Complete genome sequences of two isolates of spiraea yellow leafspot virus (genus Badnavirus) from Spiraea x bumalda 'Anthony Waterer'.

The complete genome sequences of two isolates of spiraea yellow leafspot virus (SYLSV) were determined. Spiraea (Spiraea x bumalda) 'Anthony Waterer' plants showing virus-like symptoms including yellow spotting and leaf deformation were used for sequencing. The viral genome of SYLSV-MN (Minnesota) and SYLSV-MD (Maryland) is 8,017bp in length. The sequences share 95% identity at the nucleotide level. Both isolates have the same genome organization containing three open reading frames (ORFs), with ORF3 being the largest, encoding a putative polyprotein of 232 kDa with conserved domains including a zinc finger, pepsin-like aspartate protease, reverse transcriptase (RT), and RNase H. Pairwise comparisons between members of the genus Badnavirus showed that gooseberry vein banding associated virus GB1 (HQ852248) and rubus yellow net virus isolate Baumforth's Seedling A (KM078034) were the closest related virus sequences to SYLSV, sharing 73% identity at the nucleotide level. Bacilliform virions with dimensions of 150 nm × 30 nm were observed in virus preparations from symptomatic, but not asymptomatic, plants.

Genomic characterization of cycad leaf necrosis virus, the first badnavirus identified in a gymnosperm.

A previously undescribed badnavirus was isolated from Zamia fischeri showing symptoms of chlorosis, necrosis, and ringspot. The virus has bacilliform virions 30 nm in diameter and averaging 120 nm in length. The viral genome is 9227 bp in length and contains three open reading frames characteristic of members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H. The virus is tentatively named "cycad leaf necrosis virus" (CLNV). Within the genus Badnavirus, CLNV was most closely related to sugarcane bacilliform Guadeloupe D virus (FJ439817), sharing 69% identity at the nucleotide level in the RT + RNase H region. This virus is the first badnavirus reported to infect cycads, and it has the largest genome among the currently characterized badnaviruses.

Complete genome sequence of aglaonema bacilliform virus (ABV).

Aglaonema bacilliform virus (ABV), a member of the genus Badnavirus in the family Caulimoviridae, is associated with leaf deformation and chlorosis in Aglaonema modestum. The complete genome sequence of a Minnesota isolate of ABV was determined. The ABV genome is 7,178 bp in length and similar in size and organization to those of the members of the genus Badnavirus, containing three open reading frames (ORFs) with the potential to encode three proteins of 14.92, 13.33 and 207.95 kDa, respectively. The last ORF (ORF3) encodes a putative polyprotein with conserved domains, including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H domains, in that order. Phylogenetic analysis using the amino acid sequence of the ORF3 polyprotein showed that ABV clusters with several isolates of taro bacilliform CH virus (TaBCHV). Pairwise alignment using the highly conserved RT/RNase H region reveals that ABV has the highest level of identity (71%) at the nucleotide level to a Hawaiian isolate of TaBCHV.

Complete genome sequence of a previously undescribed badnavirus occurring in Polyscias fruticosa L. (Ming aralia).

A previously undescribed badnavirus was identified in plants of Polyscias fruticosa (Ming aralia) showing symptoms of mild mosaic and leaf senescence. Characteristic bacilliform virions of the Polyscias badnavirus averaging 30 × 120 nm in size were observed by transmission electron microscopy in partially purified leaf tissue extracts from symptomatic but not asymptomatic plants collected in the USA and Nigeria. The isolate from the USA was complete sequenced. The genome is 7592 bp in length and contains three open reading frames with an arrangement similar to that of other members of the genus Badnavirus. The largest open reading frame (ORF3) encodes a putative polyprotein, with predicted domains including zinc finger, aspartic protease, reverse transcriptase (RT) and RNase H, in that order. The USA and Nigeria isolates of the virus had a high level (98%) of nucleotide sequence identity in the RT+RNase H region. Within the genus Badnavirus, these viruses were most closely related to schefflera ringspot virus (SRV), sharing 63% identity at the nucleotide level. Based on the ICTV species demarcation criteria for the genus Badnavirus (more than 20% nucleotide sequence divergence in the RT+RNase H region), the Polyscias virus is proposed to be a new member of the genus, and the name polyscias mosaic virus (PoMV) is proposed. The complete genome sequence was deposited in the NCBI GenBank database under accession no. MH475918.

A phase Ib dose allocation study of oral administration of lucitanib given in combination with fulvestrant in patients with estrogen receptor-positive and FGFR1-amplified or non-amplified metastatic breast cancer.

PURPOSE: The primary objective of this multicentric dose allocation and dose expansion study was to determine the MTD and the DLTs of the lucitanib (a tyrosine kinase inhibitor of the FGFR/VEGFR/PDFGR pathways)/fulvestrant combination. METHODS: Postmenopausal women with ER+/HER2- mBC, who have relapsed during or after treatment with fulvestrant, were eligible. The study had a dose allocation part to assess the tolerability of the combination followed by a dose expansion part. RESULTS: Eighteen patients with ER+, mBC were enrolled; median age was 66 years, 50% had a PS: 0 and all had received previous endocrine treatment. The study was prematurely terminated after 18 patients (15 in part 1 and 3 in part 2) based on preclinical experiments that failed to confirm the hypothesis that addition of lucitanib would reverse sensitivity to endocrine treatments. Based on data of global lucitanib development, it was decided to stop the dose allocation at 12.5 mg and to start the dose expansion part at 10 mg/day. The most common grade ≥ 3 toxicities (> 10% of patients) were hypertension (78%) and asthenia (22%). All patients required at ≥ 1 interruption, 13 patients (72%) required ≥ 1 dose reduction. Three patients (72%) withdrew from the study for AEs (at 10 mg). Three patients achieved a confirmed PR (10 mg n = 1; 12.5 mg n = 2). CONCLUSION: Although the combination is feasible it requires close monitoring of the patients for the management of adverse events. Further investigation is required to better understand the potential role of FGFR inhibition in reversing resistance to endocrine treatment.

Identification of Tobacco streak virus in Cranberry and the Association of TSV with Berry Scarring.

Cranberry plants bearing disfigured, scarred fruit were reported by growers in the major cranberry-growing region of central Wisconsin in July 2012. Plants bearing scarred fruit have since been observed in Massachusetts and New Jersey. Three complementary methods provided evidence of Tobacco streak virus (TSV) in symptomatic plants: (i) leaves and scarred berries tested positive for TSV by double-antibody sandwich enzyme-linked immunosorbent assay; (ii) quasi-isometric particles approximately 33 nm in diameter were extracted from leaves of symptomatic plants and visualized using transmission electron microscopy; and (iii) coat protein gene sequence analysis revealed 94 to 99% nucleotide similarity with reference TSV sequences. In newer cultivars, 99% of uprights with scarred berries tested positive for TSV. In older cultivars, 31% of uprights with scarred berries tested positive for TSV and the remaining 69% of uprights with scarred berries tested positive for Blueberry shock virus. TSV overwintered in cranberry plants, and leaves, pollen, and fruit tested positive for TSV the year following symptom occurrence. Attempts to inoculate cranberry using infected pollen or sap as inoculum failed, but several herbaceous hosts tested TSV positive following mechanical inoculation. Phylogenetic analysis of the coat protein gene of 26 TSV isolates from various cultivars of cranberry in Wisconsin, New Jersey, and Massachusetts revealed diversity. This work provides information that will be useful in understanding the epidemiology of TSV in cranberry and in the development of management strategies.

First Report of Tobacco etch virus Infection in Coleus in the United States.

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) 'Rustic Orange' obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic 'Rustic Orange' leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5'GGBAAYAATAGTGGNCAACC3') and PCPR1 (5'GGGGAGGTGCCGTTCTCDATRCACCA3') (1) and total RNA extracted from 'Rustic Orange' leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in 'Rustic Orange' was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of 'Rustic Orange' (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 'Rustic Orange' plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54. 2005. (2) D. S. Mollov et al. Plant Dis. 91:754. 2007.

First Report of Tobacco rattle virus in Sedum in Minnesota.

Sedums (Sedum spp.; Crassulaceae) are perennial landscape plants that are grown widely because they are drought tolerant and winter hardy. Plants of Sedum 'Matrona' showing faint foliar ringspot symptoms were collected at a nursery retail outlet in St. Paul, MN in July 2008 and tested for possible viral infection by transmission electron microscopic (TEM) examination of negatively stained, partially purified leaf tissue extracts (1). The only virus-like particles observed were rigid, rod-shaped particles similar to those of Tobacco rattle virus (TRV) and other tobraviruses. A random sample of 100 measurements showed particles 20 nm in diameter with two modal lengths of 115 nm and 175 nm. These virus-like particles were confirmed to be those of TRV by immunosorbent electron microscopy (1) using antiserum to TRV (ATCC PVAS 75) and by reverse transcription (RT)-PCR using total RNA extracted with the RNeasy Kit (Qiagen, Valencia, CA) and primers that yield a 462-bp amplicon from TRV RNA 1 (4). An amplicon of the expected size was obtained by RT-PCR and its nucleotide sequence (GenBank Accession No. GQ268817) had 95 to 99% identity to published TRV sequences (AAW13192 and AAB48382). Two additional amplicons generated by RT-PCR from separate plants were identical in size and nucleotide sequence to the first. On the basis of virion morphology, serological relatedness, and sequence identity, the virus associated with mild ringspot symptoms in sedum was identified as an isolate of TRV. To our knowledge, this represents the first report of TRV incidence in sedum. Although Arabis mosaic virus is the only other virus reported to occur in sedum (2), we have observed numerous, flexuous filamentous 750 to 800 nm virus-like particles in partially purified extracts of a range of sedums showing mild mosaic and/or vein-clearing symptoms in Minnesota. Similar virus-like particles were not observed by TEM in partially purified extracts from TRV-infected 'Matrona' plants, suggesting that they did not contribute to the symptoms observed. We have reported previously (3) the occurrence of TRV in a variety of widely grown perennial ornamentals that provide potential sources of inoculum for spread of this virus by nematode vectors (Trichodorus and Paratrichodorus spp.) that occur commonly in garden soil, and Sedum is now added to the list of potential TRV reservoir plants. References: (1) Y. S. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. Gera et al. Acta Hortic. 722:175, 2006. (3) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (4) D. J. Robinson. J. Virol. Methods 40:57, 1992.

Banana streak virus Identified for the First Time in Peru in Cavendish Banana (Musa AAA).

In October of 2005, a field survey was done in the province of Piura in northern Peru to determine the cause of a disease known locally as "mosaico" that was affecting organic Cavendish banana (Musa AAA) grown for the export market. Disease symptoms consisted of pronounced chlorotic and necrotic lesions on leaves of affected plants. Twenty-four farms were visited, and at each location, 10 randomly selected plants at flowering stage were evaluated for disease incidence and severity. Plants showing virus-like symptoms were observed in 18 of the 24 locations (75%). Fifty-two banana leaf samples, 27 from plants showing virus-like symptoms and 25 from asymptomatic plants, were tested for the presence of Banana streak virus (BSV), Cucumber mosaic virus (CMV), and Banana mild mosaic virus (BanMMV) by immunosorbent electron microscopy (ISEM) using partially purified leaf tissue extracts (2).The same extracts were also tested by immunocapture PCR (IC-PCR) for presence of BSV and specific BSV isolates (BSV-OL, BSV-GF, BSV-IM, and BSV-CAV) using badnavirus-specific degenerate primers and BSV isolate-specific primers, respectively (1). Seventeen of 27 leaf samples showing virus-like symptoms (63%) tested positive for BSV by ISEM and IC-PCR using badnavirus, but not isolate-specific, primers. The symptoms on the 10 samples that tested negative were not typical of BSV infection. One asymptomatic leaf sample (4%) also tested positive for BSV. To validate the PCR results, the nucleotide sequence of the amplicon from a plant showing the most prevalent foliar symptom type was determined. This sequence (GenBank Accession No. DQ674317) had ≤86% homology to the corresponding ORF III polyprotein region of BSV and other badnaviruses. Neither CMV nor BanMMV was detected in any of the 52 samples tested. From these results, it was concluded that "mosaico" disease of organic Cavendish bananas in northern Peru is associated frequently with BSV infection and that there is a high incidence of BSV infection in this area. To our knowledge, this is the first report of BSV occurrence in Peru. It was both surprising and interesting that neither BSV-OL nor BSV-GF, the two BSV isolates found most commonly in banana (Musa AAA) and plantain (Musa AAB) in South and Central America (B. E. L. Lockhart, unpublished), was detected in Cavendish banana in northern Peru. Failure to detect BSV-OL and BSV-GF suggests that field infection may be due to vertical transmission by clonal propagation rather than to horizontal transmission from local plantain and that control of "mosaico" disease could therefore be achieved by use of virus-free planting material. References: (1) A. D. W. Geering et al. Phytopathology 90:921, 2000. (2) B. E. L. Lockhart et al. Phytopathology 82:921, 1992.

Occurrence of Arabis mosaic virus in Hostas in the United States.

Hostas (Hosta spp.) are one of the most widely grown and economically important landscape perennials in the nursery industry in North America. Several viruses including Hosta virus X (HVX), Tobacco rattle virus (TRV), Tobacco ringspot virus (ToRSV), Tomato ringspot virus (TomRSV), Impatiens necrotic spot virus (INSV), and Tomato spotted wilt virus (TSWV) are known to occur in hostas (4). This report confirms the occurrence of an additional virus, Arabis mosaic virus (ArMV), in hostas in North America. This virus was first identified during the summer of 2004 in Hosta fortunei 'Sharmon' in several garden centers in Minneapolis and St. Paul, MN. Entire lots of this variety, numbering several dozen plants, showed symptoms consisting of blanching of the foliage similar to those caused by ToRSV and TomRSV infection (4). Symptoms persisted throughout the growing season. Virus-like particles, 28 to 30 nm in diameter, were observed by electron microscopy in partially purified extracts of symptomatic leaf tissue following fixation with 5% glutaraldehyde and negative staining with 2% sodium phosphotungstate, pH 7.0. Particles had an angular outline and some were penetrated by stain. No other virus-like particles were observed in these extracts. The particles were identified as those of ArMV. Identification was made using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and immunosorbent electron microscopy (ISEM) with antiserum to ArMV (PVAS-587) obtained from the American Type Culture Collection, Manassas, VA. In the spring and summer of 2005, ArMV was again identified as described above in 'Sharmon', H. undulata 'Albomarginata' samples from Minnesota, Michigan, and Nebraska, and H. 'Marion Bachman' and H. 'Touch of Class' from two wholesale nurseries in Minnesota. Symptoms in these hosta cultivars were similar to those observed in 'Sharmon' and were accompanied by stunting and leaf deformation. A portion of the coat protein (CP) gene of the ArMV isolate from 'Sharmon', designated ArMV-H, was amplified using reverse transcription-polymerase chain reaction (RT-PCR) with ArMV-specific CP primers (3) and total RNA extracted with a RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Amplicons of the expected size (220 bp) were cloned and five clones were sequenced. Nucleotide sequence identities of the ArMV-H CP sequence to corresponding ArMV databank entries varied from 94 to 88% (Genbank Accession Nos. AY017339 and D10086 and X55460 and X81815, respectively). Interestingly, the hosta ArMV isolate was not transmitted by mechanical inoculation to diagnostically susceptible indicator plants (cucumber, tobacco, and petunia) (2) or to hosta (H. undulata 'Albormarginata', H. 'Honeybells', and H. 'Royal Standard'). Testing by using ELISA and ISEM showed that 'Sharmon' source plants contained high levels of ArMV antigen and virions, and a high percentage of virions were not penetrated by negative stain, indicating that they were not empty (i.e., devoid of RNA). It appears that ArMV-H may be transmitted only vertically, (i.e., clonal propagation) and this raises some interesting questions about the molecular basis of this anomaly. An isolate of ArMV from hops was similarly reported to have a very restricted host range (1) suggesting a possibility of a common mechanism of host range restriction. References: (1) K. R. Bock. Ann. Appl. Biol. 57:431, 1966. (2) A. A. Brunt et al. Viruses of Plants. CAB Internacional Mycological Institute, Wallingford, UK, 1995. (3) P. Kominek et al. Acta Virol. 47:199, 2003. (4) B. E. L. Lockhart and S. Currier. Acta Hortic. 432:62, 1996.

Three Previously Unrecorded Viral Diseases of Astilbe, Fuschia, and Thermopsis Species in Minnesota.

Interest in virus diseases of perennial ornamentals has been increasing because of their increasing monetary value, because wholesale producers perceive an advantage in marketing disease-free stock, and because widespread international movement of these plants carries the risk of introduction of exotic viruses. In an ongoing study to identify and document viral diseases of perennial ornamentals used in the United States commercial horticultural industry, three virus-like diseases of astilbe (Astilbe chinensis), fuschia (Fuschia cv. Gartenmeister) and false lupine (Thermopsis caroliniana) occurring in Minnesota were investigated. Symptomatic plants were selected from lots in commercial greenhouses and garden centers in several locations in Minnesota. Astilbe with systemic chlorosis was found to be infected with Tobacco ringspot virus (TRSV). Fuschia with leaf mottling and leaf deformation was found to be infected with Cucumber mosaic virus (CMV), and false lupine with mosaic and leaf deformation symptoms was found to be infected with Bean yellow mosaic virus (BYMV). Identification of the three viruses was based on: 1) virion presence and morphology in partially purified leaf extracts using electron microscopy (EM) and immunosorbent electron microscopy (1); 2) enzyme-linked immunosorbent assay using crude leaf extracts; and 3) biological properties, including symptoms produced in indicator plants. Antisera to BYMV (ATCC PVAS-368), CMV (ATCC PVAS-30), and TRSV (ATCC PVAS-157) were obtained from the American Type Culture Collection, Manassas, VA. No other virus-like particles were observed with EM in partially purified leaf extracts of the three plants, no virus-like particles were observed in similar preparations from asymptomatic plants, and indicator plant tests did not indicate the presence of any other mechanically transmissible viruses. The TRSV isolate from astilbe and the BYMV isolate from false lupine produced typical symptoms on indicator plants susceptible to known isolates of these two viruses. The CMV isolate from fuschia was similar to previously described isolates of CMV (2) in most respects and was readily transmitted in a nonpersistent manner by Myzus persicae, but was unusual in that it did not infect Nicotiania benthamiana, N. glutinosa, and tomato, which are normally highly susceptible to infection by CMV. The identity of the fuschia CMV isolate was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR) amplification with CMV-specific oligonucleotide primers (3). The PCR product was of the predicted size (500 bp) and was cleaved by restriction digestion with EcoRI, suggesting that the fuschia virus is a Type II CMV isolate (3). To my knowledge, this is the first report of TRSV infection in astilbe, CMV infection in fuschia, and of a viral disease of false lupine. References: (1) Y. C. Ahlawat et al. Plant Dis. 80:590, 1996. (2) A. A. Brunt et al. Viruses of Plants. CAB International, Wallingford, UK, 1995. (3) S. Wylie et al. Aust. J. Agric. Res. 44:41, 1993.

Identification of genetic markers linked to banana streak disease expression in inter-specific Musa hybrids.

Recently-introduced inter-specific Musa hybrids, bred for improved yield and resistance to diseases, have been found to be widely infected with banana streak virus (BSV), the causal agent of banana streak disease (BSD). One hypothesis suggests: (1) that BSD occurrence in these inter-specific hybrids results from activation of BSV-Ol endogenous pararetrovirus sequences (EPRV) integrated into the Musa genome rather than from external sources of infection, and (2) that the process of genetic hybridisation may be one factor involved in triggering episomal expression of the BSV integrants. In order to test this hypothesis we carried out a genetic analysis of BSD incidence in a F1 triploid ( Musa AAB) population produced by inter-specific hybridisation between virus and disease-free diploid Musa balbisiana (BB) and tetraploid Musa acuminata (AAAA) parents. Half of the F1 progeny of this cross expressed BSV particles. Using PCR amplification to determine the presence or absence of BSV-Ol EPRVs, it was determined that this endogenous sequence was specific to the M. babisiana genome and occurred in a homozygous state. Using bulk segregant analysis, ten AFLP markers co-segregating with the absence and/or presence of BSV infection were identified in the M. balbisiana genome, but were absent from the M. acuminata genome. Seven of these markers segregated with the presence of a BSV particle and three with the absence of BSV particles. Analysis of the segregation of these markers using a test-cross configuration allowed the construction of a genetic map of the linkage group containing the locus associated with BSV infection in the F1 hybrid population. These data indicate that a genetic mechanism is involved in BSV appearance, and suggest that a monogenic allelic system confers the role of carrier to the M. balbisiana parent.

A New Badnavirus in Ribes Species, its Detection by PCR, and its Close Association with Gooseberry Vein Banding Disease.

Gooseberry vein banding disease (GVBD) affects Ribes species and cultivars worldwide. It is the second most important virus-like disease in these crops after black currant reversion disease. In this paper, we describe a bacilliform virus, Gooseberry vein banding associated virus (GVBAV), which is associated closely with GVBD, and provide evidence that GVBAV is a distinct species within the genus Badnavirus. Purified GVBAV particles were ca. 120 × 30 nm in size and contained dsDNA. The sequence of a 1.5-kb DNA fragment amplified from viral genomic DNA was similar to those of a wide range of badnaviruses and contained motifs characteristic of the RNase H domain of the badnavirus open reading frame (ORF) III polyprotein. Phylogenetic analyses suggest that GVBAV is most closely related to Spiraea yellow leaf spot virus. Using sequence derived from the polymerase chain reaction (PCR)-amplified DNA fragment, virus-specific primers were designed. These primers were used in PCR to assay for GVBAV in a range of Ribes germplasm affected with GVBD, with other unrelated virus-like diseases and viruses found in Ribes, and in healthy plants. GVBAV was detected in all of 58 GVBD-affected plants from diverse sources, but not from healthy Ribes plants nor from plants infected with other viruses.

Characterization and genomic analysis of tobacco vein clearing virus, a plant pararetrovirus that is transmitted vertically and related to sequences integrated in the host genome.

A previously undescribed caulimo-like virus was identified in the hybrid tobacco species Nicotiana edwardsonii, and was named tobacco vein clearing virus (TVCV) after the symptoms associated with its occurrence in this plant. The virions of TVCV are 50 nm in diameter and are composed of a 45 kDa capsid protein and a 7767 bp dsDNA genome. Each strand of the genome is interrupted by a site-specific discontinuity. In genome sequence and arrangement of ORFs TVCV was most similar to cassava vein mosaic virus, indicating that TVCV is a pararetrovirus. No serological relationship was detected between TVCV and any other caulimoviruses, including petunia vein clearing virus, which has similar biological properties. In N. edwardsonii TVCV was seed-transmitted to 100% of progeny plants, but was not transmitted by mechanical inoculation, grafting or Myzus persicae to any of seven other Nicotiana spp. Genomic DNA of TVCV hybridized to genomic DNA of N. edwardsonii and of N. glutinosa, its male parent, but not to genomic DNA of N. clevelandii, the female parent. TVCV has 78% sequence identity with pararetrovirus-like sequences that are present in high copy number in the N. tabacum genome, and TVCV genomic DNA hybridized to genomic DNA of N. tabacum and N. rustica. These observations suggest that the episomal form of TVCV may arise from integrated pararetroviral elements present in N. edwardsonii, that these integrants were inherited from the male parent N. glutinosa, and that these elements are related but not identical to pararetroviral elements occurring in other Nicotiana spp.

Inhibition of L-homocysteic acid and buthionine sulphoximine-mediated neurotoxicity in rat embryonic neuronal cultures with alpha-lipoic acid enantiomers.

In the present report, we have set out to investigate the potential capacity of both the oxidised and reduced forms of RS-alpha-lipoic acid, and its separate R-(+) and S-(-)enantiomers, to prevent cell death induced with L-homocysteic acid (L-HCA) and buthionine sulphoximine (BSO) in rat primary cortical and hippocampal neurons. L-HCA induced a concentration-dependent neurotoxic effect, estimated by cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, in primary neurons, but was significantly more toxic for hippocampal (EC(50)=197 microM) compared with cortical neurons (EC(50)=1016 microM) whereas D-HCA demonstrated only moderate (<20%) toxicity. On the other hand, cortical and hippocampal cultures were equally susceptible (341 and 326 microM, respectively) to the neurotoxic action of BSO. Antioxidants including butylated hydroxyanisole, propyl gallate and vitamin E protected cells against the neurotoxic effect of L-HCA and BSO. However, N-acetyl-cysteine and tert-butylphenyl nitrone, although capable of abrogating L-HCA-mediated cell death showed no protective effect against BSO-mediated toxicity. RS-alpha-lipoic acid, RS-alpha-dihydrolipoic acid and the enantiomers R-alpha-lipoic acid and S-alpha-lipoic acid protected cells against L-HCA-mediated toxicity with EC(50) values between 3.1-8.3 microM in primary hippocampal neurons and 2.6-16.8 microM for cortical neurons. However, RS-alpha-lipoic acid, RS-alpha-dihydrolipoic acid, and S-alpha-lipoic acid failed to protect cells against the degeneration induced by prolonged exposure to BSO, whereas the natural form, R-alpha-lipoic, was partially active under the same conditions. The present results indicate a unique sensitivity of hippocampal neurons to the effect of L-HCA-mediated toxicity, and suggest that RS-alpha-lipoic acid, and in particular the R-alpha-enantiomeric form is capable of preventing oxidative stress-mediated neuronal cell death in primary cell culture.

Dicentra, Epimedium, and Heuchera: New Perennial Ornamental Hosts of Tobacco rattle virus in the United States.

Yellow ringspotting and concentric line patterns in plants of Dicentra (bleeding heart), Epimedium (barrenwort), and Heuchera (coral bells) from commercial nurseries and home gardens in Minnesota, Michigan, and Massachusetts were associated with infection by Tobacco rattle virus (TRV), which was identified by particle morphology, enzyme-linked immunosorbent assay and immunosorbent electron microscopy. No other viruslike particles were observed by electron microscopy in partially purified preparations of TRV-infected leaf tissue, and TRV was not detected in asymptomatic plants. This is the first report of TRV occurrence in Dicentra in the United States and the first report of TRV occurrence in Epimedium and Heuchera. In previous reports (1,2) we have called attention to the increasing incidence of TRV in vegetatively propagated perennial ornamental plant species in the United States and to the potential for virus spread to crops such as potato, in which TRV has not been reported in the midwestern United States. It is possible that increased international trade in vegetatively propagated ornamental plants may be resulting in the introduction of TRV and other exotic viruses into the United States and elsewhere. It is also possible that the natural occurrence of TRV in North America may be actually more widespread than has been reported. References: (1) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (2) B. E. Lockhart and J. A. Westendorp. Plant Dis. 82:712, 1998.

Anemone - an Additional Perennial Ornamental Host of Tobacco Rattle Virus in the U.S.

A previous report (1) drew attention to the occurrence in the U.S. of tobacco rattle tobravirus (TRV) in several perennial ornamentals that move freely in international trade. Here we report the occurrence of TRV in an additional host plant of this type. The virus was identified in anemone (Anemone × hybrida cv. Honorine Jobert) with leaf symptoms consisting of chlorotic blotches, chlorotic line patterns, and distortion. Characteristic tobravirus-like particles with modal lengths of 50, 80, 130, and 180 nm were observed by electron microscopy in partially purified extracts of symptomatic but not of asymptomatic plants. These particles reacted specifically with antibodies to TRV and pea early-browning (PEBV) tobraviruses in immunoelectron microscopic (IEM) assays, produced typical TRV-induced symptoms in Nicotiana benthamiana and N. clevelandii, in which similar particles were detected by IEM, but, like other TRV isolates, did not infect pea (2). No other viruslike particles were observed in partially purified extracts of symptomatic anemone plants. This is the first report of TRV infection in anemone. While TRV occurs widely in Europe, it had been identified previously in the U.S. only in Oregon, Washington, and California (1,2,3). This virus is an important pathogen of several crops, including potato. Several areas of the Midwest, including Minnesota and Wisconsin, have significant seed-potato industries, and further introduction and possible dissemination of TRV pose potential regulatory and quarantine issues. This example of the movement of an exotic pathogen of potential economic importance into new areas underlines the need for closer monitoring of plant material entering international trade in an era of increasing globalization. References: (1) B. E. Lockhart et al. Plant Dis. 79:1249, 1995. (2) B. E. L. Lockhart and H. U. Fischer. Phytopathology 66:1391, 1976. (3) J. M. Crosslin and P. E. Thomas. Am. Potato J. 72:605, 1995.

The N-terminal portion of the 216-kDa polyprotein of Commelina yellow mottle badnavirus is required for virus movement but not for replication.

Commelina yellow mottle virus (CoYMV) is the type member of the badnaviruses, a genus of plant pararetroviruses. The N-terminus of the polyprotein encoded by ORF III has limited similarity to known cell-to-cell movement proteins. To test the hypothesis that the N-terminus is required for viral movement, the phenotypes caused by mutations constructed in this region were determined. Similar to mutants affected in the reverse transcriptase, mutants affected in the putative movement protein were unable to cause a systemic infection. However, when the abilities of the mutated viral genomes to direct virion assembly and replication were tested using an in vitro stem-culture system, the mutants affected in the putative movement protein were found to assemble virions, whereas the reverse transcriptase mutants were unable to do so. Moreover, the putative movement protein mutants were shown to be replication competent by detection and mapping of one of the genomic discontinuities that are the hallmark of replication by reverse transcription. Thus the N-terminal region of ORF III is required for the systemic movement but not for the replication of CoYMV.

The ORF I and II proteins of Commelina yellow mottle virus are virion-associated.

Antibodies were prepared against bacterially expressed Commelina yellow mottle badnavirus (CoYMV) proteins. Antiserum against purified virions and antiserum against the C-terminus of the putative coat protein-encoding region of ORF III detected the same virus-specific proteins, indicating that the CoYMV coat protein is encoded in ORF III. In addition to the two major forms of the coat protein (37 and 39 kDa), several high molecular weight virus-specific proteins were detected when virions were isolated without chloroform treatment. These proteins are possible ORF III polyprotein processing intermediates and might be associated with "immature" virions which are eliminated by chloroform treatment. As predicted by the genomic sequence, a 20-kDa virus-specific protein was detected by an antiserum raised against the C-terminus of the putative ORF I protein. Results of filtration experiments suggest that the ORF I protein is equally associated with virions and with plant component(s). The association between the ORF I protein and the virions was further confirmed using immunosorbent electron microscopy and immunogold labeling. The ORF I protein was not detected in virus preparations treated with chloroform, and colocalized with virions containing immature coat protein on sucrose-cesium sulfate density gradients, suggesting that it is associated with immature virions. An antiserum raised against the putative ORF II gene product detected a 15-kDa virus-specific protein whose association with the virions was unaffected by chloroform treatment. The ORF II protein was found to be sensitive to some protease(s) that copurified with the virions, and protease inhibitors preventing this degradation have been identified.

Amnesia induced in mice by centrally administered beta-amyloid peptides involves cholinergic dysfunction.

Substantial evidences suggest that the increased cerebral deposition, and neurotoxic action of the beta-amyloid peptide, the major constituent of senile plaques, may represent the underlying cause of the cognitive deficits observed in Alzheimer's disease. Herein, we attempted to verify this hypothesis by inducing a potential Alzheimer's-type amnesia after direct intracerebroventricular administration of aggregated beta 25-35-amyloid peptide in mice. In this aim, mnesic capacities were evaluated after 6-13 days, using spontaneous alternation in the Y-maze, step-down type passive avoidance and place learning in a water-maze. Pretraining administration of aggregated beta 25-35 peptide induced dose-dependent decreases in both alternation behaviour and passive avoidance, at doses of 3 and 9 nmol/mouse. A reduced but still significant impairment was observed when the peptide was not aggregated, or 'aged', by preincubation for 4 days at 37 degrees C. The beta 1-28 peptide, at 3 nmol/mouse, also induced a marked decrease in step-down latency. Posttraining, but not preretention, administration of beta 25-35 peptide also significantly impaired learning. The beneficial effects of cholinergic agents on beta 25-35-induced amnesia was examined using the cholinesterase inhibitor tacrine (THA, 1.3 and 4.3 mumol/kg i.p.) and the nicotinic receptor agonist (-)-nicotine (NIC, 0.06 and 0.2 mumol/kg i.p.). Both drugs induced a dose-dependent abrogation of the beta 25-35-induced decreases in alternation behaviour and passive avoidance. Furthermore, THA, at 1.3 mumol/kg, and NIC, at 0.2 mumol/kg, also reversed the beta 25-35-induced impairment of place learning and retention in the water-maze. Histological examination of Cresyl violet-stained brain sections indicated a moderate but significant cell loss within the frontoparietal cortex and the hippocampal formation of mice treated with aged beta 25-35 peptide (9 nmol). Examination of Congo red-stained sections in the same animals demonstrated the presence of numerous amyloid deposits throughout these brain areas. These results confirm that the deposition of beta-amyloid peptide in the brain is in some way related to impairment of learning and cholinergic degeneration and suggest that the [25-35] fragment of the beta-amyloid protein, sufficient to induce neuronal death in cultures, also induces an Alzheimer's-type amnesia in mice.