Segregation of distinct variants from Citrus tristeza virus isolate SY568 using aphid transmission.

A well-studied severe isolate of Citrus tristeza virus (CTV) known as SY568 has previously been shown to contain multiple variants of the virus which differ in their genetic and biological characters. Aphid transmission was used in an attempt to segregate some of these variants for further characterization. Resulting infections gave symptoms which varied from asymptomatic to more severe than the inoculum source. RNase protection assays (RPAs) were used to compare nine regions of the CTV genome and determine whether unique strains could be identified. Five aphid-transmitted subcultures, with fingerprints that were different from those of the inoculum sources in at least one genomic area, were then cloned, sequenced, and compared with known isolates. An asymptomatic strain was shown to be different in every area of the CTV genome when examined by RPA and sequencing of selected regions. Mixed-infection studies using graft transmission of the asymptomatic subculture and two of the more severe aphid-transmitted subcultures showed that the mild strain was not able to compete well when in the presence of any of the severe variants tested, and its titer was significantly reduced from that seen in single infection. The mild strain and a selected severe strain were singly graft inoculated into five different citrus hosts (sweet orange, grapefruit, sour orange, lemon, and lime), where they maintained their distinct biological and genetic characteristics.

First Report of Angelonia flower break virus in Nemesia spp. and Other Ornamental Plants in California.

Between June 2006 and July 2007, ornamental plant samples were collected from four counties in California (Riverside, Sacramento, San Diego, and Santa Barbara) and tested for the presence of Angelonia flower break virus (AnFBV) using ELISA (Agdia, Inc., Elkhart, IN). Tissue samples were from propagation facilities or wholesale outlets except those from Riverside County, which were from retail stores. Thirteen positive samples were found in three varieties each of Angelonia and Nemesia spp. and seven varieties of Verbena spp., with at least one positive from each county. Foliar symptoms ranged from asymptomatic to a mild mosaic with distinct flower breaking in the Angelonia spp. Results were confirmed by reverse transcription (RT)-PCR of the coat protein gene (1) and the 1,172-bp amplicons were sequenced. Viral isolates from the three varieties of the Angelonia spp. had 98 to 99% nucleotide similarity and 99 to 100% amino acid identity to the Maryland strain of AnFBV (1; GenBank Accession No. DQ221212), with 91 to 92% nucleotide similarity and 96 to 97% amino acid identity to the Israel and Florida strains (GenBank Accession Nos. DQ223771 and DQ219415). All viral isolates from the Nemesia and Verbena spp. plants had nucleotide similarities of 96 to 98% and 98% amino acid identity to the Israel and Florida strains, with 91 to 92% nucleotide similarity to the Maryland strain. AnFBV has been previously reported in Angelonia and Verbena spp. among other hosts (1,2), but not in Nemesia spp. and not in California. This recently described carmovirus appears to be well established in the state in a variety of ornamental plant species. References: (1) S. Adkins et al. Phytopathology 96:460, 2006. (2) F. Assis-Filho et al. Plant Dis. 90:1115, 2006.

Predictions of tensile strength of binary tablets using linear and power law mixing rules.

There has recently been an increased interest in predicting the tensile strength of binary tablets from the properties of the individual components. In this paper, measurements are reported for tensile strength of tablets compressed from single-component and binary powder mixtures of lactose with microcrystalline cellulose (MCC), and lactose with two types of silicified microcrystalline cellulose (SMCC and SMCC-HD), which are different in compressibility. Measurements show the tensile strength increases with the relative density for single powders, and both with the relative density and the mass fraction of cellulose in the mixtures. It was also observed, for binary mixtures compacted at 50 and 150 MPa, that there was a slight variation in porosity with the mass fraction of celluloses. The predictions of the tensile strength of binary tablets from the characteristics of the single-components was analysed with the extended Ryshkewitch-Duckworth model by assuming both linear and power law mixing rules for the determination of the parameters "tensile strength at zero porosity and bonding capacity constant". As consequence, four models were analysed and compared with measurements using criteria based on the standard deviation from the mean values. Results showed a good prediction using a linear mixing rule combined with the power law. However, as the predictions of these models depend on the powders and the porosity range for the characterization of single-components, none of them can be systematically considered as being the best to predict binary behaviour from data for individual powders.

First Report of Nemesia ring necrosis virus in North America in Ornamental Plants from California.

During the last several years, two California propagators have detected what was believed to be the tymovirus Scrophularia mottle virus (ScrMV) in several ornamental plant species on the basis of enzyme-linked immunosorbent assay (ELISA) using a ScrMV antibody system. Symptoms were generally mild, ranging from nonsymptomatic to a mild mosaic. Our laboratory confirmed the presence of a tymovirus in one Verbena sp. and two Diascia spp. cultivars on the basis of dsRNA analysis that showed bands of approximately 6,400 and 300 nucleotides representing the genomic and coat protein subgenomic RNAs, respectively. While these plants and those that were experimentally infected (Nicotiana benthamiana and N. clevelandii) also tested positive for ScrMV by ELISA, the host range did not match that published for ScrMV, notably the lack of symptoms in Chenopodium quinoa and the lack of systemic infection in Datura stramonium. A similar host range result was reported in Europe for another tymovirus that cross reacts with ScrMV antiserum, Nemesia ring necrosis virus (NeRNV) (2). Using NeRNV specific primers (1), we used reverse transcription-polymerase chain reaction (RT-PCR) to test plants that had previously tested positive for ScrMV by ELISA and had dsRNAs typical of a tymovirus. An amplicon of the appropriate size (960 bp) for NeRNV was obtained from each of five samples. Using ScrMV specific primers, the same samples failed to amplify the expected product. We have found NeRNV in three Diascia spp., one Verbena sp., and one Nemesia sp. plants in two counties in California (Riverside and San Diego). When the RT-PCR products were sequenced, they all had 99% sequence identities to NeRNV with 4 to 7 single nucleotide changes (GenBank Accession Nos. DQ648150 to DQ648154). Notably, each of the five amplicons had changes at nucleotides 5134 (G to C) and 5549 (G to T) when compared with the European isolates of NeRNV, which did not result in any amino acid changes. To our knowledge, this is the first report of NeRNV in North America and more specifically, in California. References: (1) R. Koenig et al. J. Gen. Virol. 86:1827, 2005. (2) A. L. Skelton et al. Plant Pathol. 53:798, 2004.

Electron microscopy of satellite tobacco mosaic virus crystals: metal-coated, negatively stained and stereo pairs.

Highly purified virions of satellite tobacco mosaic virus (STMV) were found to crystallize at relatively low concentrations (300-500 microg ml(-1)) in pure water. Small crystals of these preparations were examined in the transmission electron microscope after either being rotary shadowcast with metal or negatively stained with 4% uranyl acetate. Stereo views were also obtained of both types of preparations. Stereo pairs of metal-coated crystals provided good three-dimensional images. When stereo pairs of negatively stained crystals were printed from second negatives, they provided striking images although the three-dimensional aspect was not so pronounced. Images of both types of preparations were compared with a computer-generated model of the virus. This model was based on data obtained in earlier X-ray diffraction crystallographic studies. Measurements of crystal axes on the EM images were somewhat lower than those of the computer model. It is assumed the reason for this is the dehydration of crystals during preparation for electron microscopy. The EM images did verify the type of crystal lattice determined in the X-ray diffraction studies. Conversely, knowing the exact unit cell parameters and the distribution of virions in the crystal from X-ray diffraction data aids in the further interpretation of electron micrographs of virus crystals.

Heterogeneity in the 3'-terminal untranslated region of tobacco mild green mosaic tobamoviruses from Nicotiana glauca resulting in variants with three or six pseudoknots.

Isolates of tobacco mild green mosaic tobamovirus (TMGMV) were obtained from 58 plants of Nicotiana glauca in southern California and placed in one of two groups (Small type and Large type) based on the size of the subgenomic RNA for the coat protein (CP). The CP sequence differed by no more than one amino acid for the two types, and the Small type was identical to that published for TMGMV. Thirty-six of the isolates had a double-stranded (ds)RNA profile that matched that of type TMGMV, and the nucleotide sequence of the 3' untranslated region (3'UTR) of six of these isolates was similar to the published sequence of TMGMV. Twenty-two isolates had a larger dsRNA for the CP subgenomic RNA. Six of these were sequenced and all had a repeat sequence of between 147 and 165 bases in the part of the 3'UTR that is involved in the formation of pseudoknots. These novel but common isolates are predicted to have six rather than three pseudoknots. Small types (three pseudoknots=type TMGMV) yielded twice as much virus after purification as Large types (six pseudoknots). The two groups of isolates could be distinguished in N. rustica (Large type, but not Small type gave a systemic infection), and N. clevelandii (Small type but not Large type induced systemic lethal necrosis). Almost all isolates of TMGMV used in this study were initially associated with satellite tobacco mosaic virus (STMV), and both types supported STMV experimentally.

Molecular characterization of an isolate of citrus tristeza virus that causes severe symptoms in sweet orange.

The complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (CTV) isolate SY568 was determined. The genome organization is identical to that of the previously determined CTV-T36 and CTV-VT isolates. Sequence comparisons revealed that CTV-SY568, a severe stem-pitting isolate from California, has more than 87% overall sequence identity with CTV-VT, a seedling yellows isolate from Israel. Although SY568 has an overall sequence identity of 81% with CTV-T36, a quick decline isolate from Florida, the sequence identity in the 3' half of the genome is over 90% while the sequence identity in the 5' half of the genome is as low as 56%. Based on the sequence alignments of these three isolates, sequences in the 3' half of the genome are generally well conserved, while the sequences in the 5' half are relatively divergent. Sequence data of independent overlapping clones from the CTV-SY568 genome revealed two regions with highly divergent sequences. In open reading frame 1b (RNA dependent RNA polymerase), there were 118 nucleotide differences that lead to 16 amino acid changes. In the open reading frame of the divergent coat protein gene, 5 amino acid changes result from 48 nucleotide differences. Most differences occurred in the third position of the codons, and resulted in silent amino acid substitutions. RNase protection assays demonstrated that most of the clones obtained are representative of the major RNA species of this isolate. Northern analysis indicated that CTV-SY568 accumulated more viral RNA including genomic and certain subgenomic RNAs than isolates VT or T36 in sweet orange.

Specific sequence changes in the 5'-terminal region of the genome of satellite tobacco mosaic virus are required for adaptation to tobacco mosaic virus.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.

Naturally occurring variants of satellite tobacco mosaic virus.

ABSTRACT Four natural variants of satellite tobacco mosaic virus (STMV) were compared with each other and with the type strain. Differences were detected in double-stranded RNA, single-stranded RNA, and virion electrophoretic mobility patterns, while the size and antigenicity of the coat protein were similar for all. RNase protection assays detected differences in the genomes of each of the four new variants, which differed not only from each other, but also from that of type STMV. Infectious RNA transcripts were made from complementary DNA clones of one variant (STMV 10) with a genome apparently smaller than that of type STMV. A 71-base deletion in the region that contains the 6.8-kDa protein in type STMV was detected by sequence analysis of the STMV 10 clones, a result that is confirmed by the lack of a 6.8-kDa in vitro translation product for STMV 10. Only minor sequence differences exist elsewhere in the genome compared with that of type STMV. Type STMV and STMV 10 each successfully cross-protected against the other when tobacco plants were inoculated 10 days apart.

Satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV) is a small, spherical ssRNA virus common in a natural wild plant, Nicotiana glauca or tree tobacco, in southern California and is one of the best-studied satellite viruses. It is the only satellite virus that has rod-shaped viruses (tobamoviruses) for its helper. In addition to describing the general properties of STMV, this review focuses on (a) the structural properties of the virus particle including the RNA within the particle that is partially double stranded; (b) the genetic diversity within the type strain before and after serial passage and between different field isolates; (c) the effect of experimental mutation on infectivity, replication, and symptomatology; and (d) the genetic changes that occur when the satellite virus adapts to different helper tobamoviruses.

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Comparison of Detection Methods for Citrus Tristeza Virus in Field Trees During Months of Nonoptimal Titer.

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.

Cell-to-cell transport of movement-defective cucumber mosaic and tobacco mosaic viruses in transgenic plants expressing heterologous movement protein genes.

To determine if the movement proteins (MPs) of cucumber mosaic cucumovirus (CMV) and tobacco mosaic tobamovirus (TMV) are complementary in function, transgenic plants expressing genes encoding TMV or CMV MP were inoculated with movement-defective mutants of TMV and CMV. Transgenic plants expressing the MP gene of CMV strain S (subgroup II) complemented the cell-to-cell and systemic spread of a movement-defective mutant of CMV strain Fny (subgroup I) but not the local or systemic spread of a movement-defective mutant of TMV. Plants that contained the MP gene from CMV-S were not resistant to wild-type TMV infection. When inoculated with a movement-defective mutant of TMV that produced beta-glucuronidase, transgenic plants with the CMV MP gene supported only subliminal infection. Conversely, immunodetection and in situ localization techniques revealed that transgenic plants accumulating the TMV MP supported cell-to-cell spread, but not systemic transport, of a movement-defective CMV. These studies suggest that the transgenic TMV MP shares some of the functions with the CMV MP required to transport CMV, whereas the transgenic CMV MP is deficient in functions that are needed to mobilize the spread of TMV infection.

Differences in the subcellular localization of tobacco mosaic virus and cucumber mosaic virus movement proteins in infected and transgenic plants.

Our study reveals differences in the way that tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) movement proteins (MPs) partition with cellular components separated into four fractions from different aged leaves of infected and transgenic plants. Immunoblot analyses showed that TMV and CMV MPs associated predominantly with components in the cell wall fractions from leaves of transgenic plants. In infected tissue, highest levels of TMV MP were found in the organelle fractions from young and middle-aged leaves whereas most of the CMV MP was found in the detergent wash of the cell wall fraction. These results remained consistent even when plants were doubly infected with TMV and CMV. These results imply that MPs of plant viruses from different taxonomic groups differentially associate with subcellular components and that MP produced by a viral infection is targeted to additional subcellular destinations than MP produced in transgenic plants.

Characterization of deletion and frameshift mutants of satellite tobacco mosaic virus.

A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro. Deletions and frameshift mutations in the 5' open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum, although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3' untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

Mutation analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: evidence for hotspots of genetic change.

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.

A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility.

Transgenic tobacco plants that express a gene encoding a defective mutant of the tobacco mosaic virus (TMV) movement protein which are known to be resistant to several tobamoviruses were inoculated with viruses from different taxonomic groups to determine the breadth of resistance. There were significant delays in the time of appearance of disease symptoms and/or there was reduced systemic accumulation of virus in upper leaves of plants inoculated with tobacco rattle tobravirus, tobacco ringspot nepovirus, alfalfa mosaic alfamovirus, peanut chlorotic streak caulimovirus, and cucumber mosaic cucumovirus. Conversely, tobacco plants that express a gene encoding the functional tobacco mosaic virus wild-type movement protein accelerated symptom development, enhanced the severity of symptom formation, and/or increased the accumulation of these viruses and, additionally, TMV. Our results indicate that there are similar functions among the movement proteins of a number of plant viruses despite the apparent lack of sequence similarity between them.

Effect of joint position and ligament tension on the MR signal intensity of the cruciate ligaments of the knee.

Six cadaveric lower extremities were imaged with T1-weighted spin-echo pulse sequences with the knees extended and flexed to 90 degrees. Magnetic resonance signal intensities of the anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) were compared. Changing from extension to flexion resulted in decreased signal intensity in six of six ACLs and five of six PCLs. Two of the knees were then imaged with and without tension applied to the ACL. Both specimens showed a decrease in signal intensity with tension, followed by an increase in signal intensity with release of the tension. Finally, in three of the limbs the ACL was surgically reconstructed and then imaged with and without tension applied to the tendon graft. Signal intensity decreased with tension and increased with release of the tension in all three specimens. Thus, joint position and changes in ligament tension affect the signal intensity of the ACL and PCL, generally resulting in a signal intensity decrease with tension.

Satellite tobacco mosaic virus sequence variants with only five nucleotide differences can interfere with each other in a cross protection-like phenomenon in plants.

The type strain of satellite tobacco mosaic virus (STMV) contains two major variants, designated type 5 (T5) and type 6 (T6), which can be easily distinguished by RNase protection analyses. Clones containing cDNA of representative T5 and T6 STMV genomes have only five single-base differences in the entire 1059-nucleotide genome, and RNA transcribed from each clone is highly infectious when inoculated onto tobacco plants. The different RNase protection assay patterns can be used as genetic markers to identify individual STMV variants and to follow the interactions of variants and their progeny during coinfections in plants. The study described here investigated the effects of coinoculation and various delayed inoculations of T5 and T6 variants on the composition of the progeny STMV populations in systemically infected tobacco tissues. When T5 and T6 STMV RNAs were coinoculated or inoculated with 1-hr delays, the progeny from individual plants most often contained a mixture of T5 and T6 genomes. However, when there was a 24-hr delay between inoculations, the balance of T5 and T6 components in the progeny populations shifted toward predominance of the first variant inoculated. With delays of 3 or 7 days only the first variant was evident in the progeny populations, indicating that established replication of one STMV variant interferes with replication of another in a manner similar to the cross protection phenomenon.