Molecular analysis of the cucumber leaf spot virus genome.

Full-length clones of the genome of the Aureusvirus, Cucumber leaf spot virus (CLSV), have been constructed and infectious T7 polymerase derived synthetic transcripts have been produced. Mutational analysis of the genome indicates a role for p84 in viral RNA replication, the CP in systemic movement, p27 in viral cell-to-cell movement and p17 in symptom induction. A CLSV mutant lacking ORFs for the CP, p27 and p17 (CLSV YX) was capable of replication and systemic movement in transgenic Nicotiana benthamiana plants expressing the Red clover necrotic mosaic virus (RCNMV) movement protein (MP) suggesting that p25 and p84 are sufficient for viral RNA replication and that the RCNMV MP can permit CLSV cell-to-cell as well as systemic movement. Moreover, CLSV YX induced severe necrosis in both inoculated and uninoculated leaves of transgenic plants suggesting that CLSV p25 and/or p84 are important determinants of the necrotic phenotype. Another mutant similar to CLSV YX but expressing only limited amino-terminal portions of CP, p27 and p17 failed to produce necrosis or to move systemically in RCNMV MP transgenic N. benthamiana plants. These results suggest that these short translated regions or cis-acting sequences present in the CLSV CP, p27 and/or p17 ORFs suppress the necrosis induced by p25/p84 and also suppress systemic movement mediated by the RCNMV MP.

Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test.

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.

Spontaneous deletion enhances movement of a cucumber necrosis virus based chimera expressing the red clover necrotic mosaic virus movement protein genedagger.

Summary The 35-kDa movement protein (MP) gene of red clover necrotic mosaic virus (RCNMV) and 3' flanking sequence were inserted in a cucumber necrosis virus (CNV) deletion mutant lacking a large portion of the coding region for the MP. Nicotiana benthamiana plants inoculated with chimeric synthetic transcripts of the resulting hybrid cDNA clone (M5/RM2) developed both local and systemic symptoms and accumulated high levels of chimeric viral RNA. Reverse transcriptase polymerase chain reaction (RT-PCR) and sequence analysis of viral RNA extracted from systemically infected leaves of four different plants revealed that in each plant a large portion (305, 308, 315 or 127 nts) of the 3' terminus of the inserted sequence spontaneously deleted during infection. In three of the deletion derivatives, the truncated RCNMV MP open reading frame (ORF) was fused in-frame with the remaining portion of the 3' terminal region of CNV MP ORF. The movement efficiencies of M5/RM2, a cloned copy of one of the deletion derivatives (ClM5/RM2dd1), and a stop codon mutant of ClM5/RM2dd1 (ClM5/RM2dd1stop), which prevents translational fusion to the CNV MP, were compared and it was determined that deletion of RCNMV MP sequences in conjunction with fusion to CNV MP sequences increases the movement efficiency of the chimeric virus genome. Absence of the C-terminal region of the RCNMV MP in RCNMV RNA-2 abolished RCNMV movement. However, movement could be complemented in trans if cells were coinoculated with ClM5/RM2dd1. Complementation of RCNMV movement did not occur using ClM5/RM2dd1stop, suggesting a role for appended CNV MP sequences in movement of the RCNMV genome. The ability of the CNV replicase to delete unnecessary or deleterious RCNMV sequences and to append the required CNV MP sequences reinforces the role of RNA recombination in the adaptation and evolution of viral genomes.

Both RNA rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants.

The putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (CLSV) was inserted into an infectious cucumber necrosis tombusvirus (CNV) cDNA clone containing a deletion in the cell-to-cell movement protein gene. Approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric CNV/CLSV cDNA clones developed systemic symptoms 7-19 days postinoculation. Reverse transcription-polymerase chain reaction and sequence analysis of virus obtained from systemically infected leaves indicated that both point mutation and RNA rearrangement (deletion) contributed to the formation of movement competent CNV/CLSV hybrid viruses. The hybrid viruses were found to accumulate to high levels in infected plants, to form stable virions, and to be mechanically transmissible. In addition, a hybrid virus that lacked 50 amino acids at the carboxyl-terminal region of CLSV p27 was still capable of facilitating CNV movement. These data provide experimental evidence for the role of CLSV p27 in viral cell-to-cell movement and demonstrate that p27 can enable efficient movement of the CNV genome. Moreover, the data show that RNA rearrangements known to occur during CNV RNA replication can contribute to rapid evolution of the CNV genome.

A cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain.

Little is currently known regarding the specific interactions that govern transmission of plant viruses by their vectors. A cucumber necrosis virus (CNV) variant (LL5) deficient in fungal transmissibility has been isolated from mechanically passaged CNV and characterized. Although LL5 accumulates to wild-type (WT) levels, is capable of rapid systemic infection, and produces stable, highly infectious particles, it is only inefficiently transmitted by Olpidium bornovanus zoospores. The LL5 coat protein (CP) gene was amplified by RT-PCR and cloned in place of the WT CNV CP gene in an infectious CNV cDNA clone. Particles derived from this construct also failed to be efficiently transmitted. The LL5 CP gene was sequenced and found to contain two amino acid substitutions relative to WT CNV CP. One substitution (Phe to Cys) occurred in the arm region and another (Glu to Lys) in the shell domain. These amino acid changes were separately introduced into the WT CNV genome through in vitro mutagenesis and it was found that the Glu to Lys change in the LL5 CP shell domain is largely responsible for the loss of transmissibility. In vitro binding assays were developed to determine if the defect in transmissibility was due to a defect in binding zoospores. LL5 particles were found to bind less efficiently than WT CNV. Furthermore, the nontransmissible tomato bushy stunt virus did not detectably bind zoospores. These binding studies suggest that the specificity of CNV transmission by O. bornovanus occurs through specific recognition of a putative zoospore receptor.

Genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the Tombusviridae.

The complete nucleotide sequence of cucumber leaf spot virus (CLSV) has been determined and the sizes and locations of predicted viral proteins deduced. The genome consists of 4432 nucleotides and contains five long ORFs. The 5' proximal ORF encodes a 25 kDa product that terminates in an amber codon which may be readthrough to produce an 84 kDa protein (ORF 2). ORF 3 codes for the 41 kDa coat protein (CP). ORFs 4 and 5 are completely overlapping at the 3' terminus and code for 27 and 17 kDa products, respectively. The CLSV genome structure is similar to that of tombusviruses and nearly identical to pothos latent virus (PoLV), a newly proposed, atypical, member of the Tombusviridae. It is proposed that CLSV and PoLV be considered strains of a new tombusvirus species. Amino acid sequence comparisons of the CLSV CP and the CPs of several small spherical plant viruses suggest that CLSV is most closely related to melon necrotic spot carmovirus (MNSV), red clover necrotic mosaic dianthovirus (RCNMV) and cucumber necrosis tombusvirus (CNV). These viruses, like CLSV, are transmitted by the soil inhabiting fungus, Olpidium bornovanus.

Erythrocyte sedimentation rate, an underestimated tool in chronic renal failure.

BACKGROUND: Erythrocyte sedimentation rate is widely used in the general population. It has seldom been studied in patients with chronic renal failure. The purpose of this study was to assess its usefulness in haemodialysis patients. METHODS: Forty-five haemodialysis patients with no evidence of acute or chronic inflammatory illness were studied. Nine were diabetic, and 12 used a non-biocompatible membrane. Erythrocyte sedimentation rate was determined, using a modified Westergren method. Plasma fibrinogen concentration, complete blood count, and serum chemistries were also studied. RESULTS: Erythrocyte sedimentation rate was normal or mildly elevated in most of our patients, with a median of 30 mm/h. Linear analysis found positive correlation between erythrocyte sedimentation rate and fibrinogen concentration, globulin level, platelet, and white cell counts, and negative correlation with haematocrit. Fibrinogen concentration was normal in 22 patients, and moderately elevated in 14. It was significantly higher in diabetic patients, or those using a non-biocompatible membrane. The same positive correlations were found for fibrinogen concentration as for erythrocyte sedimentation rate. CONCLUSIONS: We conclude that erythrocyte sedimentation rate can be used in haemodialysis patients much in the same way as in the general population, as it is influenced by the same factors, and its baseline value is lower than previously reported. The lower concentration of fibrinogen, an independent predictor of cardiovascular risk, in patients treated with biocompatible membranes may be of clinical relevance.

Neutral-lipid transfers and cholesteryl ester transfer protein in hemodialyzed patients.

Abnormalities in cholesteryl ester transfers may play a role in the development of atherosclerosis observed in patients with end-stage renal failure treated by chronic hemodialysis. Net neutral-lipid transfers and cholesteryl ester transfer protein activity and mass were investigated in 20 hemodialyzed patients, arbitrarily divided into two groups based on fasting triglyceride levels, and compared to triglyceride-matched control groups. In the hypertriglyceridemic subjects (plasma triglyceride values > 150 mg/dl), high-density lipoprotein cholesterol was decreased, and the net cholesteryl ester transfer rates were significantly higher than the rates in normolipidemic subjects. The comparison of subjects matched for plasma triglyceride and cholesterol levels showed no significant difference in cholesteryl ester or triglyceride transfer rates between patients and controls. Our results suggest that normal or elevated net neutral-lipid transfers are not related to the renal status of the subjects, but rather to their plasma triglyceride levels.

CD19 and immunophenotype of bone marrow plasma cells in monoclonal gammopathy of undetermined significance.

AIMS: To determine whether a particular phenotype or antigen is preferentially related to monoclonal gammopathies of undetermined significance (MGUS). METHODS: Bone marrow specimens from 56 patients with MGUS were stained immunocytochemically (ABC peroxidase) for CD38, CD56, CD9, CD10, CD19, CD20, CD22, and MB2. Specimens from patients recently diagnosed with multiple myeloma and reactive bone marrow samples were studied in parallel. RESULTS: CD38 was expressed on all plasma cells from all MGUS samples tested, while 36% were positive for CD56, CD9 and MB2 were both expressed strongly; CD20 was moderately expressed, and staining for CD10 and CD22 was uncommon. For these five B cell antigens there was no clear difference between their expression in MGUS and in multiple myeloma. A great difference was found for CD19: in MGUS this antigen was expressed on 2-91% of plasma cells (mean 35%) and 77% patients had > 10% positive plasma cells; in multiple myeloma its expression was low and only 12% patients had > 10% positive plasma cells. When these results were converted to numbers of CD19 positive plasma cells per 100 nucleated bone marrow cells, reactive bone marrow and MGUS specimens had a similar number of positive plasma cells. There was no correlation between expression of any of the antigens tested. CONCLUSIONS: Many of the so-called pre-B, B or activation antigens are present on plasma cells from MGUS specimens, and expression of CD9, CD10, CD20, CD22, MB2, and CD38 in MGUS was very similar to that in multiple myeloma. CD56 was frequently expressed in MGUS. In this series CD19 was highly expressed in MGUS but not in multiple myeloma. Plasma cells bearing this antigen could represent the non-neoplastic process and determination of its expression could be useful for the diagnosis of MGUS.

Expression of apolipoprotein B epitopes in low density lipoproteins of hemodialyzed patients.

Serum and isolated low-density lipoprotein (LDL) composition abnormalities were investigated in 20 hemodialyzed patients with chronic renal failure and 15 healthy normolipidemic subjects for comparison. LDL apolipoprotein B (apo B) epitope accessibility was determined by the use of seven monoclonal antibodies (Mabs). These Mabs recognize fragments on the N-terminal part of apo B (Mabs B1, B4), on the middle part (Mab BL7), on the C-terminal (Mabs BA11, BL3), and the two remaining Mabs recognize conformational epitopes of apo B (BL5, DA7). Mab BA11 recognizes a fragment of apo B which interacts with the B/E receptor. In hemodialyzed patients, LDL content of triglycerides (P < 0.001) and apo CIII (P < 0.005) was increased, while cholesteryl esters (P < 0.005) were decreased. The accessibility of BL5 epitopes of LDL apo B was enhanced (P < 0.05), while BA11 epitope expression was decreased (P < 0.01). The conformation of patients' LDL (CRF-LDL) was probably abnormal and seemed to be related to some modification of the lipidic environment. It is important to consider a structural modification as it alters the B/E receptor recognition domain of apo B. These results confirm LDL abnormalities in hemodialyzed patients and suggest a possible modification of the recognition of the LDL by cells.

Apo AIV in plasma and dialysate fluid of CAPD patients: comparison with other apolipoproteins.

Plasma lipids and apolipoproteins were determined in 18 patients with chronic renal failure on haemodialysis (HD), 18 patients on continuous ambulatory peritoneal dialysis (CAPD), and 18 healthy controls. Significant differences were observed between the two dialysis procedures, CAPD patients showed elevated plasma cholesterol, apo AI and apoB. No significant difference in plasma apo AIV concentration was observed between the two dialysis groups. Measurement of apolipoproteins in the dialysate fluid of five patients on CAPD showed losses of all apolipoproteins (AI, AII, AIV, B, CII, CIII and E). Although daily losses of apo AI and apo AIV were similar (255 and 221 mg/day respectively), the peritoneal clearance of apo AIV was threefold that of apo AI and 12-fold that of apo B. In view of the inverse correlation between peritoneal clearance and the molecular mass of proteins, about 13% of total plasma apo AIV was lost daily as lipoprotein-free form.

Postextrasystolic sinoatrial exit block in human sick sinus syndrome: demonstration by direct recording of sinus node electrograms.

Ten patients with sick sinus syndrome having repetitive sinus node electrograms during long postpacing pauses were studied during programmed atrial stimulation. Sinus node activity was recorded using a percutaneous catheter electrode. A sinus node electrogram was recorded before the return atrial beat in seven patients; it was similar to the sinus node electrogram observed during postpacing pauses and is clearly identified because sinoatrial conduction time was markedly prolonged following the atrial extra beat. Complete sinoatrial exit block occurred in four patients. (1) Sinus node electrograms were thus validated both during postpacing pauses and during programmed atrial stimulation in most patients with sick sinus syndrome. (2) Sinoatrial conduction time was markedly prolonged after one extrasystole, accounting for supracompensatory atrial return cycles. (3) If it were cumulative following multiple extrasystoles, this effect could constitute the electrophysiologic link between an abnormal response during programmed atrial stimulation and the complete sinoatrial block recorded during the pauses that follow rapid atrial pacing.

[Application of the immunoenzyme technic in double determination of antibodies for the study of hyperlipoproteinemia]. / Application de la méthode immunoenzymatique à double détermination d'anticorps à l'exploration des hyperlipoprotéinémies.

Antibodies directed against designed apolipoprotein, were absorbed on microtiter plates, the other apolipoprotein present on the retained particles was evaluated by using corresponding peroxidase labeled antibodies. This differential antibody immunosorbent assay was applied to evaluate lipoprotein particles concentration in familial type IIa, IIb, III, IV, and in the type IV secondary to chronic renal failure. Type IIa and IIb, were characterized by the increasing plasma concentration of lipoprotein particles containing both apo B and apo E (LpE-B). Although type IIa have high level of apo CIII, the plasma concentration of lipoprotein containing both apo B and apo CIII was within the normal range. The high concentration of apo E in type III hyperlipoproteinemia, revealed the accumulation of LpB-CIII-E but mainly lipoproteins containing both apo B and apo E (LpE-B). The latter represents 0.94 +/- 0.51 g/l when compared to normolipidemic subjects: 0.29 +/- 0.06 g/l. The decrease concentration of apo AI affects essentially lipoprotein containing apo AI without apo AII (LpAI) in primary type IV hyperlipoproteinemic patients, while in chronic renal failure, both populations of apo AI (with and without apo AII) were affected. The differential antibody immunosorbent assay may be used in the future as a new approach to classify lipid transport disorders.

[Immunisation of spontaneously hypertensive rats against angiotensin I]. / Immunisation du rat spontanément hypertendu contre l'angiotensine I.

Immunization against angiotensin I has been considered in comparison with the immunization against renin, in the spontaneously hypertensive rat (SHR). Among 6 different methods of immunization, two (AI-gluta-LPH and AI-Carbo-LPH coupling) permitted to obtain high levels of antibodies against angiotensin I (higher than 1/10,000), after four injections of 50 micrograms AI at three weeks interval. The titration of the antibodies was realized in radio-immuno-assay (RIA), with the determination of the cross-reactivity with AII by the same method. Characterization of the isotypes and the affinity calculation were realized with the ELISA method. The average level of antibodies is about 1/10,000 to 1/100,000, and the cross reactivity of the antibodies for AII is about 0.1 p. 100 in RIA. In ELISA, the study of the different isotypes shows a good maturation of the immune system with a sharp elevation of the IgG1 and IgG2 alpha isotypes, after 2 or 3 injections. The affinity of the antibodies purified by affinity chromatography is about 10.3 10(-9) M. The weekly measure of the arterial pressure during 6 months does not reveal at any moment a fall of pressure during the immunizations. The average pressure of the immunized group (209.4 +/- 23.8 mmHg, n = 40) is non significantly different from the average pressure of the mock group (208.5 +/- 22.6 mmHg, n = 10).

Immunologic approaches to blockade of the renin-angiotensin system: a review.

Several immunologic approaches to blockade of the renin-angiotensin system (RAS) have been reported, involving most of the proteins and peptides of the biochemical cascade: renin, substrate, angiotensins, and converting enzyme. None as yet has involved blockade of angiotensin II receptors. Earlier and more recent studies used passive transfer of heterologous antibodies or active immunization against RAS proteins and peptides. Passive transfers have been performed with both polyclonal antibodies and now with specific monoclonal immunoglobulins. The latter are better defined in affinity, quantity, and capacity to bind and thus inhibit the biologic activity of the antigen. Active immunization produced long-term blockade of part or all of the biologic activity of the system. The immunopathologic consequences of the use of antibodies raised against a self-antigen could be of interest in defining the predominant site of storage and secretion of the relevant protein and hence the respective roles of different tissues in the production of specific proteins in, for example, the vascular pulmonary bed for converting enzyme and renal arterial tree for renin. In all cases immunologic methods offer in vivo experimental models of short- or long-term RAS blockade that could be compared with pharmacologic methods, such as converting-enzyme inhibition, angiotensin II antagonists, and renin inhibitors.

Measurement of angiotensin II concentration in rat plasma: pathophysiological applications.

A very reliable isocratic reverse phase high performance liquid chromatography (HPLC) system has been developed to separate angiotensins, which combined with a very sensitive radioimmunoassay, provided precise measurements of the endogenous angiotensin II (AII) concentration in the rat plasma in different experimental circumstances. The overall recoveries of AII were 95.2 +/- 15.8% (means +/- SD) when 10 pg of this peptide was added to 1 ml of human plasma. The coefficient of variation for within-assay precision was 10% (n = 6). The plasma AII, measured by this method, of normal male pentobarbital-anesthetized rats was 53.0-141.6 pg/ml (mean: 103.9 +/- 29.7 pg/ml). The plasma AII of rats fed a sodium deficient diet was 300.0 +/- 100.6 pg/ml, while that of rats given oral Enalapril, an angiotensin converting enzyme (ACE) inhibitor, for 1 week was 35.7 +/- 28.0 pg/ml. The plasma AII of bilaterally nephrectomized rats was 2.7 +/- 2.9 pg/ml 24 hours after nephrectomy and below the detection limit 48 hr after nephrectomy. This method, therefore, can be used to study AII in different pathophysiological states or after treatment with renin-angiotensin system inhibitors.