Tulip Virus Research Articles

Development and evaluation of one-step RT-qPCR TaqMan multiplex panels applied to six viruses occurring in lily and tulip bulbs

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics.

Complete genome sequence of tulip virus X, a Korean isolate from Tulipa gesneriana.

In this study, the presence of tulip virus X (TVX) in Korean tulips was confirmed through high-throughput RNA sequencing. Its complete genome sequence of 6,056 nucleotides was determined via Sanger sequencing, exhibiting a 99.24% nucleotide identity with TVX-J isolate. This signifies a previously unreported presence of TVX outside Japan.

First Report of Olive Mild Mosaic Virus in Imported Tulips (Tulipa gesneriana) in Korea.

Tulip cultivation in Korea primarily uses imported bulbs due to the absence of domestic production. To ensure safety and sustainability, Korean authorities have implemented strict phytosanitary measures for five viruses: arabis mosaic virus, tobacco necrosis virus, tobacco ringspot virus, tomato black ring virus, and tomato bushy stunt virus. In April 2021, 86 tulip plants presented symptoms such as chlorotic mottle, mosaic, streak, stripe, yellowing of the leaves, and color breaking on flowers. These samples were collected to investigate the incidence of viruses in four Korean provinces (Gangwon, Gyeongbuk, Gyeongnam, and Chungnam). The leaves and petals from each individual sample (10 mg each) were pooled and ground using liquid nitrogen. Total RNA was extracted using a Maxwell® 16 LEV Plant RNA Kit (Promega, Madison, USA). A cDNA library was constructed using TruSeq Standard Total RNA with Ribo - Zero (Illumina, San Diego, USA) and sequenced on an Illumina NovaSeq 6000 platform (Macrogen, Seoul, Korea) with 100-bp paired-end reads. Trinity software identified tulip breaking virus (TBV), tulip virus X (TVX), and lily symptomless virus (LSV), which are known to occur in Korea (Bak et al. 2023) by de novo assembly of 628 million reads into 498,795 contigs. The contigs were annotated as previously described (Bak et al. 2022). Moreover, a contig (ON758350) related to olive mild mosaic virus (OMMV; genus Alphanecrovirus, family Tombusviridae) was identified through BLASTn analysis. This contig had a 99.27% nucleotide (nt) identity to OMMV PPO-L190209 (KU641010), which was assembled from 201,346 reads and spanned 3,713 bp. To confirm the presence of OMMV, a primer pair (5'-GAATGTCTGGCGTTAAGCG-3'/5'-GTGTCCTGCGCATCATACAC-3') was designed to amplify a 797-bp fragment of the coat protein gene. In RT-PCR, 31.4% (27/86) of samples were positive for OMMV and coinfected with TBV or TBV+LSV. Coinfection with TBV led to chlorotic mottling and stripes, whereas triple coinfection with TBV+LSV produced distinct yellow streaks and mosaic within the lesion boundaries. In contrast, solely TBV infection did not produce such symptoms. The samples infected with OMMV were exclusively collected from Gangwon and Gyeongnam. In each province, an RT-PCR amplicon was cloned, and subsequently sequenced (Bioneer, Daejeon, Korea). The obtained sequences were named CC (OM243091) and GS (OM243092), and they shared 98.6% and 98.9% identity with PPO-L190209 (KU641010), respectively. A bioassay was conducted using a leaf infected with OMMV CC and TBV to inoculate 13 indicator species in triplicate, including Capsicum annuum, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. occidentalis, N. rustica, N. tabacum, Solanum lycopersicum, Tetragonia tetragonioides, and Tulipa gesneriana. The RT-PCR revealed positivity only for OMMV in the upper leaves of N. clevelandii, while all other species were negative with no symptoms. To our knowledge, this is the first report of OMMV occurring in tulips grown from imported bulbs in Korea, with no other known natural hosts such as olive tree (Cardoso et al. 2004), spinach (Gratsia et al. 2012), and corn salad (Verdin et al. 2018). The Korean OMMV isolates exhibited a high nt identity with the foreign isolate, and the samples were collected from farms that rely entirely on bulb imports for cultivation. These suggest that the outbreak of OMMV was likely caused by imported bulbs.

First report of Tulip Virus X infecting tulip (Tulipa gesneriana) in Korea.

Tulip virus X (tulip virus X, TVX) is a member of the genus Potexvirus (family Alphaflexiviridae) and is a positive single-stranded RNA virus. TVX was described first in Scotland (Mowat 1982), followed by several countries (Yamaji et al. 2001; Tzanetakis et al. 2005; Ward et al. 2008; Dees et al. 2011; Sochacki and Komorowska 2012; Wylie et al. 2019). In April 2021, 86 whole tulip plants showing viral symptoms in leaves (mosaic, yellowing, and malformation) and flowers (color breaking) were collected in Chilgok, Chuncheon, Goseong, Yecheon and Yesan, Korea. Furthermore, high-throughput sequencing was performed to identify viruses that infect tulips in Korea. Total RNA was extracted from pooled the leaves and petals using a Maxwell® 16 LEV Plant RNA Kit (Promega, Madison, USA). We constructed a single library using the TruSeq Stranded Total RNA LT Sample Prep Kit for Plant (Illumina, San Diego, USA). The library was 100 bp paired-end sequenced using Illumina's NovaSeq 6000 (Macrogen, Seoul, Korea) and was assembled de novo using Trinity software version trinityrnaseq_r20140717, with default parameters. The contigs were annotated as in previous study (Lee et al. 2020), revealing a single contig each related to TVX, lily symptomless virus (LSV), and tulip breaking virus (TBV) was generated from 648 million total reads. The TVX-related contig (GenBank ON205948) consisting of 6,076 bp showed 99.52% nucleotide identity (6027/6056 bp) with TVX-J (GenBank AB066288). We conducted an RT-PCR assay to validate the presence of viruses with specific primers as TVX-F5093/R5624 (5'-CTATCCGGACTCATTCTACTTC/GTGCGTTCCAGATAAGCTTG-3'), LSV-F7013/R7338 (5'-CTTGGTCGACAGGGACATAAC/GATTGGAATTGTGCTTTTCAGC-3'), and TBV-F7515/R8116 (5'-GTGTGTCATGGATGATTGTTG/CAACTGATTTGCTACCGCTAG-3'). Consequently, TVX were detected in 13 of 86 samples. Moreover, LSV and TBV were detected in 15 and 26 samples, respectively. However, the yellowing and mosaic observed in the TVX infected samples were not observed in the LSV and TBV infected samples. Subsequently, two TVX amplicons were selected, cloned and sequenced. The obtained sequences were 532 bp and were named YS24 and YS38 (GenBank LC664027 and LC664028), respectively. The Korean isolates showed 98.68% (525/532 bp) and 99.62% (530/532 bp) identity with Australian isolate (GenBank MH886522) in BLASTn analysis. To bioassay for TVX, the infected tulip leaf tissue from which YS24 was obtained was used to sap-inoculate, in triplicates, 15 species of indicator plants (Nicotiana benthamiana, N. clevelandii, N. debneyi, N. glutinosa, N. rustica, N. tabacum, Datura stramonium, Glycine max, Phaseolus vulgaris, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Cu. melo, Gomphrena globosa, and Tetragonia tetragonioides). After 14 days of inoculation, we observed distinct chlorotic spots on inoculated and upper leaves of C. quinoa, but no symptoms were observed in other indicator plants. In RT-PCR assay using TVX-specific primers, only C. quinoa showed a positive reaction. In previous studies, C. amaranticolor, C. quinoa, G. globosa, and N. benthamiana were known as the experimental host of TVX (Dees et al. 2011; Tzanetakis et al. 2005), but only C. quinoa was confirmed to be susceptible to the Korean isolate. Furthermore, transmission electron microscopy revealed typical flexuous rod-shaped viral particles in the inoculated C. quinoa. To our knowledge, this is the first report of TVX infecting tulips in Korea.

Tulip virus X

This datasheet on Tulip virus X covers Identity, Distribution.

RNA viromes of the oriental hybrid lily cultivar \u201cSorbonne\u201d

BackgroundThe lily is a perennial flowering plant belonging to the genus Lilium in the family Liliaceae. Most cultivated lily plants are propagated by bulbs. Therefore, numerous lily bulbs are frequently infected by diverse viruses causing viral diseases. To date, no study has examined the viromes of plants of one type with identical genetic backgrounds collected from different geographical regions.ResultsHere, we examined different viromes of the lily cultivar “Sorbonne” using 172 gigabytes of transcriptome data composed of 23 libraries from four different projects for the cultivar “Sorbonne.” We identified 396 virus-associated contigs from all but one library. We identified six different viruses, including Plantago asiatica mosaic virus (PlAMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tulip virus X (TVX), Lily mottle virus (LMoV), and Tobacco rattle virus (TRV). Of them, PlAMV was the most common virus infecting the lily. Scale and flower samples possessed a high number of virus-associated reads. We assembled 32 nearly complete genomes for the six identified viruses possessing the polyadenylate tails. Genomes of all six viruses were highly conserved in the lily cultivar “Sorbonne” based on mutation analysis. We identified defective RNAs from LSV, TVX, and PlAMV localized in the triple gene block region. Phylogenetic analyses showed that virus genomes are highly correlated with geographical regions and host plants.ConclusionsWe conducted comprehensive virome analyses of a single lily cultivar, “Sorbonne,” using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.

Occurrence of Plantago asiatica mosaic virus infecting oriental lily in India

Lilium is a major cut flower grown under protected cultivation in Tamil Nadu and Bengaluru provincesby importing lilium bulbs from European countries. The oriental lilium grown under protected cultivation in Nilgiris provinces expressed the symptom of viral infection. The symptoms in Oriental lily include chloroticand necrotic streaks on leaves and stunting of the infected plants. Leaf samples expressing the characteristicsymptoms of virus infection were indexed using DAC-ELISA and reverse transcription polymerase chain reaction(RT-PCR). The coat protein (CP) gene of PlAMV was amplified with an amplicon size of 722 bp and sequenceanalysis confirmed the viruses as Plantago asiatica mosaic virus (PlAMV) with 99 to 100% nucleotide and 99.5to 100% amino acid homology with other PlAMV isolates. Comparison of multiple sequence alignment analysesconfirmed the close relationship between PlAMV and Tulip virus × (TVX), which had 70% nucleotide sequenceidentity. Phylogenetic analysis of the nucleotide confirms that our PlAMV isolates formed a single subgroupwith other PlAMV isolates. The result provides important clues about spread of the virus and to the best of ourknowledge it is the first detailed study of PlAMV infecting lily in India.

The Occurrence of the Viruses in Tulip Crops in Poland

Abstract The viruses infecting tulips have a big influence on the yield and the quality of bulbs and forced flowers. Commercial bulb production is based on clonal propagation, which leads to the accumulation of viruses. Among 22 viruses occurring in tulips, the most common and the most dangerous are Tulip breaking virus, TBV; Tobacco necrosis virus, TNV; Lily symptomless virus, LSV; Cucumber mosaic virus, CMV and Tobacco rattle virus, TRV. The aim of the research was to check which viruses occur most often on Polish tulip plantations. The research was done on two tulip (Tulipa L.) cultivars ‘Strong Gold’ and ‘Leen van der Mark’ grown at 3 farms situated in different parts of Poland (Warsaw Region, Pomerania and Podlasie) during 2006-2007, and then at 2 farms located in Warsaw Region during 2008-2011. Five of the most important viruses infecting tulips (TBV, TNV, LSV, CMV, and TRV) were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in leaves and in bulbs during the period 2006-2010. In the last year of the research two different strains of TRV were detected (TRV-J and TRV-F) and Tulip virus X (TVX) as well. Search for viruses showed that most often TBV virus was detected both in the leaves and the bulbs regardless of the year and plantations. Yellow flowering cultivar ‘Strong Gold’ was infected by viruses more often than in bi-coloured (with red) ‘Leen van der Mark’, because of the difficulties with effective roguing of infected plants due to inconspicuous symptoms of virus infections on yellow coloured flowers. Other viruses were detected sporadically, however increasing occurrence of LSV and TRV was noticed from year to year. In 2011, TVX virus was detected in a few plants of ‘Strong Gold’ and it was the first case of detection of this virus in tulip in Poland.

First Report of Tulip virus X on Tulip in Poland.

Tulip virus X (TVX) was first isolated from tulips in Scotland (3). TVX is now found in all tulip-growing areas throughout the Netherlands, and other countries including Japan and New Zealand (2). In Poland, tulips are grown for bulb production in an estimated area of 450 ha. Since tulips are very popular during the winter season, bulbs are imported for forcing from the Netherlands. In spring 2011, symptoms typical of TVX infection were observed in field-grown 'Strong Gold' tulips in two farms in Mazovia Province. Leaves showed chlorotic and white, elongated streaks and spots, which are typical symptoms of TVX infection. Symptomatic leaves of four tulip plants tested positive by using TVX polyclonal antiserum in a double-antibody sandwich-ELISA assay (Bulb Quality Support B.V., Lisse, The Netherlands), whereas two plants were also infected with Tulip breaking virus. Total RNA was extracted from leaves of the same four symptomatic tulips with a silica capture method (1) and the extracts were used in a reverse transcription-PCR. Primers for the detection of TVX as well as cycling conditions were as previously reported (4). Primer pairs TVX1F-TVX1R and TVX2F-TVX2R are specific for a 219-bp fragment of the RNA-dependent RNA polymerase gene and a 189-bp fragment of the triple gene block gene-1, respectively. Amplified PCR products of the expected sizes were purified and sequenced (Genomed S.A., Warsaw, Poland). Sequence analyses showed that both fragments had 99.5 to 100% sequence identities at the nucleotide level with Genbank Accession No. AB066288. Two of the 219-bp amplicon sequences, varied by one nucleotide resulting in an amino acid substitution, were deposited in GenBank under Accession Nos. JN411683 and JN411684. To our knowledge, this is the first report of TVX in tulip in Poland. The occurrence of this virus could be important for bulb and flower production, particularly in the case of further spread because the virus can cause severe symptoms that make flowers unmarketable.

Phylogeny, coat protein genetic variability, and transmission via seeds ofHosta Virus X

The complete genome of Hosta Virus X (HVX), which is thought to be a distinct species of Potexvirus, was sequenced. Nucleotide sequences of HVX were compared with those of other members of the genus Potexvirus and phylogenetic tree was constructed. The range of identities of viral replicase open reading frame 1 (ORF1) between HVX and other potexviruses were 43.1%-55.1% and 35.9%-46.6% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis was performed according to the amino acid sequence of the replicase to determine the position of HVX in the genus Potexvirus. Results from the phylogenetic analysis demonstrated that HVX was in the same group as Cassava common mosaic virus (CsCMV), Plantago asiatica mosaic virus (PlAMV), Tulip virus X (TVX), and Hydrangea ring spot virus (HdRSV). In particular, coat protein (CP) sequences among viruses from different Hosta cultivars were revealed to be less variable than those from different isolates of Potato virus X (PVX), a Potexvirus type species. In the present study, HVX was transmissible by seeds of the Hosta "Blue Cadet" cultivar. Moreover, HVX was detected in the embryo but not in the seed coat or endosperm of the seed.

Detection of five viruses infecting dormant bulbs by TaqMan-based real-time RT-PCR

Dormant bulbs may be infected by a number of viruses which are of potential economic importance for the horticultural industry. Five important viruses in bulbs were successfully detected by TaqMan-based real-time one-step RT-PCR assays: Arabis mosaic virus (ArMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tobacco rattle virus (TRV) and Tulip virus X (TVX). Nucleic acid of appropriate quality and quantity for RT-PCR was extracted from nine bulb species using the Qiagen RNeasy plant mini kit. Depending on the assay, the sensitivity of the real-time RT-PCRs varied from being the same sensitivity to 1,000 times more sensitive than the respective conventional RT-PCRs. The reliability of the TVX real-time RT-PCR was further assessed against conventional RT-PCR using leaves and bulbs of 24 Tulipa × hybrida plants. TVX was detected in twice the number of leaf samples than bulb samples for both conventional and real-time RT-PCRs.

FIRST REPORT OF TULIP VIRUS X (TVX) IN NORWAY

A virus (isolate 10306) was isolated from bait plants (Nicotiana sp.) grown in a soil sample collected from an old orchard in the eastern part of Norway. Preliminary results from electron microscopy identified isolate 10306 as a putative member of the genus Potexvirus based on particle morphology. Molecular and biological methods were further used to determine the coat protein (CP) sequence and the host range of isolate 10306, respectively. Potexvirus-specific degenerate primers were designed and used to amplify a ~300 nucleotide fragment belonging to the RNA-dependent RNA-polymerase region of the viral genome by reverse-transcription PCR (RT-PCR). Based on the results from the sequence analysis, new specific primers were designed to amplify a fragment encompassing the CP gene. Furthermore, isolate 10306 was mechanically inoculated to different test plants and found to systemically infect eight plant species belonging to five families. CP sequence comparisons, at amino acid and nucleotide level, as well as phylogenetic analysis identified isolate 10306 as Tulip virus X (TVX). However, results from the host range studies differed from the ones previously published for this virus, suggesting that isolate 10306 could be considered a different strain of TVX. Moreover this is the first report of TVX in Norway.

First report of Tulip virus X in New Zealand

Strawberry mottle virus (SMoV) is a serious pathogen of strawberries ( Fragaria × ananassa ) and is transmitted by aphids in a semi-persistent manner. Severe strains of SMoV may reduce yield by up to 30% and losses can be up to 80% in mixed infections with other viruses (Thompson & Jelkmann, 2003; Martin & Tzanetakis, 2006). In May 2007, leaf mottling was observed on indicator plants of Fragaria vesca ‘EMK’ and ‘UC4’, and F. virginiana ‘UC12’ held in an open-shade house near Christchurch, New Zealand. No virus particles were seen using electron microscopy on crude sap preparations from mottled leaves, nor were symptoms observed when healthy plants of Chenopodium amaranticolor , C. quinoa , Cucumis sativus , Gomphrena globosa , Nicotiana benthamiana , N. clevelandii , N. occidentalis and N. sylvestris were mechanically inoculated with plant sap. RNA was extracted from affected strawberry plants and tested by RT-PCR using published SMoV-specific primers and cycling conditions (Thompson et al ., 2003). PCR amplicons of the expected size (219 bp) were obtained and an amplicon from F. vesca was sequenced (GenBank Acc. No. EU440731). A blast search in GenBank confirmed SMoV (98% nt identity with AJ311876). The plants also tested positive for SMoV using real-time RT-PCR (Ratti et al ., 2006). In July 2007, 35 plants of commercial cultivars of F. × ananassa were collected at random from 10 locations throughout New Zealand and the crowns and leaves were tested for SMoV using conventional and real-time RT-PCR. Indicator plants of F. virginiana ‘UC11’ and ‘UC12’ held in a glasshouse in Auckland were also tested. All commercial cultivars were uninfected but the indicator plants from Auckland tested positive for SMoV with both PCR methods. All plants were also tested by RT-PCR for Raspberry ringspot virus , Strawberry latent ringspot virus , Strawberry vein banding virus , Tomato ringspot virus and Tomato black ring virus and were found to be uninfected. Strawberry mottle virus (SMoV) occurs in many areas where strawberries are grown; this is the first report of the virus in New Zealand. The SMoVinfected indicator plants have been grown in New Zealand for at least 10 years. Despite the indicators in Christchurch being held in an open shade house, and the presence of at least two aphid species that can vector the virus in New Zealand ( Aphis gossypii and Chaetosiphon fragaefolii ), the virus does not appear to have become widely distributed within commercial strawberry cultivars. However, it has been decided not to impose regulatory controls to eradicate or manage the disease and therefore the disease is likely to spread in the future.

Stable recombinant alpaca antibodies for detection of Tulip virus X

For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at −20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C.

Nucleotide sequence analysis of seven Japanese isolates of Plantago asiatica mosaic virus (PlAMV): a unique potexvirus with significantly high genomic and biological variability within the species

The genomic sequences of Plantago asiatica mosaic virus (PlAMV), six lily isolates and one primrose isolate from Japan, were determined. The genomic size of all isolates was 6102 nucleotides, containing the five open reading frames typical of members of the genus Potexvirus. Pairwise comparison analyses confirmed the close relationship between PlAMV and tulip virus X. However, quite low identities were observed between different PlAMV isolates, including foreign isolates; nucleotide sequence identities of the RNA-dependent RNA polymerase (RdRp) gene between a Russian isolate (PlAMV-Ru), a Nandina isolate (PlAMV-Na) and Japanese isolates were 75-77%. These values were the lowest amongst different isolates of the same species of any potexviruses.

Tulip virus X (TVX) associated with lemon balm (Melissa officinalis) variegation: first report of TVX in the USA

Lemon balm (Melissa officinalis) has been grown for centuries as an ornamental and for its medicinal properties. One of the most popular clones shows bright variegation symptoms. Mechanical inoculations onto Gomphrena globosa, using variegated leaf tissue as the inoculum source, resulted in development of necrotic lesions about 5 days postinoculation, which suggested a viral aetiology of the variegation. Negatively stained leaf dips, examined with an electron microscope, revealed particles ≈500 × 13–14 nm in size. Double-stranded RNA was extracted from a variegated clone of M. officinalis and cloned as described elsewhere (Tzanetakis et al., 2005). Sequence data (GenBank Accession Nos AY842508–AY842510) revealed that the plant was infected with Tulip virus X (TVX). Oligonucleotide primers TVX 1F (5′-GACAYTCTAACCCCTTCGC-3′), TVX 1R (5′-GCCCTCTGTGGAAGTATCT-3′) and TVX 2F (5′-GAACAAGCACACCTCCACCA-3′), TVX 2R (5′-AGTGTGGTTTTCCCGGC-3′) were developed for detection of the virus, and were used in RT-PCR to test for TVX in four variegated clones of M. officinalis from Oregon and Washington, as well as G. globosa indicators with symptoms. The above-mentioned plants all tested positive for TVX by RT-PCR, while noninoculated G. globosa or M. officinalis plants without symptoms were negative. The amplicons were sequenced, which confirmed their identity as TVX. This is the first report of TVX in association with variegation in lemon balm and the first report of the presence of the virus in the USA.

Complete nucleotide sequence of Tulip virus X (TVX-J): the border between species and strains within the genus Potexvirus.

The complete nucleotide sequence of the genomic RNA of Tulip virus X Japanese isolate (TVX-J) has been determined. The sequence is 6056 nucleotides in length, excluding the poly(A) tail at the 3' terminus, and contains five open reading frames (ORFs) coding for proteins of Mr 153, 25, 12, 10, and 22 kDa (ORFs 1 through 5, respectively). The genome organization of TVX-J is similar to that of potexviruses, and the encoded proteins share a high degree of homology to the corresponding proteins of other potexviruses. Phylogenetic analyses based on the RNA-dependent RNA polymerase (RdRp) protein (the methyltransferase, helicase, and polymerase domains) encoded by ORF1 and the capsid protein (CP) encoded by ORF5, revealed a close relationship of TVX-J to Plantago asiatica mosaic virus (PlAMV). Pairwise comparison analyses revealed that the relationship between TVX and PlAMV is intermediate between that of strains and species, though previously they have not been considered related. Due to the relatively distant relationships of their replication apparatus and triple gene blocks, we conclude that TVX and PlAMV should be classified as distinct viruses. In addition, the borderline between species and strains of potexviruses is discussed.

Inference on binary images from binary data

The problem addressed is to reverse the degradation which occurs when images are digitised: they are blurred, subjected to noise and rounding error, and sampled only at a lattice of points. Inference is considered for the fundamental case of binary scenes, binary data and isotropic blur. The inferential process is separable into two stages: first from the lattice points to a binary image in continuous space and then the reversal of thresholding and blur. Methods are motivated by, and illustrated using, an electron micrograph of an immunogold-labelled section of tulip virus.

Inference on binary images from binary data

The problem addressed is to reverse the degradation which occurs when images are digitised: they are blurred, subjected to noise and rounding error, and sampled only at a lattice of points. Inference is considered for the fundamental case of binary scenes, binary data and isotropic blur. The inferential process is separable into two stages: first from the lattice points to a binary image in continuous space and then the reversal of thresholding and blur. Methods are motivated by, and illustrated using, an electron micrograph of an immunogold-labelled section of tulip virus.

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

SummaryLarge quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double‐antibody sandwich or plate‐trapped antigen forms of enzyme‐linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')2 ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10‐‐4 but not 10‐‐5; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S20,w, a single coat protein molecule of c. mol. wt 21 000, and a single‐stranded RNA genome of c. mol. wt 2.0 ± 106. Several dsRNA species, including the putative viral replicative form of c. mol. wt 4.1 ± 106, were isolated from virus‐infected cassava and N. benthamiana.