Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2015).

Changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2015 are listed.

De novo emergence of a novel satellite RNA of cucumber mosaic virus following serial passages of the virus derived from RNA transcripts.

Satellite RNA (satRNA) is often associated with cucumber mosaic virus (CMV); however, its origin remains unexplained and a subject for speculation. We passaged progeny of molecularly cloned CMV-Fny and CMV-LS in Nicotiana tabacum cv. Ky 14 under greenhouse conditions. A satRNA emerged after at least eight successive transfers of CMV-Fny, but no satRNA was recovered after eleven serial transfers of CMV-LS under the same conditions. The sequences of the newly emerged satRNA were determined, and an infectious cDNA clone was synthesized. Comparison of the sequences of the newly emerged satRNA with those of known CMV satRNAs showed that it is unique. This observation raises interesting questions regarding the enigmatic nature of the origin of CMV satRNAs.

Aphis glycines as a Vector of Persistently and Nonpersistently Transmitted Viruses and Potential Risks for Soybean and Other Crops.

The recently introduced soybean aphid (Aphis glycines), which is widespread in the soybean-growing regions in the United States, is the only aphid able to develop large colonies on soybean. Although its potential as a vector of plant viruses is recognized, reports on virus transmission efficiency by this aphid species are limited. In the present study, we examined the ability of A. glycines to transmit several economically important viruses. The results showed that A. glycines transmitted the potyviruses Bean yellow mosaic virus (BYMV) and Soybean mosaic virus from soybean to soybean more efficiently than Myzus persicae. However, M. persicae transmitted the alfamovirus Alfalfa mosaic virus and the potyviruses Tobacco etch virus (TEV) and Tobacco vein mottling virus (TVMV) from tobacco to tobacco more efficiently than A. glycines. This is the first report to demonstrate that the soybean aphid can vector TEV and TVMV, two economically important tobacco viruses. This also is the first report to document successful transmission of BYMV by A. glycines. All attempts to transmit the nepovirus Tobacco ringspot virus by A. glycines were unsuccessful, regardless of the length of the acquisition and inoculation feeding periods. Although the luteovirus Soybean dwarf virus (SbDV) was widely distributed in red and white clover in Kentucky, it was not detected in soybean. All transmission experiments of SbDV by A. glycines were unsuccessful. A reverse-transcription polymerase chain reaction (RT-PCR) assay was developed to detect SbDV in single aphids using a pair of primers designed to amplify a 372-bp PCR fragment in the coding region of SbDV coat protein. Although A. glycines was not a vector of SbDV, the virus was detected in 100% of tested aphids by RT-PCR after a 24- to 48-h virus acquisition access feeding. The practical applications of RT-PCR in detecting persistently transmitted viruses are discussed.

Expression of the Peanut stunt virus Coat Protein Gene Is Essential and Sufficient for Production of Host-Dependent Ribbon-Like Inclusions in Infected Plants.

ABSTRACT We previously have reported that infection of tobacco protoplasts or leaf tissue with the cucumovirus Peanut stunt virus (PSV) induced the production of unusual cytoplasmic ribbon-like inclusions. The formation of these novel inclusions is strain-specific, because infection of tobacco with subgroup II PSV strains, but not subgroup I strains, induced the production of inclusions. Furthermore, we have demonstrated that induction of the ribbon-like inclusions maps to PSV subgroup II RNA3, which codes for the coat protein (CP) and movement protein (MP). We have now extended these studies using chimeric constructs containing CP and MP open reading frames (ORFs) from PSV strains ER and W that belong to subgroups I and II, respectively. Additionally, recombinant Potato virus X (PVX) vectors containing translatable and untranslatable PSV CP ORF were constructed. Plants inoculated with infectious chimeric PSV or recombinant PVX transcripts were analyzed for CP expression by enzymelinked immunosorbent assay and reverse transcription-polymerase chain reaction and for inclusion production by electron microscopy. The results of these experiments indicated that translation of the CP ORF alone is essential and sufficient for inclusion production. In immunogold labeling experiments using an antiserum to PSV virions, abundant gold labeling of the inclusions was observed, suggesting that PSV CP is probably a major component of the inclusions. Because inclusion production is host specific, a host factor is likely to be involved. In addition to their diagnostic importance, these novel inclusions may also prove valuable in identifying the host factors that interact with PSV CP.

Soybean Seedcoat Mottling: Association with Soybean mosaic virus and Phomopsis spp. Seed Infection.

Soybean mosaic virus (SMV) infection predisposes soybean (Glycine max) seed to Phomopsis spp. seed infection and may induce seedcoat mottling. The extent of seedcoat mottling associated with the accumulation of SMV or Phomopsis spp. infection of the seedcoat was investigated in 1996 and 1997. Plants of two SMV-susceptible cultivars, 'Clark' and 'Williams', were sap inoculated with the G2 strain of SMV at growth stage R2. Control plants (SMV-resistant isolines L78-434 and L78-379, respectively) were not inoculated. Harvested seed were evaluated visually for seedcoat mottling and sorted into four categories (severe, banded, blemish, and none) according to the degree of seedcoat pigmentation. Seed infection with Phomopsis spp. was determined and SMV accumulation was assessed by enzyme-linked immunosorbent assay and nucleic acid hybridization analysis. Phomopsis spp. were isolated from seed of all mottling categories, with the incidence of infection increasing as the degree of seedcoat mottling increased. SMV was detected in both mottled and nonmottled seedcoats, with generally higher levels of SMV in mottled seedcoats. However, the relationship of SMV titer to seedcoat mottling was inconsistent across cultivars and years. Thus, the extent of seedcoat mottling was not directly related to the accumulation of SMV in the seedcoat.

Effect of Aphid Behavior on Efficiency of Transmission of Soybean mosaic virus by the Soybean-Colonizing Aphid, Aphis glycines.

The soybean aphid (Aphis glycines) was a poor vector (0.83% transmission) when the aphids were allowed overnight acquisition feed on Soybean mosaic virus (SMV)-infected soybean leaves. However, A. glycines was shown to be a very efficient vector (34.72% transmission) when individual aphids were allowed a 1-min acquisition probe on the same infected leaves used for the feeding treatment. Similar results were obtained with Myzus persicae and tobacco in transmission experiments of the potyviruses Tobacco etch virus (feeding: 1.36%; probing: 45.5%) and Tobacco vein mottling virus (feeding: 2.0%; probing: 47.5%). A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect SMV in single soybean aphids using a pair of primers designed to amplify a 469-bp PCR fragment in the coding region of SMV coat protein. In contrast to the low transmission rate obtained with the soybean aphids that acquired virus through overnight feeding, RT-PCR detected SMV in 100% of these aphids. Interestingly, the rate of SMV detection by RT-PCR in aphids that were allowed a 1-min acquisition probe (31.67%) coincided with percent transmission (34.72%). The practical application of RT-PCR in detecting nonpersistently transmitted viruses and its implications for virus epidemiology are discussed.

A novel alcohol oxidase/RNA-binding protein with affinity for mycovirus double-stranded RNA from the filamentous fungus Helminthosporium (Cochliobolus) victoriae: molecular and functional characterization.

We have cloned and sequenced a novel alcohol oxidase (Hv-p68) from the filamentous fungus Helminthosporium (Cochliobolus) victoriae that copurifies with mycoviral double-stranded RNAs. Sequence analysis revealed that Hv-p68 belongs to the large family of FAD-dependent glucose methanol choline oxidoreductases and that it shares significant sequence identity (>67%) with the alcohol oxidases of the methylotrophic yeasts. Unlike the intronless alcohol oxidases from methylotrophic yeasts, a genomic fragment of the Hv-p68 gene was found to contain four introns. Hv-p68, purified from fungal extracts, showed only limited methanol oxidizing activity, and its expression was not induced in cultures supplemented with methanol as the sole carbon source. Northern hybridization analysis indicated that overexpression of Hv-p68 is associated with virus infection, because significantly higher Hv-p68 mRNA levels (10- to 20-fold) were detected in virus-infected isolates compared with virus-free ones. We confirmed by Northwestern blot analysis that Hv-p68 exhibits RNA binding activity and demonstrated that the RNA-binding domain is localized within the N-terminal region that contains a typical ADP-binding beta-alpha-beta fold motif. The Hv-p68 gene, or closely similar genes, was present in all species of the genus Cochliobolus but absent in the filamentous fungus, Penicillium chrysogenum, as well as in two nonmethylotrophic yeasts examined. This study represents the first reported case that a member of the FAD-dependent glucose methanol choline oxidoreductase family, Hv-p68, may function as an RNA-binding protein.

Resistance to Bean pod mottle virus in Transgenic Soybean Lines Expressing the Capsid Polyprotein.

ABSTRACT Transgenic fertile soybean plants were generated from somatic embryos of soybean (Glycine max) cv. Jack transformed via particle bombardment with the capsid polyprotein (pCP) gene of Bean pod mottle virus(BPMV). The plant transformation vector (pHIG/BPMV-pCP) utilized in these experiments contained the BPMV-pCP coding sequence, an intron-containing GUS gene, and the hygromycin phosphotransferase gene. Southern blot hybridization analysis showed that 19 transgenic soybean plants selected for resistance to hygromycin contained the genes for GUS and BPMV-pCP. The progeny of five of these transgenic soybean plants (plants 137, 139, 157, 183, and 186) were characterized in detail. An additional transgenic plant (plant 200) contained the intron-GUS and hygromycin resistance genes, but lacked the BPMV-pCP gene and was used as a negative control. Southern blot hybridization analysis of the five transgenic plants showed the presence of three copies of the T-DNA in a similar banding pattern suggesting that they were derived from a single transformation event. Western and northern blot analyses showed that the expression levels of BPMV-pCP and pCP transcript were high in these five pCP plants. Infectivity assays with detached leaves demonstrated that all five pCP plants exhibited resistance to virus infection because they accumulated lower levels of BPMV compared with plant 200 and nontransformed controls. Unlike the T(2) progeny of line 183-1 that segregated with respect to the pCP gene and, consequently, to BPMV resistance, the T(2) progeny of the homozygous line 183-2 showed little or no symptoms in response to rub-inoculation with virions of a severe strain of BPMV. Although BPMV accumulation was evident in leaves on which viruliferous beetles were allowed a 72-h inoculation access period, the upper noninoculated leaves of the T(2) progeny of line 183-2 plants were symptomless and accumulated little or no virus. Because the progeny of this homozygous transgenic line exhibited systemic resistance, they could potentially be useful in generating commercial cultivars resistant to BPMV.

A cellular protein with an RNA-binding activity co-purifies with viral dsRNA from mycovirus-infected Helminthosporium victoriae.

A cellular protein that co-purifies with mycoviral dsRNA was isolated from the plant pathogenic fungus Helminthosporium victoriae (telomorph: Cochliobolus victoriae) infected with two viruses, the totivirus Helminthosporium victoriae 190S virus and the chrysovirus-like Helminthosporium victoriae 145S virus (Hv145SV). The cellular protein, which was, designated Hv-p68, accumulated to higher levels in virus-infected isolates compared to virus-free ones. The majority of the Hv145S dsRNAs were found in association with Hv-p68 and not packaged in virions. Hv-p68 could also be detected as a minor component of the virus capsid. Evidence is presented that Hv-p68 occurs in vivo as an octamer and that it possesses RNA-binding activities. Based on partial amino acid sequence analysis, Hv-p68 was shown to share significant sequence identity with alcohol oxidases from methylotrophic yeasts. Hv-p68 is proposed to play a role in viral RNA packaging/replication and in regulating viral pathogenesis.

Expression of the Totivirus Helminthosporium victoriae 190S virus RNA-dependent RNA polymerase from its downstream open reading frame in dicistronic constructs.

The undivided double-stranded RNA (dsRNA) genome of Helminthosporium victoriae 190S virus (Hv190SV) (genus Totivirus) consists of two large overlapping open reading frames (ORFs). The 5'-proximal ORF encodes a capsid protein (CP), and the downstream, 3'-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and Schizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

Molecular characterization of an atypical old world strain of Peanut stunt virus.

Northern blot analyses of RNAs from an Iranian strain of Peanut stunt virus (PSV-I Cucumovirus) using cloned cDNA probes to the genomic RNAs from two PSV strains, PSV-ER (subgroup I) and PSV-W (subgroup II), indicated that PSV-I RNA-3 is derived from a subgroup II strain. No hybridization signals, however, were detected with PSV-I RNAs 1 and 2 suggesting they are distinct from both subgroups I and II. Nucleotide (nt) sequence analysis of cloned cDNA fragments (1-1.5 kbp in size) representing PSV-I RNAs 1 and 2 showed that PSV-I is more closely related to subgroup II strains than to any other sequenced cucumovirus. The percent nt identity between RNA-1 sequences from PSV-I and PSV-ER or between PSV-I and PSV-W were 79.1% and 88.8%, respectively. The corresponding values for RNA-2 were 77.5% and 86.7%. Sequence comparison analyses of deduced amino acid sequence of cloned partial sequences of PSV-I RNAs 1 and 2 indicated that PSV-I 1a and 2a proteins are most closely related to those of subgroup II strains (93% identity). PSV-I supported the replication and encapsidation of PSV G-satellite RNA (G-satRNA), but not cucumber mosaic virus WL1-satRNA. PSV-I may be perceived as an Old World derivative of subgroup II strains, or it may represent a natural reassortment between a subgroup II (RNA-3) and an as yet uncharacterized subgroup of PSV strains (RNAs 1 and 2).

Complete nucleotide sequence of bean pod mottle virus RNA1: sequence comparisons and evolutionary relationships to other comoviruses.

The complete nucleotide sequence of bean pod mottle comovirus (BPMV) RNA 1 was determined. It is 5983 nucleotides long, excluding the poly(A) tail, and encodes a polyprotein of 1850 amino acid (aa) residues. Multiple alignments of the deduced aa sequence of BPMV polyprotein with those of cowpea mosaic virus (CPMV), red clover mottle virus (RCMV) and cowpea severe mosaic virus (CPSMV) indicated that BPMV RNA1 encodes the predicted set of five mature proteins: the equivalent of CPMV 32K protease cofactor, 58K putative helicase, VPg, 24K protease and 87K putative RNA-dependent RNA polymerase. Of the four proposed cleavage sites in BPMV RNA1 polyprotein, the one at the 32K/58K site (Q/A) is distinct for BPMV polyprotein and those at the 58K/VPg and VPg/24K junctions (Q/S and Q/M, respectively) are identical in all four comovirus polyproteins. Sequence comparison and phylogenetic analysis revealed that BPMV RNA1 is more closely related to CPSMV than to CPMV or to RCMV.

Production of infectious RNA transcripts from full-length cDNA clones representing two subgroups of peanut stunt virus strains: mapping satellite RNA support to RNA1.

Full-length cDNA clones from which infectious transcripts could be generated were constructed from the genomic RNAs of two distinct strains of peanut stunt cucumovirus (PSV), PSV-ER and PSV-W. PSV-ER, a subgroup I strain, is known to support efficient replication of satellite RNA (satRNA) in infected plants, whereas PSV-W, a subgroup II strain, does not support satRNA replication. Although artificial reassortants (pseudorecombinants) of all possible combinations of infectious transcripts representing RNA1, RNA2 and RNA3 were infectious, only those having RNA1 from PSV-ER supported the replication of satRNA. These results demonstrate conclusively that support of PSV satRNA replication maps to RNA1. Comparisons of secondary structure predictions of the C-terminal helicase-like domain of the 1a proteins of four PSV strains belonging to two subgroups did not reveal any obvious differences between strains that differ in satRNA support. The complete nucleotide sequence of RNA1 from strains PSV-ER and PSV-W were determined and found to be 79% identical. Sequence comparison analysis of RNA1 sequences of cucumoviruses confirmed the placement of the PSV strains into two distinct subgroups.

Origin, adaptation and evolutionary pathways of fungal viruses.

Fungal viruses or mycoviruses are widespread in fungi and are believed to be of ancient origin. They have evolved in concert with their hosts and are usually associated with symptomless infections. Mycoviruses are transmitted intracellularly during cell division, sporogenesis and cell fusion, and they lack an extracellular phase to their life cycles. Their natural host ranges are limited to individuals within the same or closely related vegetative compatibility groups. Typically, fungal viruses are isometric particles 25-50 nm in diameter, and possess dsRNA genomes. The best characterized of these belong to the family Totiviridae whose members have simple undivided dsRNA genomes comprised of a coat protein (CP) gene and an RNA dependent RNA polymerase (RDRP) gene. A recently characterized totivirus infecting a filamentous fungus was found to be more closely related to protozoan totiviruses than to yeast totiviruses suggesting these viruses existed prior to the divergence of fungi and protozoa. Although the dsRNA viruses at large are polyphyletic, based on RDRP sequence comparisons, the totiviruses are monophyletic. The theory of a cellular self-replicating mRNA as the origin of totiviruses is attractive because of their apparent ancient origin, the close relationships among their RDRPs, genome simplicity and the ability to use host proteins efficiently. Mycoviruses with bipartite genomes (partitiviruses), like the totiviruses, have simple genomes, but the CP and RDRP genes are on separate dsRNA segments. Because of RDRP sequence similarity, the partitiviruses are probably derived from a totivirus ancestor. The mycoviruses with unencapsidated dsRNA-like genomes (hypoviruses) and those with bacilliform (+) strand RNA genomes (barnaviruses) have more complex genomes and appear to have common ancestry with plant (+) strand RNA viruses in supergroup 1 with potyvirus and sobemovirus lineages, respectively. The La France isometric virus (LIV), an unclassified virus with multipartite dsRNA genome, is associated with a severe die-back disease of the cultivated mushroom. LIV appears to be of recent origin since it differs from its host in codon usage.

First Report of Peanut Stunt Cucumovirus Naturally Infecting Desmodium sp.

Plant species in the genus Desmodium (Fabaceae) are used as forage and cover crops and include a number of common weeds such as beggarweed (D. tortuosum) and beggarlice (D. intortum). Accessions of the genus are part of the plant genetic resources collection maintained at Griffin, GA. Peanut stunt cucumovirus (PSV) was detected in naturally infected plants of Desmodium sp. PI 322505 (from Brazil) in a germ plasm regeneration plot by a direct antigen coating-enzyme-linked immunosorbent assay (DAC-ELISA) with an antiserum against PSV strain ER (subgroup I) originally isolated from cowpea in Georgia. The infected plants showed mild mosaic symptoms. Indicator host studies in the greenhouse revealed symptoms characteristic of PSV on Nicotiana tabacum cv. Burley 21 (ringspots and oak leaf pattern), Chenopodium album subsp. amaranticolor (chlorotic local lesions), and Vigna unguiculata (chlorotic spots followed by systemic mild mosaic). These symptomatic indicator plants tested positive for PSV by DAC-ELISA. Greenhousegrown plants of D. incanum (kaimi-clover) and D. uncinatum (Spanish tick-clover) were inoculated with the field isolate and the plants were tested for PSV by DAC-ELISA (10 infected of 10 tested and 3 infected of 9 tested, respectively). The PSV isolate infecting Desmodium spp. was found to contain satellite RNA and it generated the predicted products in reverse transcription-polymerase chain reactions (RT-PCRs) with primers based on specific PSV-ER sequences. The RT-PCR products were confirmed by restriction-enzyme digestion (1). This is the first report of PSV naturally infecting a member of the genus Desmodium. Because some members of this genus may grow as perennial weeds near peanut, cowpea, or other host crops, this genus may serve as an alternate/overwintering host for the virus. Reference: (1) R. A. Naidu et al. Phytopathology 85:502, 1995.

Characterization of a Severe Strain of Cucumber Mosaic Cucumovirus Seedborne in Cowpea.

A new seedborne strain of cucumber mosaic cucumovirus (CMV) that induces severe symptoms on many cowpea genotypes was detected in Georgia in 1994. This strain, designated CMV-Csb, is asymptomatic on tobacco, but it produces more severe cowpea stunt symptoms when present in combination with blackeye cowpea mosaic potyvirus than do the more prevalent CMV isolates. The new strain is seedborne in cowpea (1.5 to 37%), has no associated satellite RNA, and is classified as a member of subgroup I of CMV strains based on nucleic acid hybridization assays.

Unusual Cytoplasmic Inclusions Induced in Tobacco by Peanut Stunt Virus Subgroup II Strains Map to RNA3.

ABSTRACT Infection of tobacco protoplasts or leaf tissues with peanut stunt virus (PSV) subgroup II strains induced the production of unusual cytoplasmic ribbon-like inclusions. The inclusion structures appeared as long, thin, densely staining sheets that were prevalent within the cytoplasm, accumulating most commonly near vacuoles. Numerous virions and ribosomes could be seen adjacent to the inclusion surfaces. The formation of these novel inclusions appeared to be subgroup specific, since infection of tobacco with PSV strains W and B (subgroup II), but not strains ER, V, and J (subgroup I), induced the inclusions. Furthermore, inclusion formation was shown to be host specific, because the inclusions were not detected in either of two leguminous host species infected with PSV subgroup II strains. Using tobacco protoplasts electroporated with various assortments of infectious RNA transcripts derived from cDNA clones of genomic RNAs of PSV-ER and PSV-W, we demonstrated that induction of the unusual ribbon-like inclusions maps to PSV-W (subgroup II) RNA3. This conclusion is consistent with the finding that PSV strain BV-15, a natural intraspecific reassortant that derives its RNA2 and RNA3 from a subgroup I strain, did not induce inclusion formation.

Molecular Evidence That Strain BV-15 of Peanut Stunt Cucumovirus Is a Reassortant Between Subgroup I and II Strains.

ABSTRACT In Northern hybridization assays, RNA1 of peanut stunt virus (PSV) strain BV-15 hybridized strongly with a cloned cDNA probe to RNA1 from strain PSV-W (subgroup II). Cloned probes to PSV-W RNA2 and RNA3, however, did not hybridize with the corresponding RNAs from strain BV-15. The complete nucleotide sequence of PSV-BV-15 RNA2 has been determined, and sequence comparison analysis showed that it is closely related to PSV subgroup I strains; the percent nucleotide sequence identity between PSV-BV-15 RNA2 and RNA2 sequences from PSV subgroup I and II strains were 90.5 and 75%, respectively. The possibility that PSV-BV-15 RNA2 may contain short regions derived from a subgroup II strain (i.e., represent a mosaic structure indicative of recombination) was investigated. Results indicated, however, that the entire PSV-BV-15 RNA2 sequence is derived from a subgroup I strain. PSV-BV-15 RNA3 has previously been shown to belong to subgroup I strains. These results thus establish that PSV strain BV-15 is a natural reassortant between PSV subgroups I and II strains. A reverse transcription-po-lymerase chain reaction assay is proposed for differentiating between this reassortant strain and PSV strains in subgroups I and II.

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3.

Strains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80%, the corresponding value between strains ER and J was 91%, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic, encoding a putative movement protein (3a protein) and a coat protein (CP). The intercistronic and 5' untranslated region (UTR) sequences of PSV strains, unlike those of cucumber mosaic cucumovirus (CMV) strains, are highly conserved and thus not useful for distinguishing the two subgroups. However, the 3' UTR sequences of PSV strains, like those of CMV strains, can discriminate between the two subgroups since strains within the same subgroup are 95% identical in their 3' UTRs whereas those in different subgroups are only 74-78% identical. PSV-W and PSV-ER RNA4s were determined to be 994 and 1006 nucleotides long, respectively. PSV 3a and CP genes have higher percentage nucleotide sequence identities to those of tomato aspermy cucumovirus than to those of CMV.