Tobacco rattle virus genome alterations in the Hosta hybrid 'Green Fountain' and other plants: reassortments, recombinations and deletions.

Tobacco rattle virus from a Hosta hybrid contained one RNA1 (Ho-1) and two RNA2 species (Ho-2a, Ho-2b). Whereas Ho-1 resembles TRV Al RNA1 from Alstroemerias, Ho-2a and Ho-2b resemble TRV TpO1 RNA2 from a potato field. Ho-2a has a complete RNA2-specific sequence, whereas that of Ho2-b carries a large deletion. The short RNA1-related 3' end of Ho-2a is distinct from that of Ho-1, whereas the longer one of Ho-2b is identical to that of Ho-1. TRV RNA2 molecules may apparently become associated with different TRV RNA1 molecules, from which they can acquire 3'ends of various lengths while often losing large portions of their RNA2-specific sequences.

Deletions and recombinations with the RNA1 3' ends of different tobraviruses have created a multitude of tobacco rattle virus TCM-related RNA2 species in Alstroemeria and tulip.

In vegetatively propagated Alstroemeria plants that showed pronounced stunting and necrotic leaf spots, a tobravirus infection was diagnosed in which one tobacco rattle virus (TRV, strain AL) RNA1 species was associated with seven different RNA2 species. The latter differed considerably in size and in the types of their 3' RNA1-related sequences. The 5' RNA2-specific part of all these RNA2 molecules showed almost 100% sequence identity with that of RNA2 of the TRV isolate TCM from tulip, but in some of these RNA2 molecules it was shorter than in the TCM isolate, whereas in others it was longer. One of the TRV AL RNA2 molecules, i.e. TC3'PE-a, contained the full set of three full-length RNA2-specific ORFs (ORF2a, -2b and -2c), whereas the previously analysed TCM sequence contained only ORF2a and -2b. In four of these TRV AL RNA2 molecules, i.e. those that had a relatively short RNA2-specific part, the 3' end was identical to that of the cognate TRV AL RNA1, but in the other three, which had a long RNA2-specific part, it was closely related to that of pea early browning virus (PEBV) RNA1, which was not detected in the infected plants. A comparison with previously described TRV/PEBV RNA2 recombinants suggested that the various TRV AL RNA2 molecules may represent various steps and side steps in an evolutionary process, which is apt to open the wide host range of TRV also to PEBV-derived RNA2 species.

Host Range, Purification, and Genetic Variability in Sweet potato chlorotic fleck virus.

Sweet potato chlorotic fleck virus (SPCFV) has recently been classified as a putative new member of the genus Carlavirus (family Flexiviridae) on the basis of its molecular properties. In this study, SPCFV was characterized in terms of host range, physical and biological characteristics, and genetic variability. In addition to sweet potato, SPCFV infected some plant species in the families Convolvulaceae, Chenopodiaceae, and Solanaceae. Limited numbers of virus particles were observed in the assimilation parenchyma cells of infected plant tissues; some cells had a distorted and enlarged endoplasmic reticulum though without any cytoplasmic and amorphous inclusions. The normal length of SPCFV particles was determined to be approximately 800 nm. In enzyme-linked immunosorbent assays, polyclonal antibodies raised against purified SPCFV virions were able to detect the virus in infected sweet potato and indicator plant tissues. In immunoelectron microscopy, SPCFV particles were all strongly decorated when reacted with homologous antiserum. Comparison of the 3' terminal part of the genome of a range of geographically diverse isolates revealed a high level of genetic diversity. The amino acid sequence identity in the coat protein and the nucleic acid binding protein ranged from 89 to 99.7% and from 75.9 to 99.2%, respectively. Phylogenetic analysis of both proteins showed a geographically associated clustering into two genogroups.

Daphne decline: what is the causal agent(s)?

A double infection of Daphne mosaic virus (DapMV) and an associated bacilliform virus was observed in the samples of diseased Daphne mezereum shrubs that showed mosaic patterns, precocious leaves reddening, defoliation, repeated flowering with subsequent declining. Extensive aggregations of bacilliform particles (166-370 x 65 nm and 169-233 x 68-78 nm) occurred in the nucleus or perinuclear space of root and leaf tissues suggesting that the virus might belong to the genus Nucleorhabdovirus, family Rhabdoviridae. However, the single bacilliform virions appeared occasionally in the cytoplasm of the leaf cells. We supposed that occurrence of the mixed virus infection could be the cause of D. mezereum decline in the Czech Republic.

Chickpea chlorotic stunt virus: A New Polerovirus Infecting Cool-Season Food Legumes in Ethiopia.

ABSTRACT Serological analysis of diseased chickpea and faba bean plantings with yellowing and stunting symptoms suggested the occurrence of an unknown or uncommon member of the family Luteoviridae in Ethiopia. Degenerate primers were used for reverse transcriptase-polymerase chain reaction amplification of the viral coat protein (CP) coding region from both chickpea and faba bean samples. Cloning and sequencing of the amplicons yielded nearly identical (96%) nucleotide sequences of a previously unrecognized species of the family Luteoviridae, with a CP amino acid sequence most closely related (identity of approximately 78%) to that of Groundnut rosette assistor virus. The complete genome (5,900 nts) of a faba bean isolate comprised six major open reading frames characteristic of polero-viruses. Of the four aphid species tested, only Aphis craccivora transmitted the virus in a persistent manner. The host range of the virus was confined to a few species of the family Fabaceae. A rabbit antiserum raised against virion preparations cross-reacted unexpectedly with Beet western yellows virus-like viruses. This necessitated the production of murine monoclonal antibodies which, in combination with the polyclonal antiserum, permitted both sensitive and specific detection of the virus in field samples by triple-antibody sandwich, enzyme-linked immunosorbent assay. Because of the characteristic field and greenhouse symptoms in chickpea, the name Chickpea chlorotic stunt virus is proposed for this new member of the genus Polerovirus (family Luteoviridae).

Serotype A and B strains of bean common mosaic virus are two distinct potyviruses.

The serological relationships among strains of bean common mosaic virus (BCMV) (genus Potyvirus, family Potyviridae) were investigated by testing 13 isolates of the 10 known BCMV pathotypes with two monoclonal antibodies and six antisera to BCMV strains. In addition, other properties of serologically distinct BCMV strains were compared. Two groups of BCMV strains were obtained by ELISA and Western blot serology: serotype A contained the BCMV strains NL3, NL5, and NL8 and serotype B contained the BCMV strains NL1, NL2, NL4, NL6, US4, NL7, NY15, and Fla. SDS polyacrylamide gel electrophoresis and Western blotting of freshly purified preparations, and of extracts from leaves infected with eleven BCMV strains showed that the apparent molecular mass of the capsid protein of the serotype A isolates NL3, NL5, and NL8 are lower (about M(r) 33,000) than those of the serotype B isolates (M(r) 34,500 to 35,000). The normal lengths of the particles of the serotype A isolates were shorter (810-818 nm) than those of most isolates (except NL6 and NY15) of serotype B (847-886 nm). All isolates studied induced cytoplasmic pinwheel and scroll inclusions. Cells infected with serotype A isolates contained a specific type of proliferated endoplasmic reticulum which was never found in cells infected with serotype B isolates. The capsid protein gene of a representative member of each serotype was cloned and sequenced. Molecular mass calculations based upon nucleotide sequence-derived amino acid sequences yielded M(r) of 29,662 and 32,489 for the capsid proteins of the serotype A isolate NL8 and the serotype B isolate NL4, respectively. Comparison of the coat-protein sequences showed considerable differences at the N-termini whereas the core regions and the C-termini appeared to be highly conserved. Marked differences were also observed within the 3' non-coding regions of cloned cDNAs of NL 4 and NL 8. The striking differences between the two serotypes of BCMV strongly suggest that they be classified as two distinct potyviruses which naturally infect Phaseolus beans.

Differentiation of the four viruses of the sugarcane mosaic virus subgroup based on cytopathology.

A cytological comparison has been made of representative isolates of johnsongrass mosaic (JGMV), maize dwarf mosaic (MDMV), sorghum mosaic (SrMV) and sugarcane mosaic (SCMV) viruses. These four viruses now encompass the complex of virus strains which were formerly considered as strains of sugarcane mosaic and/or maize dwarf mosaic viruses. The structure of the cytoplasmic cylindrical inclusions induced by these viruses, together with other cytological alterations, allow the four viruses to be distinguished. Pinwheels, scrolls and laminated aggregates were produced only by SCMV whereas JGMV, MDMV, and SrMV produced only pinwheels and scrolls. SrMV produced amorphous cytoplasmic inclusions which are not produced by JGMV and MDMV. The latter two were rather similar in cytological effects except that the SCMV-JG (U.S.A.) isolate of MDMV produced aggregates of needle-like structures in the cytoplasm which were not found with JGMV and the other MDMV isolates. The specific cytological effects induced by these viruses thus corroborate the recent classification of these viruses based mainly on the properties of the coat-protein gene, the 3' noncoding nucleotide sequences, and host reactions.

Use of protein A-gold complex for specific labelling of antibodies bound to plant viruses. I. Viral antigens in suspensions.

Colloidal gold particles 5 nm in diameter complexed with protein A (pAg) were bound specifically to rabbit antibodies which had reacted with antigens of 12 different plant viruses previously adsorbed to electron microscope (EM) support films. Conditions for highly specific labelling of antigens combined with a low non-specific background were assessed. In antiserum dilution tests, pAg labelling increased by up to four two-fold dilution steps the sensitivity of detecting low amounts of antibodies on virus particles. The pAg complex allowed detection of small antigens having no distinct morphology. The combination of dense antibody coating and pAg labelling substantially increased virus particle dimensions and contrast. This effect was further increased by double-antibody coating of the particles using rabbit anti-virus IgG followed by goat anti-rabbit IgG, prior to pAg incubation. Screening for elongated virus particles at low concentration in crude sap was then facilitated at very low EM magnifications. The pAg technique could be combined with trapping steps by using F(ab')2 fragments instead of complete antibodies for coating the grids.

Further Evidence that Shallot Yellow Stripe Virus (SYSV) Is a Distinct Potyvirus and Reidentification of Welsh Onion Yellow Stripe Virus as a SYSV Strain.

ABSTRACT An antiserum to shallot yellow stripe virus (SYSV) was raised and used in combination with a range of other antisera to potyviruses of Allium spp. in electron microscopic decoration experiments. The serological results corroborated an earlier finding that the type isolates of SYSV and Welsh onion yellow stripe virus (WoYSV) are closely related to each other and only distantly related to onion yellow dwarf (OYDV) and leek yellow stripe (LYSV) viruses, the two other major potyviruses infecting Allium spp. Moreover, the decoration results indicated that Japanese potyviruses named OYDV and Wakegi yellow dwarf virus are isolates of SYSV. Sequence analysis of the 3'-terminal regions of the SYSV and WoYSV ge-nomes revealed coat protein (CP) amino acid and 3'-nontranslated region (3'-NTR) nucleotide sequence identities of 95 and 89%, respectively. The CP amino acid and 3'-NTR nucleotide sequences of these viruses differed from those of OYDV and LYSV by >25 and >67%, respectively. The serological and molecular studies showed that SYSV and WoYSV are different strains of a potyvirus distinct from OYDV and LYSV. For priority reasons, we propose that these strains together with the Wakegi-type isolates of OYDV described in Japan be referred to as SYSV and that SYSV isolates from Allium spp. other than shallot be designated as the Welsh onion strain of SYSV (SYSV-Wo).

Occurrence of Petunia Vein-Clearing Virus in the U.S.A.

Petunia vein-clearing virus (PVCV), a tentative member of the caulimovirus group of plant pararetroviruses, was first identified in petunia (Petunia hybrida Vilm.) in Germany in the hybrid cv. Himmelröschen (1). A similar virus was recently identified in Minnesota in the petunia cv. Fantasy Pink grown from seed in commercial greenhouses. This virus has spherical particles 46 to 47 nm in diameter in preparations negatively stained with 1% uranyl acetate or sodium phosphotungstate, pH 7.0, and which contain a double-stranded DNA genome approximately 7.3 kb in size. The virus was shown by immunoelectron microscopy (IEM) to be closely related serologically to PVCV. No serological relationship to any other caulimoviruses was detected. Like PVCV, which is transmitted only by seed and grafting, the Minnesota virus isolate was not transmitted by mechanical inoculation to petunia or any other indicator plant. Symptoms associated with infection by PVCV in cv. Fantasy Pink consisted of mild vein clearing to severe vein yellowing, and reduction in leaf size and internode length. Symptoms were most frequently expressed when plants were under water and nutrient stress. Vigorously growing plants usually showed no symptoms, and no virions were detected by IEM in partially purified extracts from such plants. This suggests that infection of petunia hybrids by seed-borne PVCV may possibly be more widespread, but may go unnoticed because virus-induced symptoms may not be elicited in plants growing under favorable conditions. References: (1) D. Lesemann and R. Casper. Phytopathology 63:1118, 1973.

Occurrence of Lily mottle virus in Escarole.

Lily mottle virus (LMoV), genus Potyvirus, an important virus of lily that also causes flower-breaking in tulip (1), is considered to have a natural host range restricted to the family Liliaceae. In 1996, escarole (Cichorium endivia L. var. latifolium LAM) plants growing in fields close to Torino, Italy, and showing mosaic and necrotic spots on outer leaves were infected by a potyvirus related to LMoV. The virus was identified by immunoelectron microscopy (IEM) done on experimentally infected Nicotiana benthamiana and Chenopodium quinoa. The virus isolated from escarole (LMoV-E) had an experimental host range similar to that of lily isolates of LMoV, although species within the Liliaceae were not tested. LMoV-E systemically infected all nine escarole cultivars and six of seven endive cultivars (C. endivia L. var. crispum LAM) but did not infect any of six lettuce and two chicory cultivars (C. intybus L. var. foliosum HEGI). Symptoms ranged from mild to severe mosaic and were generally more severe on escarole than on endive. Some of the same escarole, endive, and lettuce cultivars were inoculated with a typical LMoV isolate from lily (Le97/49, from A. F. L. M. Derks, the Netherlands), which induced mild systemic infections in only one escarole and one endive cultivar. Both cultivars were also susceptible to LMoV-E. LMoV-E was purified from N. benthamiana, and an antiserum was prepared. IEM decoration titer experiments were done with LMoV-E and four other LMoV isolates from Liliaceae and their homologous antisera. Heterologous titers ranging from identity to serological differentiation index values of 2 to 4 were obtained, confirming the identity of the escarole isolate as a LMoV strain and indicating considerable serological variability among LMoV isolates. In a field survey of endive and escarole crops in 1998, in the area where LMoV-E was first identified, the virus was again detected by enzyme-linked immunosorbent assay in 4 of 80 escarole plants tested. LMoV-E appears to be a LMoV strain particularly adapted to escarole. To our knowledge, this is the first report of LMoV identified in a naturally infected host outside monocotyledonous plants. Reference: (1) E. L. Dekker et al. J. Gen. Virol. 74:881, 1993.

First Record of Pea enation mosaic virus Naturally Infecting Chickpea and Grasspea Crops in Syria.

Virus-like symptoms not commonly encountered on most chickpea (Cicer arietinum L.) and grasspea (Lathyrus sativus L.) genotypes were noticed at the ICARDA farm near Aleppo, Syria, during April and May 2001. Primary symptoms included stunting, accompanied by leaf mottling and yellowing. The causal agent was transmitted by the pea aphid (Acyrthosiphon pisum Harris) in a persistent manner. Efficiency of transmission was 100% when aphids acquired the virus from grasspea and then inoculated lentil, whereas transmission efficiency was 21% when aphids acquired the virus from chickpea and then inoculated lentil. Samples of symptomatic chickpea and grasspea reacted strongly with the antiserum prepared against a Dutch isolate (E154) of Pea enation mosaic virus (PEMV), provided by L. Bos (Wageningen, the Netherlands) (1), using tissue blot immunoassay (2). Negatively stained preparations from chickpea and grasspea revealed typical PEMV-like isometric particles ≍30 nm in diameter. With immunoelectron microscopy, these particles were effectively trapped and strongly decorated with PEMV antibodies (immunoglobulin G diluted 1:10) provided by M. Musil (Bratislava, formerly Czechoslovakia) (4). The virus capsid protein was 22 kDa based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, typical of the PEMV coat protein, and reacted strongly with PEMV antiserum (E154) in western blots. This is the first report of PEMV naturally infecting chickpea and grasspea in Syria and, to our knowledge, the first report in West Asia. PEMV reached epidemic levels on lentil in Syria for the first time in 1994 (3). Field symptoms observed in May 2001 suggest that PEMV may also seriously affect lentil, chickpea, and grasspea crops in Syria. References: (1) K. Mahmood and D. Peters. Neth. J. Plant Pathol. 79:138, 1973. (2) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994. (3) K. M. Makkouk et al. Plant Dis. 83:303, 1999. (4) M. Musil et al. Acta Virol. 14:285, 1970.

First Report of Chickpea Chlorotic Dwarf Virus Infecting Spring Chickpea in Syria.

During May 2003, a high incidence of symptoms suggestive of virus infection in spring chickpea were observed in many fields in Al-Ghab Valley, Syria, the ICARDA farm (near Aleppo, Syria), as well as in other locations in northern Syria, including the Idleb governorate. Symptoms observed were yellowing, stunting, and necrosis. A total of 1,345 chickpea samples with these symptoms (331 from Al-Ghab Valley, 269 from the ICARDA farm, and 745 from the Idleb governorate) were collected and tested for the presence of five viruses with tissue-blot immunoassay (TBIA) (4) at the Virology Laboratory of ICARDA, using the following antisera: monoclonal antibodies for Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) (1); Bean leafroll virus (BLRV, family Luteoviridae) (4B10) (3); Beet western yellows virus (BWYV, genus Polerovirus, family Luteoviridae [ATCC PVAS-647, American Type Culture Collection, Manassas, VA]); and Soybean dwarf virus (SbDV, family Luteoviridae, [ATCC PVAS-650]) and polyclonal antibodies for Chickpea chlorotic dwarf virus (CpCDV, genus Mastrevirus, family Geminiviridae, provided by H. J. Vetten, BBA, Braunschweig, Germany). The most common virus present was BWYV (detected in 54.1% of samples tested), followed by CpCDV (19.2%), BLRV (10.2%), and FBNYV (5.5%). SbDV was not detected in any of the samples tested. Using immunosorbent electron microscopy, infected chickpea samples revealed low numbers of geminivirus-like particles after 15 min of incubation on CpCDV antiserum-coated grids. When CpCDV was purified from infected chickpea plants, the virus coat protein was 32 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis typical of CpCDV coat protein (2) and reacted strongly with CpCDV antiserum in western blots. The CpCDV vector in Syria was found to be Orosius albicinctus Distant, and is thought to be similar to Orosius orientalis (Matsumura), the reported vector of CpCDV (2). FBNYV, BWYV, and BLRV infection of chickpea have been previously reported from Syria, but to our knowledge, this is the first report of CpCDV infecting chickpea in Syria. References: (1) A. Franz et al. Ann. Appl. Biol. 128:255, 1996. (2) N. M. Horn et al. Ann. Appl. Biol. 122:467, 1993. (3) L. Katul. Characterization by serology and molecular biology of bean leaf roll virus and faba bean necrotic yellows virus. Ph.D. thesis. University of Gottingen, Germany, 1992. (4) K. M. Makkouk and A. Comeau. Eur. J. Plant Pathol. 100:71, 1994.

First Report of Pepino Mosaic Virus on Tomato.

Early in 1999 a new viral disease occurred in protected tomato (Lycopersicon esculentum) crops in the Netherlands. Infected plants showed yellow leaf spots and mosaic. Transmission electron microscopic analysis revealed particles typical of potexviruses. Only three potexviruses have been reported to infect solanaceous crops: Pepino mosaic virus (PepMV), Potato aucuba mosaic virus (PAMV), and Potato virus X (PVX). Inoculation of test plants and serological tests showed that the new virus clearly differed from PAMV and PVX. Immuno-electron microscopy with antiserum to PepMV (1), the original PepMV isolate, and the virus from tomato showed decoration titers of 1:800 (homologous) and 1:400, respectively. Neither virus reacted with antiserum to PVX, nor did PVX react with antiserum to PepMV. Results of host plant analysis with 17 plant species mostly resembled those expected for PepMV. Nucleotide sequence alignment of DNA fragments obtained by reverse-transcriptase polymerase chain reaction with a specific primer set for potexviruses, directed against the RNA polymerase region, showed 93% identity between PepMV and the virus from tomato, while homologies with PVX, PAMV, and other potexviruses were <60%. Results indicate that the potexvirus in tomato is PepMV. PepMV was first found in pepino (Solanum muricatum) in Peru in 1974 and described by Jones et al. in 1980 (1). This is the first report of a natural infection of tomato by PepMV. Reference: (1) R. Jones et al. Ann. Appl. Biol. 94:61, 1980.

Daphne mosaic virus (DapMV), a new potyvirus from Daphne mezereum in the Czech Republic.

Daphne shrubs with light green rings and mosaic on leaves contained flexuous filamentous virions (696 x 13 nm) and cylindrical inclusions typical of the subdivision III of Edwardson's classification for inclusions induced by members of the family Potyviridae. Decoration tests using antisera to 67 potyviruses revealed distant serological relations among chilli veinal mottle virus, Colombian datura virus, papaya ringspot virus, tobacco vein mottling virus and yam mosaic virus. The 3' terminal region of the virus genome was amplified by RT-PCR using primers specific for cloned and sequenced members of the family Potyviridae. The most similar sequences in the GenBank were those of isolates of wild potato mosaic virus (WPMV) and yam mild mosaic virus (YMMV), originating from Peru and Guadeloupe, respectively. The new sequence had 63.2% and 61.9% nucleotide identity to WPMV and YMMV in the coat protein gene. The results suggest that the Czech isolate from daphne should be regarded as a new member of the genus Potyvirus. The name daphne mosaic virus (DapMV) is suggested for this virus.

The phylogenetic structure of the cluster of tobamovirus species serologically related to ribgrass mosaic virus (RMV) and the sequence of streptocarpus flower break virus (SFBV).

Ribgrass mosaic virus (RMV), turnip vein-clearing virus (TVCV) and Youcai mosaic virus (YoMV; formerly designated as oilseed rape mosaic virus; ORMV) belong to the genus Tobamovirus and are arranged in one out of three subgroups because of their common host range, serological cross-reactivity and amino acid composition of their coat proteins. The recently defined species Wasabi mottle virus (WMoV) is closely related to the same subgroup. The distinction of the four species is difficult and the lack of sequence information of a wide range of isolates has led to an unclear nomenclature. To clarify this situation we sequenced the coat protein genes from 18 isolates which were serologically related to members of the species of this cluster. The size of the coat protein was conserved with the exception of one isolate which revealed an N-terminal extension due to the mutation of three stop-codons. Phylogenetic analysis of these CP ORFs resulted in a tree with three clusters each containing at least one of the approved species RMV, TVCV and 1ptYoMV/WMoV in which our isolates were distributed. The tree was congruent and did support the present taxonomic status of species within this subgroup. For practical purpose we developed a subgroup 3 specific primer pair and a species differentiating restriction fragment length polymorphism (RFLP). Sequencing of the genome of Streptocarpus flower break virus (SFBV) which is serologically distantly related to the subgroup 3 viruses revealed a distinct genome organization. Therefore we propose that this virus should be regarded as a member of a species not belonging to any of the subgroups so far established.

Zygocactus virus X-based expression vectors and formation of rod-shaped virus-like particles in plants by the expressed coat proteins of Beet necrotic yellow vein virus and Soil-borne cereal mosaic virus.

Expression vectors were constructed from 35S promoter-containing full-length cDNA clones of Zygocactus virus X (ZVX). The expression of foreign genes was driven by the ZVX coat protein (cp) subgenomic promoter. It was successful only when the variable region downstream of the conserved putative promoter region GSTTAAGTT(X(12-13))GAA was retained. Most of the ZVX cp gene, except for a short 3' part, was replaced by the corresponding sequence of the related Schlumbergera virus X (SVX) and its cp subgenomic promoter to enable encapsidation of the transcribed RNA by an SVX/ZVX hybrid cp. Vector-expressed cp of Beet necrotic yellow vein virus (BNYVV) assembled in Chenopodium quinoa, Tetragonia expansa and Beta vulgaris leaves into particles resembling true BNYVV particles. The virus produced from these constructs retained its ability to express BNYVV cp in local infections during successive passages on C. quinoa. This ability was lost, however, in the rarely occurring systemic infections.

Molecular characterization of isolates of anagyris vein yellowing virus, plantago mottle virus and scrophularia mottle virus -- comparison of various approaches for tymovirus classification.

The complete nucleotide sequences were determined for the genomic RNAs of three tymoviruses, i.e. isolates of anagyris vein yellowing virus (AVYV), plantago mottle virus (PlMoV) and scrophularia mottle virus (SrMV) which are all serologically closely related to ononis yellow mosaic virus (ibid) and to Nemesia ring necrosis virus (NeRNV), a recently described recombinant virus which is widely spread in commercially grown ornamental plant species belonging to the Scrophulariaceae. Total nucleotide and coat protein amino acid sequence identities revealed similar groupings in the genus tymovirus as serological studies did. The latter, however, tended to suggest much closer relationships than the molecular data and may fail to recognise the distinctiveness of new tymovirus species. The usefulness of various species demarcation criteria for the classification of tymoviruses is discussed.

Nemesia ring necrosis virus: a new tymovirus with a genomic RNA having a histidylatable tobamovirus-like 3' end.

The complete nucleotide sequence of the genomic RNA of the new virus Nemesia ring necrosis virus (NeRNV), which is widespread in various ornamental plant species belonging to the Scrophulariaceae and Verbenaceae, has been determined. Based on its gene content, the folding properties of its 5'-untranslated region and in vitro translation experiments, NeRNV RNA is a typical tymovirus RNA. Its 3' end, however, differs greatly from those of the valine-specific tymoviral RNAs that have been analysed previously. It can be folded into an upstream pseudoknot domain and a histidine-specific tRNA-like structure, a combination that, so far, has been found only in tobamoviral RNAs. The identity elements found in NeRNV RNA for recognition by yeast histidyl-tRNA synthetase are more similar to those of yeast tRNAHis than the ones found in tobacco mosaic virus RNA. As a result NeRNV RNA can be charged with histidine even more efficiently than tobacco mosaic virus RNA.

Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing.

Subcellular localization of the Poa semilatent virus cysteine-rich gammab protein was studied by using different approaches. In infected tissue, gammab was detected mainly in the P30 fraction as monomers, dimers and oligomers. Green fluorescent protein-fused gammab was found to localize in punctate bodies in the cytoplasm. Colocalization with marker proteins demonstrated that these bodies represent peroxisomes. Immunoelectron microscopy revealed that gammab was localized in the peroxisomal matrix and that localization of gammab in peroxisomes required the C-terminal signal tripeptide SKL. An SKL-deletion mutant exhibited a diffuse localization, but retained the protein's ability to suppress RNA silencing, determine infection phenotype and support virus systemic spread. These data indicate that gammab functions are not associated with the protein's localization to peroxisomes.