Molecular characterization of the plant virus genus Ourmiavirus and evidence of inter-kingdom reassortment of viral genome segments as its possible route of origin.

Ourmia melon virus (OuMV), Epirus cherry virus (EpCV) and Cassava virus C (CsVC) are three species placed in the genus Ourmiavirus. We cloned and sequenced their RNA genomes. The sizes of the three genomic RNAs of OuMV, the type member of the genus, were 2814, 1064 and 974 nt and each had one open reading frame. RNA1 potentially encoded a 97.5 kDa protein carrying the GDD motif typical of RNA-dependent RNA polymerases (RdRps). The putative RdRps of ourmiaviruses are distantly related to known viral RdRps, with the closest similarity and phylogenetic affinity observed with fungal viruses of the genus Narnaviridae. RNA2 encoded a 31.6 kDa protein which, expressed in bacteria as a His-tag fusion protein and in plants through agroinfiltration, reacted specifically with antibodies made against tubular structures found in the cytoplasm. The ORF2 product is significantly similar to movement proteins of the genus Tombusviridae, and phylogenetic analysis supported this evolutionary relationship. The product of OuMV ORF3 is a 23.8 kDa protein. This protein was also expressed in bacteria and plants, and reacted specifically with antisera against the OuMV coat protein. The sequence of the ORF3 protein showed limited but significant similarity to capsid proteins of several plant and animal viruses, although phylogenetic analysis failed to reveal its most likely origin. Taken together, these results indicate that ourmiaviruses comprise a unique group of plant viruses that might have evolved by reassortment of genomic segments of RNA viruses infecting hosts belonging to different eukaryotic kingdoms, in particular, fungi and plants.

The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation.

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

Variability of the coat protein gene of Citrus psorosis virus in Campania, southern Italy.

Variability of the Coat protein (CP) gene of Citrus psorosis virus (CPsV) was assessed serologically, and by sequence analyses of two genomic regions located in the 3' (region C) and 5' (region V) halves of the gene. Analysis of 53 psorosis field sources from Campania, Italy, with 23 monoclonal antibodies revealed nine serogroups and at least ten different epitopes. Sequence analysis of 19 of these sources showed limited nucleotide diversity of the CP gene in the population. Diversity was slightly higher in region V than in region C. Phylogenetic analysis of the V and C regions of the CP showed that the Campania sources of CPsV were clearly separated from the CPsV-4 isolate from Florida. For C region, most of the CPsV sources clustered together, whereas two clusters were observed for region V. The ratio between nonsynonymous and synonymous substitutions for regions C (0.083) and V (0.345) indicated negative selective pressure for amino acid changes, more intense in the C region. No correlation was found between serogroups and specific aminoacid sequences, field location or citrus cultivar.

The partial sequence of RNA 1 of the ophiovirus Ranunculus white mottle virus indicates its relationship to rhabdoviruses and provides candidate primers for an ophiovirus-specific RT-PCR test.

A 4018 nucleotide sequence was obtained for RNA 1 of Ranunculus white mottle virus (RWMV), genus Ophiovirus, representing an incomplete ORF of 1339 aa. Amino acid sequence analysis revealed significant similarities with RNA polymerases of viruses in the family Rhabdoviridae and a conserved domain of 685 aa, corresponding to the RdRp domain of those in the order Mononegavirales. Phylogenetic analysis indicated that the genus Ophiovirus is not related to the genus Tenuivirus or the family Bunyaviridae, with which it has been linked, and probably deserves a special taxonomic position, within a new family. A pair of degenerate primers was designed from a consensus sequence obtained from a relatively conserved region in the RNA 1 of two members of the genus, Citrus psorosis virus (CPsV) and RWMV. The primers, used in RT-PCR experiments, amplified a 136 bp DNA fragment from all the three recognized members of the genus, i.e. CPsV, RWMV and Tulip mild mottle mosaic virus (TMMMV) and from two tentative ophioviruses from lettuce and freesia. The amplified DNAs were sequenced and compared with the corresponding sequences of CPsV and RWMV and phylogenetic relationships were evaluated. Assays using extracts from plants infected by viruses belonging to the genera Tospovirus, Tenuivirus, Rhabdovirus and Varicosavirus indicated that the primers are genus-specific.

Nucleotide sequence, genome organisation and phylogenetic analysis of Indian citrus ringspot virus. Brief report.

The sequence of the single-stranded RNA genome of Indian citrus ringspot virus (ICRSV) consists of 7560 nucleotides. It contains six open reading frames (ORFs) which encode putative proteins of 187.3, 25, 12, 6.4, 34 and 23 kDa respectively. ORF1 encodes a polypeptide that contains all the elements of a replicase; ORFs 2, 3 and 4 compose a triple-gene block; ORF5 encodes the capsid protein; the function of ORF6 is unknown. Phylogenetic analysis of the complete genome and each ORF separately, and database searches indicate that ICRSV, though showing some similarities to potexviruses, is significantly different, as in the presence of ORF6, the genome and CP sizes, and particle morphology. These differences favour its inclusion in a new virus genus.

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

Consultant outreach, 1991 to 1998. An update and extension on its distribution in Scotland.

OBJECTIVE: To assess the extent and distribution of consultant outreach in Scotland between 1991 and 1998. DESIGN: The paper has three parts. First a description of the trends in consultants and consultant activity provides the background. This is followed by the results of an update of the 1991 survey of all health centres in Scotland and its extension to all GP premises considered suitable to hold consultant clinics. Finally, binary regression analysis of outreach is used to test the importance of total list size, distance to alternative provision and deprivation. Fourteen of the most common consultant specialties are studied. SETTING AND SUBJECTS: Scotland-wide data on consultants and consultant activity using annual data over the 1990s; and a Scotland-wide survey of 231 health centres and 312 GP premises over the period July to December 1998. RESULTS AND CONCLUSIONS: Consultant full time equivalents (ftes) increased and, with minor exceptions, consultant activity did so too. In respect of outreach, the increase was largely at GP premises and for psychiatry. For only two specialties of the fourteen studied, obstetrics and general psychiatry, could outreach be considered important. Such outreach provision as was made went where the total list size was largest and alternative provision farthest distant. The evidence that deprivation had an influence on outreach varies with specialty and is qualified.

Indian citrus ringspot virus: a proposed new species with some affinities to potex-, carla-, fovea- and allexiviruses.

An isolate of Indian citrus ringspot virus from Kinnow mandarin in northern India had flexuous particles with evident cross-banding and a modal length of 650 nm. It was mechanically transmitted to five herbaceous hosts including Phaseolus vulgaris cv Saxa, in which it became systemic. In thin sections, virus particles were observed in the cytoplasm of parenchyma cells but no specific inclusions were seen. The virus was purified from infected Saxa bean leaves and an antiserum prepared. There was no serological cross-reaction with representative allexi-, capillo-, potex- and trichoviruses, except a faint one-way reaction with Potato virus X. Purified virus yielded a major band, the presumed coat protein (CP), of about 34 kDa, and a single ssRNA of about 7.5 kb, which was infectious. Two ORFs encoding putative proteins of 34 kDa and 23 kDa were located in the 3' part of the RNA. The product of the 34 kDa ORF was confirmed as the CP by expression in E. coli. The derived amino acid sequence of the CP contained some short motifs similar to those of potex-, fovea-, carla- and allexiviruses but otherwise there was no strong similarity to any of these. The 23 kDa ORF contained a zinc finger-like sequence, as in similar ORFs in carla- and allexiviruses but overall amino acid homology with these was low. The virus does not appear to fall into any known genus. A new species is proposed. Serological and molecular diagnostic reagents were prepared.

An Ophiovirus isolated from lettuce with big-vein symptoms.

Big-vein is a widespread and damaging disease of lettuce, transmitted through soil by the chytrid fungus Olpidium brassicae, and generally supposed to be caused by Lettuce big-vein virus (LBVV; genus Varicosavirus). This virus is reported to have rigid rod-shaped particles, a divided double-stranded RNA genome, and one capsid protein of 48 kD, but has not been isolated or rigorously shown to cause the disease. We provide evidence that a totally different virus, here named Mirafiori lettuce virus (MiLV), is also very frequently associated with lettuce showing big-vein symptoms. MiLV was mechanically transmissible from lettuce to Chenopodium quinoa and to several other herbaceous test plants. The virus was partially purified, and an antiserum prepared, which did not react with LBVV particles in decoration tests. As reported for LBVV, MiLV was labile, soil-transmitted and had a single capsid protein of 48 kD, but the particles morphologically resembled those of ophioviruses, and like these, MiLV had a genome of three RNA segments approximately 8.5, 1.9 and 1.7 kb in size. MiLV preparations reacted strongly in Western blots and in ISEM with antiserum to Tulip mild mottle mosaic virus, an ophiovirus from Japan also apparently Olpidium-transmitted. They reacted weakly but clearly in Western blots with antiserum to Ranunculus white mottle virus, another ophiovirus. When lettuce seedlings were mechanically inoculated with crude or partially purified extracts from MiLV-infected test plants, many became systemically infected with MiLV and some developed big-vein symptoms. Such plants did not react in ELISA using an LBVV antiserum or an antiserum to tobacco stunt virus, and varicosavirus-like particles were never seen in them in the EM after negative staining. We conclude that MiLV is a hitherto undescribed virus assignable to the genus Ophiovirus. The cause or causes of lettuce big-vein disease and the properties of LBVV may need to be re-evaluated in light of our results.

First Report of Anemone Plants Infected by Ranunculus white mottle virus.

Ranunculus white mottle virus (RWMV) (1), genus Ophiovirus, has been reported in crops of several cultivars of commercial ranunculus (Ranunculus asiaticus hybrids) during the 1990s in Liguria in Northwest Italy. Symptoms associated with RWMV in ranunculus are not clear-cut owing to the presence of mixed viral infections. During autumn 1999, a severe disease in commercial crops of anemone (Anemone coronaria) was noted in the same area. Plants appeared stunted with young leaves showing curling, deformation, and necrotic spotting. Disease incidence in some fields reached 40 to 50%. DAS- and TAS-enzyme-linked immunosorbent assays (ELISAs) for presence of RWMV and for the viruses most frequently infecting anemone in Italy were run on 24 field samples. Seven proved to be infected by RWMV in mixed infection with Cucumber mosaic virus subgroup II or with Tobacco necrosis virus. Ophiovirus-like particles were detected by negative staining and electron microscopy from sap extracts of field plants that were RWMV-positive by ELISA. Sap from these plants was also mechanically inoculated to indicator plants. Total RNAs were extracted from RWMV-infected field samples and from inoculated Nicotiana benthamiana and N. clevelandii and used in molecular tests. A DIG-DNA probe targeting the 1.8-kb RNA2 of RWMV was used in Northern blots and dot blots of total RNAs, confirming the infection in field samples and multiplication of the virus in test plants, unfortunately still in mixed infection. At present, it is difficult to evaluate RWMV symptomatology in anemone, but the presence of this virus in mixed infection seems to produce serious effects. This is the first report of RWMV in anemone. Reference: (1) A. M. Vaira et al. Arch. Virol. 142:2131, 1997.

Functional expression in bacteria and plants of an scFv antibody fragment against tospoviruses.

BACKGROUND: Recombinant antibodies expressed in plants ('plantibodies'), directed against crucial antigens and addressed to the right cell compartment, may be able to protect against viral diseases. Moreover, antibody fragments produced in bacteria or plants may provide low cost reagents for immunodiagnosis. OBJECTIVES: In an attempt to develop genetic immunisation against tomato spotted wilt tospovirus (TSWV), we engineered an scFv fragment starting from a monoclonal antibody (mAb) able to recognise an epitope of the glycoprotein G1 conserved among a large number of tospoviruses. After establishing functional expression in bacteria, we aimed to drive expression of this molecule in the secretory pathway of plants. STUDY DESIGN: An antibody phage display expression system was used to isolate the correct VH and VL binding regions from the hybridoma secreting the original mAb. To assess functional expression in plant, we first used an epichromosomal expression vector derived from potato virus X (PVX). In this vector the scFv gene was cloned to produce a cytosolic or a secretory protein. For secretion, the signal sequence derived from the polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris was used. Subsequently, the gene encoding the secretory scFv, was used to transform Nicotiana benthamiana plants. RESULTS: High expression levels of fully active molecule were obtained in Escherichia coli. The engineered molecule retained the binding specificity and dissociation rate constant (k(off)) of the cognate monoclonal antibody. Both PVX-infected and transformed plants expressed fully functional scFv molecules in the secretory pathway. CONCLUSION: This engineered scFv may be valuable for inexpensive diagnosis, for studying the role of the glycoproteins in virus transmission and, possibly, for a 'plantibody'-mediated resistance to tospoviruses.

The decline of fertility in Malta: the role of family planning.

PIP: This study examines the nature and extent of contraceptive use in Malta and determines what factors affected the shift from traditional to modern methods. Data were obtained from the 1993 Survey of Family Planning in Malta of 98 general medical practitioners and up to 15 clients per practitioner and a similar 1971 survey. The total fertility rate in Malta declined from 2.25 in 1967 to 2.00 in 1985. Changes occurred such that the influence of the Church declined, formal education increased, legislation supported family planning, and income increased. Both Malta surveys indicated a proportion who did not attempt to space or limit births and some who relied on more than one method. During 1971-93, choice of contraceptive method changed from traditional methods to oral pills and condoms. Government family planning clinics were set up in 1981 and provided information that included natural methods. Diaphragms were provided free of charge. Abortion was, and still is, illegal. Sterilization is also illegal; male sterilization is more accessible. The probability of a woman using some form of contraception is modeled. Findings suggest that age has little impact. Women with 2-3 children and more highly educated women had higher probabilities of using contraception. Older married women and better educated women had a higher probability of use and use of the rhythm method. Family size was unrelated to use of the rhythm method. Age and marital duration were unrelated to use of coitus interruptus. Better educated women were unlikely to rely on coitus interruptus. Condom use was unrelated to age, education, family size, or marriage duration. Younger women were more likely to use the pill and more effective methods of contraception. Effective contraceptive use was related to better female education, larger family size, and longer marriage duration.^ieng

Partial characterization of a new virus from ranunculus with a divided RNA genome and circular supercoiled thread-like particles.

An undescribed virus, here named ranunculus white mottle virus, was isolated in Italy from cultivated ranunculus showing mottle and distortion of leaves. The virus was mechanically transmissible to several herbaceous hosts. In negative stain, the particles appeared as circularised supercoiled threads 3 nm in diameter of different contour lengths; in some conditions the circles collapsed to form linear pseudobranched structures 9 nm in diameter. Immunolabeling of thin sections showed that viral antigen was widely distributed in the cytoplasm of parenchyma cells. The virus was not serologically related to the morphologically similar tenuiviruses, citrus psorosis-ringspot virus and tulip mild mottle mosaic virus. A major 43 kDa protein was present in purified preparations and in infected plant tissue, as also was a minor 28 kDa protein, serologically related to the major one. Nucleic acids extracted from purified particles consisted of at least three RNAs, of approximately 7.5, 1.8 and 1.5 kb, which appeared partly in single- and partly in double-stranded form. Purified preparations, but not viral RNAs, when mechanically inoculated, were infectious. Host range, tissue tropism, particle morphology and coat protein size place the virus closest to citrus psorosis-ringspot and tulip mild mottle mosaic viruses. These three viruses in turn show similarities with the Tenuiviruses and Bunyaviridae.

Purification of tomato yellow leaf curl geminivirus.

Attempts were made to find a good purification procedure for tomato yellow leaf curl virus (TYLCV), a dangerous and continuously spreading whitefly-transmitted germinivirus, up to now only partially purified. Electron microscopy, serology and spectrophotometry were used to evaluate different procedures. The scheme finally adopted was the following: collect leaves and stems from Nicotiana benthamiana graft-infected 45-60 days previously (5-10 g/plant); homogenize with 0.5 M phosphate buffer pH 6 containing 2.5 mM NaEDTA, 10 mM Na2SO3, 0.1% 2-mercaptoethanol, 1% Triton X-100 and 0.1% Driselase (3-4 ml of buffer for each g of material); incubate overnight on ice with gentle agitation; filter; emulsify with 15% cold chloroform; centrifuge at low speed; ultracentrifuge supernatant; resuspend pellets in 0.5 M phosphate buffer pH 7 containing 2.5 mM NaEDTA; centrifuge at low speed; repeat resuspension of the pellets and low-speed centrifugation; ultracentrifuge the pooled supernatant on a Cs2SO4 gradient (e.g. for 5 h at 41,000 rpm); collect the virus band and dialyse or ultracentrifuge the virus. The virus yield was 5-10 mg per kg of tissue.

The closely related citrus ringspot and citrus psorosis viruses have particles of novel filamentous morphology.

Some properties of the particles of citrus ringspot virus (CtRSV) and the related citrus psorosis-associated virus (CPsAV) are described. The particles of CtRSV have been reported to be sinuous linear structures about 10 nm in diameter and of two lengths (300 to 500 nm and 1500 to 2500 nm) representing 'top' and 'bottom' sedimentation components. We show that these particles are collapsed double-stranded forms of nucleocapsid-like, highly flexuous open circles formed of filaments 3 to 4 nm in diameter. Top-component filaments had contour lengths of 600 to 1000 nm, i.e. twice that reported for the corresponding collapsed form. Bottom-component filaments had contour lengths about four times longer than those of top-component filaments. The structures suggest that CtRSV represents a new genus (possibly family) related to the tenuiviruses. However, we failed to demonstrate any serological relationship between CtRSV and several tenuiviruses; moreover, the capsid protein sizes and host ranges are quite different. We offer the name Ophiovirus for the proposed new genus.

The chief scientist reports... Consultant out-patient services: provision at health centres in Scotland.

Hospital based consultant (out-patient) services are most likely to be found in the community at health centres. By 1991 some 45% of the Scottish population had access to services provided at these health centres. The clinical specialties most commonly provided were psychiatry, obstetrics, paediatrics and gynaecology, and they accounted for some 10% or more of all attendances. Regular consultant visits in these and other specialties were more common at the larger health centres and at those further distant from alternative provision.

Non-price allocative procedures: Scottish solutions to a National Health Service problem.

Except for a few NHS services, the allocation of resources depends on administrative-cummedical decision-making. At one level the Scottish Home and Health Department allocates funds between the fifteen health boards, at another level clinicians allocate resources between patients. We examine experience at a level intermediate between these two, and focus on the provision of two services--diagnostic radiology and ECG--at health centres. A benefit: cost framework is used to test three hypotheses about how the two services have been allocated. The three hypotheses relate to the benefits from provision and are characterised as 'medical excellence', 'equity' and 'market' orientated. Data on health centre list size and distance to alternative provision are used to test the hypotheses. The conclusions are as follows. The equity and market models are equally valid descriptions for ECG, a service provided by general practitioners. A combination of the equity and/or market model with the medical model is a valid description for diagnostic radiology, a service provided by health boards and the Scottish Home and Health Department.

The impact of teaching on hospital costs: a budgetary approach to non-market institutions.

NHS hospitals contribute to medical education, training nurses and research, as well as to the care of patients. In the past they have been funded largely on the basis of resources employed, with additional funding for medical education and training nurses. The intellectual basis for the funding of medical education is a single econometric study of English hospitals in the financial year 1969-70. The methodology used has since been criticized, and it has been suggested that actual expenditure has been very much less than that earmarked by the health departments. New estimates are obtained using Scottish data for the financial year 1985-86. The method used is to proceed in a two-stage fashion, identifying via regression techniques variables measuring hospital activity and resources which contribute significantly to hospital costs. We then assess the significance of medical education, nurse training and hospitals' teaching status against this background. Our conclusions include: (1) actual expenditure on medical education was probably less than the funding formula allowed, but the error of margin is too large to suggest overfunding; (2) training nurses incurs significant financial costs, even after the explicit allowances made; and (3), major teaching hospitals tended to cost more, but not significantly more than their non-teaching counterparts. These financial implications for NHS hospitals should be borne in mind given the current NHS review.

In vitro synthesis of double-stranded RNA by carnation cryptic virus-associated RNA-dependent RNA polymerase.

Partially purified carnation cryptic virus (CarCV) preparations possessed RNA-dependent RNA polymerase activity which was absent in comparable preparations from virus-free carnations. Enzyme activity was dependent upon the presence of virus particles, Mg2+, and the four ribonucleoside triphosphates, and was insensitive to inhibitors of DNA-dependent RNA polymerases. The 32P-labeled enzyme reaction products were largely dsRNAs as indicated by resistance to S1 nuclease and RNase A at high but not low ionic strength. The in vitro synthesized dsRNAs hybridized specifically with CarCV genomic dsRNAs, and the radioactive products present in the polymerase reaction mixture sedimented with the virus particles in sucrose density gradients. The data suggest that the RNA-dependent RNA polymerase associated with CarCV particles is a replicase which catalyzes the synthesis of copies of the genomic dsRNAs.