Genus Ilarvirus Research Articles

Spinach latent virus Infecting Tomato in Virginia, United States.

Plants in a single field of commercial tomato (Solanum lycopersicum) of unidentified cultivars in Virginia in July, 2012, were observed showing stunting, leaf distortion, twisting and thickening, discoloration, and color streaking and ringspots on fruits. Serological tests were negative for Cucumber mosaic virus, Groundnut ringspot virus, Tomato spotted wilt virus, Tomato chlorotic spot virus, Impatiens necrotic spot virus, Tobacco mosaic virus, and Tomato bushy stunt virus (Agdia, Inc., Elkhart, IN). Using a membrane-based macroarray (3), hybridization was observed to 8 of 9 70-mer oligonucleotide probes of Spinach latent virus (SpLV; genus Ilarvirus, family Bromoviridae). To confirm the hybridization results, complementary DNA (cDNA) was synthesized using random hexamers and MMLV reverse transcriptase (Promega, Madison, WI), followed by PCR amplification using ilarvirus degenerate primers (4). Fragments of approximately 380 bp were amplified and directly sequenced (GenBank Accession KC_466090); a BLAST search showed a 99% identity to the SpLV RNA 2 reference genome (NC_003809). Primers for SpLV RNA1 (SpLVRNA1f-GGTGTCACCATGCAAACTGG, SpLVRNA1r-AGCTCTTCGTAATAGGCCTGC) and SpLV RNA3 (SpLVCPf-GAAGTCTTTCCCAGGTGAGCA, SpLVCPr-AGGTGGGCATATGGACTTGG) were designed and cDNA was amplified using the IQ supermix (Biorad, Hercules, CA) with thermocycling of 94°C for 4 min, 35× (94°C 45 s, 55°C 45 s, 72°C 45 s), and 72°C for 10 min. The resulting fragments of 538 bp for RNA1 (KC_466088) and 661 bp for RNA3 (KC_466089) showed 100% identity to reference genome sequences for SpLV (NC_003808 and NC_003810, respectively). To demonstrate virus transmissibility, Chenopodium quinoa plants were mechanically inoculated using tomato leaf material (same source described above) ground in 30 mM Na2HPO4 buffer, pH 7.0. Necrotic spots developed on the inoculated leaves 10 dpi. Younger, non-inoculated leaves showed yellow mottling and tested positive for SpLV by RT-PCR (two of two plants tested). The detection of SpLV is rarely reported, with only one record from the United States (2). Although SpLV is described as a latent virus, it has been found associated with tomato fruit symptoms in New Zealand (1). It is not known if the fruit ringspot and other symptoms on the Virginia samples were due to virus infection. Since SpLV is seed-transmissible and seed production takes place in different parts of the world, it has the potential to spread with germplasm and become more widespread in North America.

Simultaneous detection of Apple Chlorotic Leaf Spot Virus and Apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR

Apple Chlorotic Leaf Spot Virus (ACLSV; family Betaflexiviridae, genus Trichovirus) and Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) are economically important viruses of apple (Malus×domestica Borkh.). A duplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed for simultaneous detection of both ACLSV and ApMV along with an internal control (NADH dehydrogenase subunit 5 gene). The internal control was used to minimize the risk of getting false negative results. Specific primers were designed against coat protein gene of ApMV while for ACLSV & internal control previously reported primer sets were used. Firstly the uniplex RT-PCR assay was standardized for the detection of each virus independently. Then duplex RT-PCR protocol for simultaneous detection of ACLSV and ApMV was standardized by using primer pairs ACLSV-CP1F & ACLSV-CP1R and ApMV-CP1F & ApMV-CP5R, respectively, along with primer set for internal control. Robustness of the technique was further validated wherein; duplex RT-PCR was carried out to detect both viruses in crab apples, rootstocks and different popular cultivars of apple. Results reveal that the duplex RT-PCR assay has detection sensitivity as that of uniplex RT-PCR assay for respective viruses. This optimized duplex RT-PCR provides a simple, rapid, sensitive and convenient way for simultaneous detection of ACLSV & ApMV by reducing the time and cost of the supplies.

A new ilarvirus isolated from Viola × wittrockiana and its detection in pansy germoplasm by qRT-PCR

An infectious agent was transmitted mechanically from samples of Viola spp. showing white mosaic and leaf deformation to Nicotiana benthamiana. dsRNA extracted from the N. benthamiana plants migrated as four specific bands that were absent in non-inoculated plants. Sequence analysis of cDNA clones generated from the second-smallest dsRNA showed the greatest similarity to the RNA3 of prune dwarf virus (PDV) (genus Ilarvirus, family Bromoviridae). However, because of differences in molecular, biological, and serological properties between this virus isolate and PDV, a new ilarvirus species, named "Viola white distortion associated virus" (VWDaV) is proposed. Specific oligonucleotides and a TaqMan(®) probe were designed for diagnostic purposes. The possible association between the virus and the original white distortion symptoms is discussed.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn(2+) ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

The 2b protein of Asparagus virus 2 functions as an RNA silencing suppressor against systemic silencing to prove functional synteny with related cucumoviruses

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1–3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.

Genetic variability of tobacco streak virus in South India based on the analysis of coat protein gene

Tobacco streak virus (TSV), a member of the genus Ilarvirus, family Bromoviridae is an important viral pathogen in peanut and other crops in South India. Fifteen TSV isolates naturally infecting groundnut, sunflower, onion, black gram, green gram, jute, tagetes, calotropis, pumpkin, watermelon and kenaf plants were collected from fields in different regions of Andhra Pradesh, Tamil Nadu and Karnataka. Virus was identified as TSV by direct antigen coating enzyme linked immunosorbent assay using TSV antiserum. The CP gene from each isolate was amplified using TSV coat protein specific primers. About 700 bp product was amplified, cloned, sequenced and determined its length as 717 nucleotides and codes for 239 amino acids. The sequence analysis revealed that the CP gene shared 91–100% and 91–99% sequence identity with TSV at nucleotide and amino acid level, respectively. The phylogenetic relationship based on the nucleotide sequence of these isolates from different geographical regions was also analysed in this study.

Ilarviruses ofPrunusspp.: A Continued Concern for Fruit Trees

Prunus spp. are affected by a large number of viruses, causing significant economic losses through either direct or indirect damage, which results in reduced yield and fruit quality. Among these viruses, members of the genus Ilarvirus (isometric labile ringspot viruses) occupy a significant position due to their distribution worldwide. Although symptoms caused by these types of viruses were reported early in the last century, their molecular characterization was not achieved until the 1990s, much later than for other agronomically relevant viruses. This was mainly due to the characteristic liability of virus particles in tissue extracts. In addition, ilarviruses, together with Alfalfa mosaic virus, are unique among plant viruses in that they require a few molecules of the coat protein in the inoculum in order to be infectious, a phenomenon known as genome activation. Another factor that has made the study of this group of viruses difficult is that infectious clones have been obtained only for the type member of the genus, Tobacco streak virus. Four ilarviruses, Prunus necrotic ringspot virus, Prune dwarf virus, Apple mosaic virus, and American plum line pattern virus, are pathogens of the main cultivated fruit trees. As stated in the 9th Report of the International Committee on Taxonomy of Viruses, virions of this genus are "unpromising subjects for the raising of good antisera." With the advent of molecular approaches for their detection and characterization, it has been possible to get a more precise view of their prevalence and genome organization. This review updates our knowledge on the incidence, genome organization and expression, genetic diversity, modes of transmission, and diagnosis, as well as control of this peculiar group of viruses affecting fruit trees.

FIRST REPORT ON THE OCCURRENCE OF TOBACCO STREAK VIRUS IN SUNFLOWER IN IRAN

Sunflower (Helianthus annuus), an important oilseed crop grown in Iran and many other countries, is a frequent host of Tobacco streak virus (TSV, genus Ilarvirus), which infects a wide range of crops and weeds. To study the occurrence and distribution of TSV in Iran, 1,272 samples were collected from sunflower fields in Kerman, Golestan, Isfahan, Mazandaran, Qom, Azarbayejan-Gharbi, Markazi, Hamedan and Tehran provinces during 2006 to 2008. TSV was detected by DAS-ELISA in 20.9% of the samples. The characterization of two Iranian virus isolates 43R (from Tehran) and IR (from Isfahan) showed that both have isometric particles 26 to 35 nm in diameter, and coat protein (CP) subunits with a molecular weight of 30.9 kDa. In mechanical inoculation assays, the Iranian TSV isolate induced local lesions on a number of plant species of the families Chenopodiaceae and Solanaceae. Vicia faba and sunflower reacted with systemic symptoms. A fragment of 747 nt in length from the CP gene of each isolate was amplified, cloned and sequenced. BLAST analysis of the CP sequences showed that both isolates are similar to Sudanese TSV isolates and belong to the same group. This is the first report on the detection of TSV in Iran.

First Report of Apple mosaic virus in Alaska.

Apple mosaic virus (ApMV; family Bromoviridae, genus Ilarvirus) is one of the oldest and most economically important viruses of apples (Malus × domestica Borkh.) (1,3). Yield losses may vary from negligible to as much as 50%, depending on the affected cultivar. Although ApMV is found worldwide and occurs naturally in more than 65 plant species (1), it has not been reported to occur in Alaska. In July 2011, noticeably bright yellow mosaic leaves were observed on apple 'Valentine' and its rootstalk 'Ranetka' from an apple orchard in Wasilla, AK. Leaves were collected and assayed by reverse transcription (RT)-PCR using ApMV-specific primers (2) and total RNA extracted with buffer modifications to RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Briefly, 50 mg of leaf tissue was ground in liquid nitrogen and 450 μl of SE buffer (0.14 M NaCl, 2 mM KCl, 2 mM KH2PO4, 8 mM Na2HPO4·2H2O [pH 7.4], 0.05% vol/vol Tween-20, 2% wt/vol polyvinylpyrrolidone 40, 0.2% wt/vol ovalbumin, 0.5% wt/vol bovine serum albumin, and 0.05% wt/vol sodium azide) was added, and after vigorous vortexing, 80 μl of the mixture was added to 400 μl of RLT buffer supplied by the kit and then processed as directed by the manufacturer (4). Direct sequencing of the predicted ~260-bp PCR product resulted in 97 to 98% nucleotide identities to ApMV accessions in GenBank when analyzed by BLAST. To determine the distribution and incidence of infection in the Wasilla orchard, all 118 apple trees (99 cultivars) were then sampled and assayed serologically by double-antibody sandwich-ELISA with ApMV antiserum according to the manufacturer's protocol (Agdia, Inc., Elkhart, IN). Apple 'Geneva Early' and the same 'Valentine' tree and its rootstock tested positive for ApMV by ELISA and RT-PCR. Strong diagnostic ApMV symptoms were not apparent on the infected 'Geneva Early', which is typical for most commercially grown apples. No leaves were available on the 'Ranetka' rootstock of ApMV-infected 'Geneva Early' for virus indexing. An additional 21 apple trees with no symptoms from an orchard in Talkeetna, AK tested negative to ApMV by ELISA. Limited natural spread of ApMV to other plants may be by pollen and seed transmission. The most prevalent mode of transmission is from ApMV-infected rootstock and grafts. It is important to obtain new propagation plant material from certified virus tested nurseries and to avoid grafting plant material containing ApMV. To my knowledge, this is the first report of ApMV in Alaska.

Complete Nucleotide Sequence of the RNA 3 of Bacopa chlorosis virus and Production of Polyclonal Antibodies to a Recombinant Coat Protein

AbstractThe complete sequence of the RNA 3 of a virus causing chlorosis in Impatiens in Germany was determined and identified as an isolate of Bacopa chlorosis virus (BaCV, genus Ilarvirus). BaCV has previously only been reported from bacopa in the USA, but no coat protein (CP) sequence has been previously available. Both RNA 3 encoded proteins, CP and movement protein, showed highest sequence identity to Parietaria mottle virus, a subgroup 1 ilarvirus. Attempts to purify BaCV failed, so an antiserum was raised against a recombinant CP. The polyclonal antiserum so produced allowed specific detection of BaCV but showed no serological cross‐reaction with other ilarviruses and was unsuitable for immunoelectron microscopy. The host range includes many important flowering plant species, highlighting the potential threat BaCV might pose for the horticultural industry. This is the first report of BaCV occurring in Germany and outside the US.

First Report of Strawberry necrotic shock virus in China.

Strawberry necrotic shock virus (SNSV) is an economically important viral pathogen that infects Fragaria and Rubus spp. SNSV was first identified in the 1950s and early studies indicated that SNSV was a strain of Tobacco streak virus (TSV). Recently, it was shown that SNSV was a distinct virus based on molecular characterization (2). Currently, SNSV is a tentative member of the Ilarvirus genus in the Bromoviridae family. In 2008, a small sampling survey for SNSV was done in Heilongjiang Province of China, and 15 strawberry samples were collected from symptomless strawberries in a home garden that had more than 5 years of strawberry cultivation history. Total nucleic acid was extracted from strawberry leaflets by modified cetyltrimethylammoniumbromide methods (3). Reverse transcription (RT)-PCR was operated with the published primer pair CPbeg F/CPend R (2). Amplified DNA fragments with the predicted size were obtained only in one strawberry sample, which was further cloned and sequenced. The sequence (GenBank Accession No. HQ830017) was closely related and highly homologous (89.7 to 98.5% identity) to that of viral isolates (GenBank Accession Nos. AY363228-AY363242) from Fragaria and Rubus spp. Phylogenetic analysis based on nucleotide sequence of the coat protein gene was done with the neighbor-joining method of MEGA 4.0 software. The result showed that all the isolates of SNSV fell into two distinct clades. The Chinese isolate formed one small clade with Japanese isolate 1291. The isolate was also transmitted to Chenopodium quinoa by mechanical inoculation in the greenhouse, and the symptom of chlorotic mottling could be found in C. quinoa and detected by RT-PCR. To determine whether the sample was infected by other strawberry viruses, RT-PCR assays with the published primer pairs SVBVdeta/SVBVdetb, SMoVdeta/SMoVdetb, and SMYEVdeta/SMYEVdetb were also performed for detection of Strawberry vein banding virus, Strawberry mottle virus, and Strawberry mild yellow edge virus using total nucleic acid extracted from the SNSV-positive sample as a template (1). The result indicated that it had been also infected by Strawberry mild yellow edge virus, although no visible symptoms were observed. To our knowledge, this is the first report of SNSV in strawberry in China. Additional work is needed to elucidate the biological characterization and significance of the finding.

First Report of Potato yellowing virus (Genus Ilarvirus) in Solanum phureja from Ecuador.

A bacilliform virus, named Potato yellowing virus (PYV), causing chlorosis of leaves was reported in 1992 in potato (Solanum tuberosum) fields in Peru (1) and symptomless wild potatoes (S. fernandezianum) in Chile (4). PYV is reported as an alfamo-like virus (1) (family Bromoviridae) but no sequence information is available for this virus, making its taxonomic position inside the Bromoviridae uncertain (currently this family is organized into five genera: Alfamovirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus). Herein we report the presence of PYV in native potatoes (Solanum phureja) collected from Ecuador where the crop constitutes an important source of income in rural communities. Forty accessions of S. phureja collected in Ecuador in June 1986 and maintained in vitro at the International Potato Center (CIP) germplasm bank were analyzed by double-antibody sandwich (DAS)-ELISA with antiserum raised against a Peruvian isolate of PYV (1). PYV was detected in six accessions (15% of the material) corresponding to cultivars Chaucha Tomate and Chaucha Blanca (from the province of Cañar), Chaucha Negra Ojona and Chaucha Amarilla (Loja Province), and Cuica and Chaucha (Azuay Province). Mechanical inoculation of the indicator plant Physalis floridiana with leaf extracts of these six plants, a PYV isolate from Peru (1) (positive control), and an additional four plants testing negative for PYV (negative controls) induced symptoms of mosaic and leaf deformation only with the six clones from Ecuador and the PYV isolate from Peru. To further confirm the presence of the virus, we used universal PCR primers designed for the Bromoviridae (Ilar1F5: 5'-GCNGGWTGYGGDAARWCNAC-3' and Ilar1R7: 5'-AMDGGWAYYTGYTYNGTRTCACC-3') that target the helicase motif (RNA1) (3). Total RNA was extracted from 200 mg of leaf material (from potato and mechanically inoculated P. floridiana) using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's instructions and cDNA was synthesized using random hexamer primers. We obtained a reverse transcription-PCR amplified band only from samples that were DAS-ELISA positive to PYV. To identify the virus at the genus level, we cloned the PCR fragments (265 nucleotides) from four of the samples from Ecuador and the Peruvian isolate into plasmid vectors (pGEM-T Easy Vector cloning system; Promega, Madison, WI) for Sanger sequencing (Macrogen, Seoul, Korea). Phylogenetic analysis grouped PYV sequences with those of the genus Ilarvirus. Among the ilarviruses, Fragaria chiloensis latent virus (2) was the closest relative of PYV, with which it shares 77% nucleotide and 85% amino acid sequence identity. PYV isolates from Ecuador split into two different variants (91% identity) that shared 93% nucleotide and 99% amino acid sequence identity with the Peruvian isolate. Collectively, the data suggest that PYV is a distinct ilarvirus and that it is more widely spread among South American potatoes than previously suggested. The GenBank Accession Numbers for the sequences described are: HQ141053 (Loja1), HQ141054 (Azuay), HQ141055 (Cañar), and HQ141056 (Loja2) for the isolates from Ecuador and HQ141057 (PYV-Cañete) for the isolate from Peru.

First report of Tobacco streak ilarvirus infecting onion (Allium cepa)

Tobacco streak ilarvirus (TSV) is a member of the genus Ilarvirus (family Bromoviridae ) and consists of non-enveloped, isometric particles, approximately 25 to 35 nm in diameter. TSV has a wide host range, infecting more than 200 plant…

Sequence analysis of RNA 2 and RNA 3 of lilac leaf chlorosis virus: a putative new member of the genus Ilarvirus

RNA 2 and RNA 3 of lilac leaf chlorosis virus (LLCV) were sequenced and shown to be 2,762 nucleotides (nt) and 2,117 nts in length, respectively. RNA 2 encodes a putative 807-amino-acid (aa) RNA-dependent RNA polymerase associated protein with an estimated M (r) of 92.75 kDa. RNA 3 is bicistronic, with ORF1 encoding a putative movement protein (277 aa, M (r) 31.45 kDa) and ORF2 encoding the putative coat protein (221 aa, M (r) 24.37 kDa). The genome organization is similar to that typical for members of the genus Ilarvirus. Phylogenetic analyses indicate a close evolutionary relationship between LLCV, ApMV, and PNRSV.

Genomic sequences of blackberry chlorotic ringspot virus and strawberry necrotic shock virus and the phylogeny of viruses in subgroup 1 of the genus Ilarvirus

Three members of subgroup 1 of the genus Ilarvirus: blackberry chlorotic ringspot (BCRV), strawberry necrotic shock (SNSV), and tobacco streak viruses (TSV), may infect Rubus and Fragaria species. All cause symptoms similar to those previously attributed to infection by TSV alone. Although similarities exist among the genomic sequences of the three, phylogenetic analysis shows them to be distinct viruses. These viruses and Parietaria mottle virus, the other currently accepted member of subgroup 1, appear to have evolved from a common ancestral virus, share conserved motifs in the products of the genomic RNAs, and constitute a distinct subgroup within the genus.

PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae

A PCR assay was developed for the universal detection of ilarviruses using primers designed to the RNA-dependent RNA polymerase gene in RNA2. The assay detected 32 isolates of 15 definite and 2 tentative ilarvirus species using a one-step RT-PCR. The assay was more specific, and at least as sensitive as a commercial assay, and allowed direct sequencing of amplicons. No cross-reaction was observed with neither healthy plants of 15 host species nor from isolates in other genera of the Bromoviridae. A further PCR assay targeting the helicase motif of RNA1 was able to detect all species tested within the family Bromoviridae, including members of the Alfamovirus, Anulavirus, Bromovirus, Cucumovirus and Ilarvirus. The assays provide a sensitive and cost-effective way for detecting and characterising members of the Bromoviridae and can be used for quarantine and certification programmes.

Characterisation of a novel ilarvirus causing grapevine angular mosaic disease

Unusual symptoms were observed on ‘Baresana’ x ‘Baresana’ Vitis vinifera hybrid vines in the Grapevine Variety Collection of the Grapevine Institute, Athens. The affected vines showed sharp angular mosaic on leaves, along the veins and in vein angles, malformations, abortive flowers or very few berries with smaller, wrinkled and non-germinating seeds, as well as gradual decline, severe stunting and death of the vine. Serological tests on diseased vines for the presence of 13 known grapevine viruses gave negative results. An infectious agent was transmitted mechanically to several herbaceous indicator plants. Koch’s Postulates were fulfilled, and the agent, proven to be a virus, was named Grapevine angular mosaic virus (GAMV). Serological tests have been developed for the virus. The most conserved polymerase region showed significant similarity of GAMV with members of subgroup 1 of the Ilarvirus genus; however ML phylogenetic analysis could not support its clustering within this subgroup. GAMV differs serologically and in particle morphology from Grapevine line pattern virus (GLPV) a putative member of the Ilarvirus genus that infects grapevine. It is proposed that GAMV is a novel member of the Ilarvirus genus.

The nucleotide sequences of the RNA 1 and RNA 2 of asparagus virus 2 show a close relationship to citrus variegation virus

Asparagus virus-2 (AV-2) is a member of the genus Ilarvirus and infects the vegetable Asparagus officinalis L. The virus is transmitted through seed. Even though the presence of AV-2 causes reductions in vigor and productivity, plants grown from infected seed show no obvious symptoms. The virus has been reported from Europe, North America, and Japan. However, because of the ease with which the virus is seed borne, it has probably been moved to wherever seed lots of asparagus have been distributed [20]. Other ilarviruses are also seed borne and, in addition, have been shown to be transmitted naturally through pollen and the feeding activity of thrips [11]. Ilarviruses are members of the family Bromoviridae. They share traits at the molecular level with the extensively studied cucumoviruses and bromoviruses and have a particularly close relationship to the alfamovirus alfalfa mosaic virus [1]. Ilarviruses possess a tripartite genome of positive-sense ssRNAs. The RNA 1 is monocistronic, coding for a viral replicase. The RNA 2 is bicistronic in some members of the genus but monocistronic in others. The open reading frame (ORF) proximal to the 5 0 UTR codes for the viral polymerase. The second, smaller ORF found in some members of the genus is located toward the 3 0 terminus of the molecule and expressed through a subgenomic RNA that codes for a 2b protein. The initial ORF of RNA 3 codes for the movement protein (MP), and the second ORF of RNA 3 codes for the coat protein (CP). The CP gene is expressed via a subgenomic RNA 4 [7]. AV-2 is a member of subgroup 2 of the ilarviruses, having been shown to be related to the other members of this subgroup: citrus leaf rugose virus (CiLRV), citrus variegation virus (CVV), elm mottle virus (EMoV), spinach latent virus (SpLV), and tulare apple mosaic virus (TAMV), both serologically and at the molecular level [19]. The complete sequence for the RNA 3 of AV-2 has been described [17], as have fragments of sequence for the RNA 1 and RNA 2 [GenBank accessions U93601, U93602, U93603]. Here, we present the complete sequences for the RNA 1 and RNA 2 of AV-2 and discuss the close relationship between this virus and another member of subgroup 2, citrus variegation virus.

Recombination structure and genetic relatedness among members of the family Bromoviridae based on their RNAs 1 and 2 sequence analyses

In determining putative recombination events and their evolution rates in the RNAs 1 and 2 of currently the known members of the family Bromoviridae, a detailed study comprising 107 accessions retrieved from the international databases, has been carried out by using RECCO and RDP v3.31beta algorithms. These programs allowed the detection of potential recombination sites in all the five virus genera composing the family Bromoviridae with various degrees of consistency. The RNAs 1 and 2 showed inferred phylogenies fully congruent and clearly delineated five clusters representing the five studied virus genera. In this respect, we proposed to classify the Ilarviruses in three distinct subgroups instead of 10 as mentioned in several reports of the International Committee on Taxonomy of Viruses where its suggestions were based on antigenic differences. Moreover, we confirmed that Alfalfa mosaic virus should be considered as a component of the Ilarvirus genus instead of being the unique representative of Alfamovirus genus. In addition, Pelargonium zonate spot and Olive latent 2 viruses fully deserve their affiliation to the family Bromoviridae.

Partial nucleotide sequences of the RNA 1 and RNA 2 of lilac ring mottle virus confirm that this virus should be considered a member of subgroup 2 of the genus Ilarvirus

Partial sequences of the RNA 1 and RNA 2 of LRMV are described. These confirm that LRMV is most closely related to members of the subgroup 2 ilarviruses. However, these sequences also show that LRMV is sufficiently different from other members of subgroup 2 so as to explain why oligonucleotide primers that had been used to amplify related subgroup 2 viruses did not amplify corresponding products from LRMV.