection limits and specificity. Molecular hybridization with a multi-riboprobe (‘clotri’) containing partial sequences of PBNSPaV, LChV-1 and LChV-2, allowed the detection of each of the three viruses in Prunus hosts. The sensitivity of single riboprobes and “clotri” was comparable.

Yellowing disease of tomato caused by Tomato chlorosis virus newly recognized in Japan

In 2008, virus-like symptoms of yellowing, interveinal chlorosis, leaf curling and necrotic fleck were observed on greenhouse-tomato plants (Solanum esculentum) in Tochigi Prefecture. The symptomatology and the characteristics of the causal agent such as whitefly transmissibility and particle morphology are similar to those for Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV), species of the genus Crinivirus in the family Closteroviridae. Sequencing of reverse transcription-polymerase chain reaction (RT-PCR) products using the degenerate primers for heat shock protein 70 homolog genes of closteroviruses and specific primers for TICV and ToCV indicated that the virus was ToCV, that has not previously been reported in Japan.

The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome.

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3' half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla, a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3' nontranslated region (NTR) (nucleotides 18 394-19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3' NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease.

First Report of Grapevine leafroll-associated virus 4 and 5 in Grapevines in China.

Grapevine leafroll disease (GLD) is one of the most important diseases of grapevines worldwide. Nine serologically distinct viruses in the Closteroviridae family are associated with GLD. Previous studies reported that Grapevine leafroll-associated virus (GLRaV) -1, -2, -3, and -7 were present in grapevines in China with GLRaV-1 and -3 the predominant viruses associated with GLD (1). To confirm if GLRaV-4 and -5 were also present in China, 36 dormant canes from individual vines of 29 cultivars that showed GLD leaf symptoms during the growing season were collected from the germplasm collection plot of the Research Institute of Pomology, Chinese Academy of Agricultural Sciences. Total RNA extracted by a silica capture protocol (2) from phloem-enriched bark of 36 samples was tested separately for GLRaV-4 and -5 by reverse transcription (RT)-PCR using virus-specific primers. Primers LR4F (5'-ACATTCTCCACCTTGTGCTTTT-3') and LR4R (5'-CATACAAGCGAGTGCAATTAC-3') (4) were used to amplify a 321-bp fragment corresponding to a partial region of the HSP70 gene from GLRaV-4. One sample from cv. Autumn Royal was infected by GLRaV-4. The amplicon was cloned and a single clone was sequenced (GenBank Accession No. GQ246624) that showed 99% nucleotide identity with a corresponding region of a GLRaV-4 isolate from the United States (Accession No. AF039553). Since antiserum specific to GLRaV-4 was unavailable, a second pair of primers, LR4CP-F (5'-GGTGTCCAGCGCTTCCAA-3') and LR4CP-R (5'-GCCAGAGAAGCATCGTAA-3'), was designed on the basis of the sequence of GLRaV-4 from Chile (Accession No. EU746620) that amplified a 300-bp fragment specific to the coat protein gene of GLRaV-4. The amplicon was cloned and a single sequence (Accession No. GQ479041) was compared with a corresponding nucleotide sequence of GLRaV-4 from Chile (Accession No. EU746621) showing 99% identity. A sample from cv. Malaga Rose was positive when tested by ELISA with antibodies specific to GLRaV-5 (Neogen Europe, Ltd. Scotland, UK) and this was confirmed by amplification of a 690-bp fragment corresponding to the GLRaV-5 coat protein gene using virus-specific primers LR5F (5'-CCCGTGATACAAGGTAGGACA-3') and LR5R (5'-CAGACTTCACCTCCTGTTAC-3') (3). The amplicon was cloned and a single clone was sequenced (Accession No. GQ246625) that showed 95% nucleotide identity with the CP gene sequence of GLRaV-5 from Argentina (Accession No. EU815935). To our knowledge, this is the first report of GLRaV-4 and -5 in grapevines in China. Confirmation of these viruses in China is very important for producing virus-free plants and this information also will be helpful in developing a multiplex RT-PCR assay to simultaneously detect multiple GLRaVs and helpful with studies on the molecular variability of these viruses.

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus ( Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.

First Report of Tomato chlorosis virus in Tomato in Costa Rica

In early 2007, severe yellowing and chlorosis symptoms were observed in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants in Costa Rica. Symptoms resembled those of the genus Crinivirus (family Closteroviridae), and large populations of whiteflies, including the greenhouse whitefly Trialeurodes vaporariorum (Westwood), were observed in the fields and on symptomatic plants. Total RNA was extracted from silica gel-dried tomato leaf tissue of 47 representative samples (all were from symptomatic plants) using TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed separately with each of the four primer sets with the Titan One-Tube RT-PCR Kit (Roche Diagnostics Corp., Chicago IL). Specific primers used for the detection of the criniviruses, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), were primer pair ToCV-p22-F (5'-ATGGATCTCACTGGTTGCTTGC-3') and ToCV-p22-R (5'-TTATATATCACTCCCAAAGAAA-3') specific for the p22 gene of ToCV RNA1 (1), primer pair ToCVCPmF (5'-TCTGGCAGTACCCGTTCGTGA-3') and ToCVCPmR (5'-TACCGGCAGTCGTCCCATACC-3') designed to be specific for the ToCV CPm gene of ToCV RNA2 (GenBank Accession No. AY903448) (2), primer pair ToCVHSP70F (5'-GGCGGTACTTTCGACACTTCTT-3') and ToCVHSP70R (5'-ATTAACGCGCAAAACCATCTG-3') designed to be specific for the Hsp70 gene of RNA2 of ToCV (GenBank Accession No. EU284744) (1), and primer pair TICV-CP-F and TICV-CP-R specific for the coat protein gene of TICV (1). Amplified DNA fragments (582 bp) were obtained from nine samples, four from the greenhouse and five from the open field, with the ToCV-p22 specific primers and were cloned into the pCRII TOPO cloning vector (Invitrogen, Carlsbad, CA). Nucleotide sequence analysis of all purified RT-PCR products verified their identity as ToCV, sharing 99.5 to 100% sequence identity among themselves and 96% to 98% sequence identity with previously reported ToCV p22 sequences from Florida (Accession No. AY903447), Spain (Accession No. DQ983480), and Greece (Accession No. EU284745). The presence of ToCV in the samples was confirmed by additional amplification and sequence analysis of the CPm (449-bp fragment) and Hsp70 (420-bp fragment) genes of ToCV RNA2 and sharing 98 to 99% sequence homology to Accession Nos. AY903448 and EU284774, respectively. One representative sequence of the p22 gene of the Costa Rican isolate was deposited at GenBank (Accession No. FJ809714). No PCR products were obtained using either the TICV-specific primers nor from healthy tomato tissue. The ToCV-positive samples were collected from a region in the Central Valley around Cartago, Costa Rica. To our knowledge, this is the first report of ToCV in Costa Rica. The economic impact on tomato has not yet been determined. Studies are underway to determine the incidence of ToCV in Costa Rica field-grown and greenhouse tomatoes.

Two Crinivirus-specific proteins of Lettuce infectious yellows virus (LIYV), P26 and P9, are self-interacting

Interactions of Lettuce infectious yellows virus (LIYV)-encoded proteins were tested by yeast-two-hybrid (Y2H) assays. LIYV-encoded P34, Hsp70h, P59, CP, CPm, and P26 were tested in all possible pairwise combinations. Interaction was detected only for the P26–P26 combination. P26 self-interaction domains were mapped using a series of N- and C-terminal truncations. Orthologous P26 proteins from the criniviruses Beet pseudoyellows virus (BPYV), Cucurbit yellow stunting disorder virus (CYSDV), and Lettuce chlorosis virus (LCV) were also tested, and each exhibited strong self-interaction but no interaction with orthologous proteins. Two small putative proteins encoded by LIYV RNA2, P5 and P9, were also tested for interactions with the six aforementioned LIYV proteins and each other. No interactions were detected for P5, but P9–P9 self-interaction was detected. P26- and P9-encoding genes are present in all described members of the genus Crinivirus, but are not present in other members of the family Closteroviridae. LIYV P26 has previously been demonstrated to induce a unique LIYV cytopathology, plasmalemma deposits (PLDs), but no role is yet known for P9.

Molecular characterization of a closterovirus from carrot and its identification as a German isolate of Carrot yellow leaf virus

A high-molecular-weight dsRNA (approximately 15 kbp) was isolated from chlorotic leaves of a carrot plant and used for determining the entire nucleotide sequence of a closterovirus. The complete genome of this carrot closterovirus (CCV) was 16.4 kb in length and contained ten open reading frames (ORFs). The genome organization of CCV resembled that of beet yellow stunt virus, but ORF2 and ORF3 were in a reversed order. Based on Hsp70h sequences, CCV is most closely related to carnation necrotic fleck virus and mint virus 1, two viruses of the genus Closterovirus (family Closteroviridae). The major coat protein gene of CCV was expressed in Escherichia coli for raising an antiserum. This permitted routine detection of CYLV by DAS-ELISA and immunoelectron microscopy and was used for demonstrating the bipolar nature of the CCV virion. Moreover, the antiserum gave a Western blot reaction with a reference sample of a Carrot yellow leaf virus (CYLV) isolate from the Netherlands, suggesting that CCV is a German isolate of CYLV.

Synergistic interaction between the Potyvirus, Turnip mosaic virus and the Crinivirus, Lettuce infectious yellows virus in plants and protoplasts

Lettuce infectious yellows virus (LIYV), the type member of the genus Crinivirus in the family Closteroviridae, is specifically transmitted by the sweet potato whitefly ( Bemisia tabaci) in a semipersistent manner. LIYV infections result in a low virus titer in plants and protoplasts, impeding reverse genetic efforts to analyze LIYV gene/protein functions. We found that synergistic interactions occurred in mixed infections of LIYV and Turnip mosaic virus (TuMV) in Nicotiana benthamiana plants, and these resulted in enhanced accumulation of LIYV. Furthermore, we examined the ability of transgenic plants and protoplasts expressing only the TuMV P1/HC-Pro sequence to enhance the accumulation of LIYV. LIYV RNA and protein titers increased by as much as 8-fold in these plants and protoplasts relative to control plants. LIYV infections remained phloem-limited in P1/HC-Pro transgenic plants, suggesting that enhanced accumulation of LIYV in these plants was due primarily to increased replication efficiency, not to greater spread.

‘Kwanzan Stunting’ syndrome: Detection and molecular characterization of an Italian isolate of Little cherry virus 1

Evident stunting was observed for the first time on Prunus serrulata ‘Kwanzan’ indicator trees in Southern Italy during the indexing of two sour cherry accessions from cultivars ‘Marasca di Verona’ and ‘Spanska’. Bud break and shooting were delayed and the developing leaves remained small. During the third year many Kwanzan plants died, regardless of the indexed cultivar. Electrophoretic analysis showed the presence of dsRNA pattern in extracts of stunted Kwanzan with a similar size to that of viruses of the family Closteroviridae. An identical pattern of more abundant dsRNA bands was obtained from GF305 seedlings grafted with the same sour cherry accessions. Observations by electron microscopy revealed the presence of long flexuous virus particles in both indicators (Kwanzan and GF305), characteristic of closteroviruses. Subsequent cloning work, starting from the dsRNA extracts of cultivar Marasca di Verona grafted on GF305 indicator, yielded 7 different clones, all showing high identity to the Little cherry virus 1 genome. Full sequencing of this virus isolate (ITMAR) was then done resulting in a complete genome composed of 16,936 nt. Primers designed on the obtained sequences for RT-PCR detection confirmed the presence of Little cherry virus 1 in Kwanzan and GF305 trees, inoculated with both sour cherry cultivars. Phylogenetic analysis of the minor coat protein grouped virus isolates into two clusters: one including Italian isolates of sweet cherry, Japanese plum, peach and almond, together with German sweet cherry UW1 isolate, and a second one containing the Italian isolates of sour cherry (ITMAR and ITSPA), that were found associated with strong symptoms of ‘Kwanzan Stunting’.

The complete nucleotide sequence of the RNA2 of the crinivirus tomato infectious chlorosis virus: isolates from North America and Europe are essentially identical

The complete nucleotide sequences of the RNA2 of two isolates of Tomato infectious chlorosis virus (TICV, genus Crinivirus, family Closteroviridae) from the United States and Spain, respectively, were determined. The sequences of both isolates were found to be nearly identical. TICV RNA2 consisted of 7,914 nucleotides in both isolates and contains eight open reading frames that encompass the Closteroviridae hallmark gene array represented by a heat shock protein 70 family homologue, a protein of 59 kDa, the major coat protein, and a divergent copy of the coat protein. Phylogenetic analysis suggested that TICV is most similar to Lettuce infectious yellows virus (LIYV), the type species of the genus Crinivirus.

Genome organization and phylogenetic relationship of Pineapple mealybug wilt associated virus-3 with family Closteroviridae members

The nucleotide sequence of Pineapple mealybug wilt associated virus-3 (PMWaV-3) (Closteroviridae: Ampelovirus), spanning seven open reading frames (ORFs) and the untranslatable region of the 3' end was determined. Based on the amino acid identities with orthologous ORFs of PMWaV-1 (54%-73%) and PMWaV-2 (13%-35%), we propose PMWaV-3 is a new species in the PMWaV complex. PMWaV-3 lacks an intergenic region between ORF1b and ORF2, encodes a relatively small, 28.8 kDa, coat protein, and lacks a coat protein duplicate. Phylogenetic analyses were used to analyze seven different domains and ORFs from members of the family Closteroviridae. Two distinct clades within the recognized genus Ampelovirus were observed; one that includes PMWaV-3 and PMWaV-1 and several GLRaVs and another that includes PMWaV-2 and GLRaV-3, the type member of the genus Ampelovirus.

新規クリニウイルスによるメロン退緑黄化病（新称）の発生

In 2004, an unidentified disease causing chlorotic yellows on the leaves of melon (Cucumis melo) plants was found in Kumamoto Prefecture. A few flexuous filamentous structures (ca. 900 nm in length) were observed with transmission electron microscopy. RT-PCR and subsequent nested PCR amplified a DNA fragment, which had 75% and 73% similarities with a corresponding sequence for Cucurbit yellow stunting disorder virus and Beet pseudo-yellows virus, respectively. Both Bemisia tabaci Q-biotype and B. tabaci B-biotype (syn. B. argentifolii) acquired and transmitted the virus, and the infected melon plants developed the original symptoms of the disease. These results demonstrate that the virus is a new species in the genus Crinivirus (family Closteroviridae), and we propose the name Cucurbit chlorotic yellows virus.