Late Survival in Nonoperated Patients with Infrarenal Abdominal Aortic Aneurysm.

OBJECTIVE/BACKGROUND: Historical studies report high rupture rates in patients with nonoperated abdominal aortic aneurysms (AAAs) of > 5.5 cm diameter, although a recent audit has questioned this. METHODS: This was a retrospective review of 138/764 (18%) patients with AAAs evaluated in a preassessment anaesthetic clinic (PAC) between 2006 and 2012, who either did not undergo elective AAA repair or who underwent deferred repair. The remaining 626 underwent repair. Patients with severe comorbidities (dementia, advanced malignancy, life-expectancy < 1 year) and not referred to PAC were excluded. RESULTS: At a median of 27 months, 71 (52%) died, 36 (51%) following rupture. Cumulative survival, free from rupture or surgery for acute symptoms, was 96% at 1 year, 84% at 3 years, and 64% at 5 years, where baseline AAA diameters were 5.5-6.9 cm. For diameters ≥ 7 cm, survival, free from rupture, was 65% at 1 year, 29% at 3 years, and 0% at 5 years. Median interval to rupture was 47 months (AAA diameter 5.5-6.9 cm) and 21 months where baseline diameters were ≥ 7 cm. Rupture accounted for 32% of late deaths in patients with AAAs of 5.5-5.9 cm diameter, 46% in those with AAAs measuring 6.0-6.9 cm in diameter, and 71% in patients with AAA measuring ≥ 7 cm in diameter. CONCLUSION: Approximately half of all late deaths in this nonoperated cohort were not AAA related, suggesting that even had repair been undertaken, it would not have prolonged patient survival. The incidence of rupture in "high-risk" patients with an AAA < 7 cm diameter was < 5% at 1 year, thereby giving ample time to optimise risk factors and improve pre-existing medical conditions prior to undertaking a deferred intervention. Even if these patients did not undergo surgical repair, the risk of late rupture was relatively low. By contrast, nonoperated patients with AAAs ≥ 7 cm in diameter face a very high risk of rupture and will probably benefit from elective surgery, with the caveat that a higher procedural risk might have to be incurred.

Factors influencing short- and long-term mortality after lower limb amputation.

Mortality after lower limb amputation is high, with UK 30-day mortality rates of 9-17%. We performed a retrospective analysis of factors affecting early and late outcome after lower limb amputation for peripheral vascular disease or diabetic complications at a UK tertiary referral vascular centre between 2003 and 2010. Three hundred and thirty-nine patients (233 male), of median (IQR [range]) age 73 (62-79 [26-92]) years underwent amputation. Thirty-day mortality was 12.4%. On regression modelling, the risk of 30-day mortality was increased in patients of ASA grade ≥ 4 (OR 4.23, 95% CI 2.07-8.63), p < 0.001 and age between 74 and 79 years (OR 3.8, 95% CI 1.10-13.13), p = 0.04 and older than 79 years (OR 4.08, 95% CI 1.25-13.25), p = 0.02. Peri-operative (30-day) mortality for these groups was 23.2%, 13.7% and 18.8%, respectively. Survival and Cox regression analysis demonstrated that long-term mortality was associated with: age 74-79 years (HR 2.15, 95% CI 1.38-3.35), p = 0.001; age > 79 years (HR 2.78, 95% CI 1.82-4.25), p < 0.001; ASA grade ≥ 4 (HR 2.04, 95% CI 1.51-2.75), p < 0.001; out-of-hours operating (HR 1.51, 95% CI 1.08-2.10), p = 0.02; and chronic kidney disease stage 4-5 (1.57, 95% CI 1.07-2.30), p = 0.02. Anaesthetic technique was associated with long-term mortality on survival analysis (p = 0.04), but not when analysed using regression modelling. Mortality after lower limb amputation relates to patient age, ASA, out-of-hours surgery and renal dysfunction. These data support lower limb amputations' being performed during daytime hours and after modification replace with 'of ' correctable risk factors.

Genomic sequences of blackberry chlorotic ringspot virus and strawberry necrotic shock virus and the phylogeny of viruses in subgroup 1 of the genus Ilarvirus.

Three members of subgroup 1 of the genus Ilarvirus: blackberry chlorotic ringspot (BCRV), strawberry necrotic shock (SNSV), and tobacco streak viruses (TSV), may infect Rubus and Fragaria species. All cause symptoms similar to those previously attributed to infection by TSV alone. Although similarities exist among the genomic sequences of the three, phylogenetic analysis shows them to be distinct viruses. These viruses and Parietaria mottle virus, the other currently accepted member of subgroup 1, appear to have evolved from a common ancestral virus, share conserved motifs in the products of the genomic RNAs, and constitute a distinct subgroup within the genus.

Viruses in subgroup 2 of the genus Ilarvirus share both serological relationships and characteristics at the molecular level.

Sequence data have been determined for 5 members of subgroup 2 of the genus Ilarvirus. These data support the known serological relationships among accepted members of this group and indicate that the ilarvirus Hydrangea mosaic virus (HdMV) is an isolate of Elm mottle virus (EMoV). The close relationships between members of this subgroup, exhibited through the coat proteins coded on RNA 3, extend to the other genomic molecules. Primers designed from the sequences of RNA 1 and RNA 2 of EMoV amplified fragments from all other subgroup 2 viruses but not from other ilarviruses. Although closely related, members of this subgroup occur naturally in distinctly different host species. The possible origins of the viruses are discussed in relation to similarities among the genomic molecules, in particular RNA 3.

First Report of Tomato ringspot virus in Butterfly Bush (Buddleia davidii).

Leaves displaying bright yellow or light green line pattern symptoms were collected from individual, large, mature buddleias in a home garden in Clemson, SC, a botanical garden in Knoxville, TN, and a container-grown plant on sale in a retail home and garden store in Seneca, SC. Buddleias grown in the southeastern United States frequently display virus-like symptoms, but the line pattern symptom displayed by these plants was atypical of the mosaic, mottling, and leaf deformation seen when buddleias are infected with Alfalfa mosaic virus (AMV) or Cucumber mosaic virus (CMV) (2,4). Line pattern symptoms are frequently seen in woody species infected by ilarviruses or nepoviruses (2). No ilarviruses are reported to infect buddleia and only the nepovirus, Strawberry latent ringspot virus, which is restricted mainly to Europe, is reported to infect this species (1,2). The nepoviruses Tomato ringspot virus (ToRSV) and Tobacco ringspot virus (TRSV) are frequently found infecting plants of many species in the southeastern United States (3). Total RNA was extracted from the three symptomatic plants and used in reverse transcription-polymerase chain reactions (RT-PCR) to detect ToRSV and TRSV using primer pairs developed in this laboratory, which amplify regions around the amino terminus of the coat protein of the respective viruses. The expected amplification product for ToRSV of 327 base pairs was obtained from samples tested from each plant, and the nucleotide sequence of the product showed 96% identity with the corresponding fragment of GenBank Accession No. NC_003839 that the primers were designed to amplify. Repeated attempts to isolate a virus from symptomatic leaves using sap inoculation to Chenopodium amaranticolor Coste & Reyne, C. quinoa Willd, Nicotiana clevelandii Gray, and N. tabacum L. have failed. Repeated testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of leaves from the plant growing in Clemson consistently produced absorbance values at 405 nm in the range of 0.47 to 0.55 (mean of 8 separate samples per test) for symptomatic and asymptomatic leaves. The range of values for the positive control (ToRSV-G growing in N. clevelandii) was 1.3 to 1.5. The ranges of values for the noninfected controls (noninfected N. clevelandii and leaf tissue from a buddleia known to be infected with AMV and CMV but in which ToRSV or TRSV had never been detected by RT-PCR) were 0.102 to 0.104 and 0.102 to 0.106, respectively. The extraction buffer produced absorbance readings in the range of 0.098 to 0.102. RT-PCR of RNA extracted from other portions of the leaves used in the ELISA consistently amplified the 327-bp product from symptomatic leaves and from the positive control but not from noninfected control tissues. RNA from asymptomatic leaves on the infected plant also produced the 327-bp product in RT-PCR. Isolation of viruses from woody hosts is frequently difficult, and although, we have yet to succeed to confirm the association between the observed symptom and ToRSV, the evidence from PCR and ELISA would indicate ToRSV is present in these plants. To our knowledge, this is the first report of ToRSV, a member of the genus Nepovirus, in buddleia. References: (1) J. Albouy and J. C. Devergne. Maladies á Virus des Plants Ornementales. INRA Editions, Paris, 1998. (2) J. I. Cooper. Virus Diseases of Trees and Shrubs. 2nd ed. Chapman and Hill, London, 1993. (3) J. R. Edwards and R. G. Christie. Pages 352-353 in: Handbook of Viruses Infecting Legumes. CRC Press, Boca Raton, FL, 1991. (4) C. J. Perkins and R. G. T. Hicks. Plant Pathol. 38:443, 1989.

The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3'-UTR of the genomic RNA 3.

In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3' termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3' untranslated terminal region (3'-UTR) of the genomic RNA 3.

Differences between the coat protein amino acid sequences of English and Scottish serotypes of Raspberry ringspot virus exposed on the surface of virus particles.

The region of the RNA 2 coding for the putative helper/movement protein and the coat protein (CP) of each of six isolates of Raspberry ringspot virus was sequenced and these sequences were compared with the published sequence of the Scottish type isolate. Minimal differences were detected among the putative translations of the helper/movement proteins, however, multiple alignment and phylogenetic analysis of the putative CPs separated the English and Scottish serotypes into two distinct clades. Superimposing the amino acid sequences of the CPs of these two serotypes on the 3D model for the CP of a comovirus/nepovirus, showed that eight of the differences identified between the two serotypes occurred on the surface of the protein. Inspection of the recently reported structure of the capsid protein of Tobacco ringspot virus, the type member of the genus Nepovirus, indicated identical locations for these differences. The change of H (Scottish isolates) to R (English isolates) at position 219 in the amino acid sequences of the viruses occurred on an exposed, erect surface loop. The potential role of this change, and other unique differences between the amino acid sequences of the two serotypes, in the specificity of nematode transmission of the virus is discussed.

The sequence of RNA 1 and RNA 2 of tobacco streak virus: additional evidence for the inclusion of alfalfa mosaic virus in the genus Ilarvirus.

Here we describe the complete sequence of RNA 1 and 2 of the WC isolate of tobacco streak virus (TSV). These two sequences complete the information on the genome of TSV, the type member of the genus Ilarvirus, and are the first sequences described for the RNA 1 and RNA 2 of a member of subgroup 1 of this genus. The sequences have a similar organization to those reported for the corresponding RNAs of other ilarviruses. However, the putative translation products of these two molecules differ sufficiently from previously sequenced ilarviruses so that TSV should remain in a subgroup on its own. Phylogenetic comparison of sequence data for RNA 1 with that of other ilarviruses and alfalfa mosaic virus (AMV) reveals two distinct clusters (TSV, CiLRV, and SpLV) and (AMV, PDV, and ApMV). These data support the suggestion [16] based on data for RNA 3 of ilarviruses that AMV should be included as a true ilarvirus.

The complete sequence of the genomic RNAs of spinach latent virus.

We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus-CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C2H2 type "zinc finger"-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus.

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.

The nucleotide sequence of hydrangea mosaic virus RNA 3 exhibits similarity with the RNA 3 of tobacco streak virus.

The complete nucleotide sequence of the RNA 3 of hydrangea mosaic virus (HdMV) was determined. It consists of 2268 nucleotides and contains two open reading frames (ORF). ORF 1 encodes for a putative translation product of 293 amino acids which shared 64.9% identity with the 3a protein of tobacco streak virus (TSV). ORF 2 encodes for a putative translation product of 220 amino acids which shared 54.2% identity with the coat protein of TSV. The relationship between the proteins of HdMV and the corresponding proteins of ilarviruses other than TSV was more distant. No zinc-finger-like motif was found in the coat protein of HdMV but the N-terminus of the protein was rich in basic amino acids. Both terminal, non-coding regions of HdMV RNA 3 contained repeated sequences with corresponding homologous fragments in the RNA 3 of TSV. On the basis of the similarities between HdMV and TSV that we detected, we propose that HdMV be included in subgroup 1 of the genus Ilarvirus together with TSV.

The nucleotide sequence of citrus leaf rugose virus RNA 1.

The nucleotide sequence of citrus leaf rugose virus (CiLRV) RNA 1 consists of 3404 nucleotides and contains one open reading frame (ORF) which encodes a putative translation product of 1051 amino acids with a calculated M(r) of 118339. Both the nucleotide sequence of CiLRV RNA 1 and its translated polypeptide share similarities with those of the RNA 1 of alfalfa mosaic virus. However, the relationship is not as close as that which exists between the polymerase signatures of the two viruses, which are found on RNA 2. This is the first report of the full-length sequence for the RNA 1 of an ilarvirus and completes the first sequence for an entire ilarvirus genome. If it is typical of members of the genus then, as has long been speculated, the genomic organization of ilarviruses is identical to that of other genera in the family Bromoviridae.

The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses.

Complete sequence data for the RNA 3 of both citrus leaf rugose (CiLRV) and citrus variegation (CVV) ilarviruses have been determined. The RNAs are 2289 nt (CiLRV) and 2309 nt (CVV) in length and both contain the typical Bromoviridae arrangement of two open reading frames (ORFs) which, when translated, code for proteins that correspond to the Mr 32,000 (32K) putative movement proteins (ORF 1) and the coat proteins (ORF 2) of the respective viruses. The 3' termini of both viruses can be folded to form a secondary structure similar to those reported for other ilarviruses. These are the first complete nucleotide sequences for RNA 3 of members of subgroup 2 of the ilarviruses. The two viruses share substantial homology in nucleic acid sequence, code for identically sized coat proteins and share high levels of identity in the translated products of both ORFs. Although related, these viruses differ sufficiently to be considered distinct. The RNA 3s of CiLRV and CVV appear to be distinct from those of other ilarviruses for which comparable sequence data are available and also from the closely related alfalfa mosaic virus.

The nucleotide sequence of citrus leaf rugose ilarvirus RNA-2.

The nucleotide sequence of citrus leaf rugose ilarvirus (CiLRV) RNA-2 consists of 2990 nucleotides and contains one open reading frame (ORF) which encodes a deduced translation product of 832 amino acids with a calculated M(r) of 95,501 (95K). The 5' terminus of the RNA has a m7Gppp cap. Both the nucleotide sequence of CiLRV RNA-2 and its translated polypeptide share homologies with the nucleotide sequence and translated polypeptide, respectively, of RNA-2 of alfalfa mosaic virus (AlMV). The homology occurs in the central region of both the nucleic acid sequence and the polypeptide. Homologies between either CiLRV or AlMV and other Bromoviridae (cucumber mosaic virus--CMV, brome mosaic virus--BMV and cowpea chlorotic mottle virus--CCMV) were far less. Alignment of the 104 amino acid region (polymerase signature) of the 95K protein against 10 other 'alpha-like' plant viral polymerase signatures showed that CiLRV and AlMV are more closely related to each other than to CMV, BMV or CCMV. This is the first report of the full-length sequence of RNA-2 of an ilarvirus.

The complete nucleotide sequence of prune dwarf ilarvirus RNA 3: implications for coat protein activation of genome replication in ilarviruses.

The complete nucleotide sequence of prune dwarf ilarvirus (PDV) RNA 3 has been determined from cloned viral cDNAs. The PDV RNA 3 is 2129 nucleotides and contains two large open reading frames (ORFs) separated by an intergenic region of 72 nucleotides. The 5' proximal ORF (ORF-1) is 882 nucleotides, encoding a gene product which shares homology with putative cell-to-cell movement proteins of related viruses, including tobacco streak virus (TSV) and alfalfa mosaic virus (AIMV). The downstream ORF (ORF-2) is 657 nucleotides and encodes a gene product which shares primary sequence homology and structural features with AIMV coat protein. Furthermore, when expressed in bacteria, this ORF produces a polypeptide which comigrates with authentic PDV coat protein and reacts with PDV coat protein antiserum. Hybridization data suggest that the genomic organization of PDV RNAs 3 and 4 is similar to that of TSV, the only other ilarvirus for which sequence information is published. The 3' untranslated region (UTR) of PDV RNA 3 is similar to that of TSV and AIMV, containing a potential stem-loop structure followed by the sequence AUGC, a structure which may signal binding of coat protein and activation of genome replication. However, a striking feature of the deduced PDV coat protein sequence is the absence of a "zinc-finger" motif thought to function in binding of the coat protein to the 3'-UTR in ilarviruses and AIMV. This result suggests that the zinc-finger motif is not a required aspect of coat protein activation of replication in ilarviruses.

Tests for Transmission of Prunus Necrotic Ringspot and Two Nepoviruses by Criconemella xenoplax.

In two of three trials, detectable color reactions in ELISA for Prunus necrotic ringspot virus (PNRSV) were observed for Criconemella xenoplax handpicked from the root zone of infected peach trees. Criconemella xenoplax (500/pot) handpicked from root zones of peach trees infected with PNRSV failed to transmit the virus to cucumber or peach seedlings. The nematode also failed to transmit tomato ringspot (TomRSV) or tobacco ringspot viruses between cucumbers, although Xiphinema americanum transmitted TomRSV under the same conditions. Plants of peach, cucumber, Chenopodium quinoa, and Catharanthus roseus were not infected by PNRSV when grown in soil containing C. xenoplax collected from root zones of PNRSV-infected trees. Shirofugen cherry scions budded on Mazzard cherry seedling rootstocks remained symptomless when transplanted into root zones of PNRSV-infected trees. Virus transmission was not detected by ELISA when C. xenoplax individuals were observed to feed on cucumber root explants that were infected with PNRSV and subsequently fed on roots of Prunus besseyi in agar cultures. Even if virus transmission by C. xenoplax occurs via contamination rather than by a specific mechanism, it must be rare.

Use of experimental designs with quantitative ELISA.

Precise use of enzyme-linked immunosorbent assay (ELISA) as a quantitative technique depends on repeatability of color development and its measurement. Variation in measured response among wells, within and among microtiter plates, often precludes such precision. For example, plates with all wells treated uniformly exhibited unacceptable optical density differences in excess of 0.35 and 0.25 O.D. U among row and column averages, respectively. Arrangement of samples on plates according to classical experimental designs, with compact blocking features and two-dimensional control over spatial patterns, provides a possible remedy. Analysis of variations over uniformly treated plates demonstrated the potential for increased precision when such designs are used instead of random arrangements. Retrospective analysis of more than 100 tests performed with various experimental designs confirmed that this potential was realized when using Youden square and lattice square designs. Several designs appropriate for microtiter plates are presented and their conduct described.