The structure of foot-and-mouth disease virus.

Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural targets.

A multi-centre randomised controlled trial of minimally invasive direct coronary bypass grafting versus percutaneous transluminal coronary angioplasty with stenting for proximal stenosis of the left anterior descending coronary artery.

OBJECTIVES: To compare the clinical- and cost-effectiveness of minimally invasive direct coronary artery bypass grafting (MIDCAB) and percutaneous transluminal coronary angioplasty (PTCA) with or without stenting in patients with single-vessel disease of the left anterior descending coronary artery (LAD). DESIGN: Multi-centre randomised trial without blinding. The computer-generated sequence of randomised assignments was stratified by centre, allocated participants in blocks and was concealed using a centralised telephone facility. SETTING: Four tertiary cardiothoracic surgery centres in England. PARTICIPANTS: Patients with ischaemic heart disease with at least 50% proximal stenosis of the LAD, suitable for either PTCA or MIDCAB, and with no significant disease in another vessel. INTERVENTIONS: Patients randomised to PTCA had local anaesthetic and underwent PTCA according to the method preferred by the operator carrying out the procedure. Patients randomised to MIDCAB had general anaesthetic. The chest was opened through an 8-10-cm left anterior thoracotomy. The ribs were retracted and the left internal thoracic artery (LITA) harvested. The pericardium was opened in the line of the LAD to confirm the feasibility of operation. The distal LITA was anastomosed end-to-side to an arteriotomy in the LAD. All operators were experienced in carrying out MIDCAB. MAIN OUTCOME MEASURES: The primary outcome measure was survival free from cardiac-related events. Relevant events were death, myocardial infarction, repeat coronary revascularisation and recurrence of symptomatic angina or clinical signs of ischaemia during an exercise tolerance test at annual follow-up. Secondary outcome measures were complications, functional outcome, disease-specific and generic quality of life, health and social services resource use and their costs. RESULTS: A total of 12,828 consecutive patients undergoing an angiogram were logged at participating centres from November 1999 to December 2001. Of the 1091 patients with proximal stenosis of the LAD, 127 were eligible and consented to take part; 100 were randomised and the remaining 27 consented to follow-up. All randomised participants were included in an intention-to-treat analysis of survival free from cardiac-related events, which found a non-significant benefit from MIDCAB. Cumulative hazard rates at 12 months were estimated to be 7.1 and 9.2% for MIDCAB and PTCA, respectively. There were no important differences between MIDCAB and PTCA with respect to angina symptoms or disease-specific or generic quality of life. The total NHS procedure costs were 1648 British pounds and 946 British pounds for MIDCAB and PTCA, respectively. The costs of resources used during 1 year of follow-up were 1033 British pounds and 843 British pounds, respectively. CONCLUSIONS: The study found no evidence that MIDCAB was more effective than PTCA. The procedure costs of MIDCAB were observed to be considerably higher than those of PTCA. Given these findings, it is unlikely that MIDCAB represents a cost-effective use of resources in the reference population. Recent advances in cardiac surgery mean that surgeons now tend to carry out off-pump bypass grafting via a sternotomy instead of MIDCAB. At the same time, cardiologists are treating more patients with multi-vessel disease by PTCA. Future primary research should focus on this comparison. Other small trials of PTCA versus MIDCAB have now finished and a more conclusive answer to the original objective could be provided by a systematic review.

The internal ribosome entry site (IRES) of hepatitis C virus visualized by electron microscopy.

Translation of hepatitis C virus (HCV) RNA is initiated via the internal ribosome entry site (IRES), located within the 5' untranslated region. Although the secondary structure of this element has been predicted, little information on the tertiary structure is available. Here we report the first structural characterization of the HCV IRES using electron microscopy. In vitro transcribed RNA appeared as particles with characteristic morphology and gold labeling using a specific oligonucleotide confirmed them to be HCV IRES. Dimerization of the IRES by hybridization with tandem repeat oligonucleotides allowed the identification of domain III and an assignment of domains II and IV to distinct regions within the molecule. Using immunogold labeling, the pyrimidine tract binding protein (PTB) was shown to bind to domain III. Structure-function relationships based on the flexible hinge between domains II and III are suggested. Finally, the architecture of the HCV IRES was seen to be markedly different from that of a picornavirus, foot-and-mouth disease virus (FMDV).

Reversible dilated cardiomyopathy following treatment of atopic eczema with Chinese herbal medicine.

Chinese herbal medicines are increasingly being used as an alternative treatment for chronic skin disease. Most patients and many doctors remain insufficiently aware of their potential toxicity. We report a patient with eczema who developed a severe cardiomyopathy following a 2-week course of Chinese herbal medicine. The connection between the two conditions was not made until 2 weeks after presentation when the patient was specifically asked if she had ingested any unusual substances. The belief that herbs, as natural products available without prescription, are harmless, is commonplace and patients may not consider them worthy of mention during a standard medical history.

Specific interactions between human integrin alpha v beta 3 and chimeric hepatitis B virus core particles bearing the receptor-binding epitope of foot-and-mouth disease virus.

Purified integrin alpha v beta 3 was used in solid-phase binding studies with chimeric hepatitis B cores which carry the RGD-containing loop of VP1 protein of the foot-and-mouth disease virus (FMDV). High levels of specific binding between the integrin and the particles were detected by enzyme-linked immunosorbent assays. The binding was Mn2+ cation dependent and could be competed with fibronectin, vitronectin, and the peptide GRGDSPK. Particles in which the RGD motif had been mutated to RGE failed to bind, indicating that the chimeric cores bound specifically to the ligand binding site of integrin alpha v beta 3. Electron micrographs showed several individual alpha v beta 3 molecules bound to the surface of each chimeric particle. Collectively, these data constitute firm evidence that the RGD-containing loop of FMDV is critical for binding to alpha v beta 3 and provide support for identification of alpha v beta 3 as a potential cellular receptor for FMDV.

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.

Association of conventional and exertional coronary heart disease risk factors in 5,000 apparently healthy men.

BACKGROUND AND HYPOTHESIS: The aim of this study was to define the prevalence of previously undetected coronary heart disease among asymptomatic males, aged 30-65 years, by means of resting and exercise electrocardiography in conjunction with an analysis of conventional and exertional coronary risk factors. METHODS: Between January 1985 and December 1989 we examined 5,000 clinically asymptomatic subjects. A detailed case history was obtained for each individual, followed by a complete physical examination, comprehensive blood (including lipid) profile, lung function tests, chest x-ray, a resting 12-lead electrocardiogram (ECG), and a maximal treadmill exercise ECG. Whenever possible, on-line computerized respiratory analysis (Beckman Metabolic Measurement Cart) was carried out during the exercise tests. Conventional and exertional coronary heart disease risk factors were also recorded. RESULTS: A total of 162 persons (3.2%) showed abnormal S-T segment responses during the exercise or recovery period. Of these, 92 subjects underwent further investigations: coronary angiography (79), 201thallium scanning (13), 201thallium scanning followed by coronary angiography (7). Of the 86 patients who proceeded to coronary angiography, 19 (22%) had either normal coronary artery anatomy or only insignificant disease. Among the 67 (78%) of patients with significant angiographically demonstrable disease, 26 received coronary artery bypass grafting, 7 underwent coronary angioplasty, and the remainder continued on medical management. CONCLUSIONS: These results are discussed in relation to a variety of conventional and exertional coronary risk factors.

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.

Longitudinal study of fetal middle cerebral artery flow velocity waveforms preceding fetal death.

OBJECTIVE: To assess longitudinally fetal cerebral vasodilatation in small-for-gestational age fetuses to investigate whether intrauterine death might be predictable. DESIGN: Prospective observational study. SETTING: Ultrasound department in a university hospital. SUBJECTS: Five pregnancies with ultrasonographically confirmed small fetuses (abdominal circumference less than the 3rd centile) monitored longitudinally until time of intrauterine death. MAIN OUTCOME MEASURE: Time between last ultrasound examination and diagnosis of intrauterine death, and variation in middle cerebral artery pulsitility index prior to death. RESULTS: Two of the five fetuses showed a reversal of adaptation (as indicated by an elevation of the middle cerebral artery pulsitility index) within 48 hours of intrauterine death. The other three had their final ultrasound examination 3 to 7 days before death and showed no such reversal of adaptation. CONCLUSION: Reversal of adaptation in fetal hypoxaemia as indicated by a rise in the middle cerebral artery pulsitility index may be a predictor of intrauterine death within 48 hours. Whether delivery after reversal of adaptation would result in salvage of neurologically intact babies needs to be investigated.

Induction of T-helper cell response to hepatitis B core antigen in chronic hepatitis B: a major factor in activation of the host immune response to the hepatitis B virus.

The T helper (Th) cell response to hepatitis B core antigen (HBcAg) was analyzed in 76 chronic hepatitis B virus (HBV) carriers with varying degrees of hepatic inflammation and HBV replication. Fifty-five patients had active viral replication, 28 with minimal histological changes and normal alanine transaminase (ALT) and 27 with active hepatic inflammation and elevated ALT. The remaining 21 chronic hepatitis B surface antigen (HBsAg) carriers had undetectable HBV replication, minimal histological activity, and normal ALT. In addition, 34 chronic HBV carriers were studied prospectively during treatment with alpha-interferon. The HBcAg-specific Th cell response was evaluated by a proliferative assay using 3H-thymidine uptake and gamma-interferon production by peripheral blood mononuclear cells. The proliferative response and gamma-interferon production of patients with active hepatic inflammation were significantly higher than in patients with minimal histological changes and in controls. In the longitudinal analysis during alpha-interferon treatment, 22 of 34 patients sustained an ALT flare accompanied by a parallel, significant Th cell response, which preceded or coincided with the ALT flare. The elevation in the Th cell response and the ALT flare were followed by a significant rise in the serum immunoglobulin (Ig) M anti-HBc index. Ten of twenty-two patients with an enhanced Th cell response and an ALT flare seroconverted after alpha-interferon treatment. The Th cell activity in the 10 responders rapidly subsided after hepatitis B e antigen (HBeAg) to anti-HBe seroconversion, whereas in the 12 nonresponders it remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Rhinoviruses and cells: molecular aspects.

Recent developments in our understanding of the molecular structure and functioning of the human rhinoviruses are briefly reviewed. The determination of virus structure at near atomic resolution has provided a wealth of information that can be used to help interpret dynamic properties of the virus. The mechanisms involved in capsid assembly, attachment to cell surfaces, uncoating, and the initiation of infection and antibody-mediated neutralization of infectivity can all be examined afresh in the light of the structural data. In addition, molecular techniques for manipulating and analyzing the biological properties of nucleic acids and proteins are providing a wealth of information on details of the replication machinery of the viruses. Probably one of the least understood aspects of viral replication is the precise nature of the interactions between host and viral components during infection. Unfortunately, despite all of these impressive scientific advances the cure for the common cold is still elusive.

Drug-induced Sweet's syndrome (acute febrile neutrophilic dermatosis) associated with hydralazine.

We report a case of Sweet's syndrome associated with hydralazine. The association of Sweet's syndrome with hydralazine, and with the oral contraceptive, minocycline, and trimethoprim/sulphamethoxazole, has been reported previously. We suggest that a drug aetiology should be sought in cases of Sweet's syndrome.

In vitro cleavage of hepatitis C virus polyprotein substrates by purified recombinant NS3 protease.

The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

Monoclonal antibodies to a peptide of human rhinovirus type 2 with different specificities recognize the same minimum sequence.

Monoclonal antibodies (MAbs) raised against a synthetic peptide including residues 156-170 of protein VP2 of human rhinovirus type 2 (HRV2) have previously been shown to be of differing specificities. The basis for these differences has now been examined in greater detail by ELISA, radioimmunoprecipitation and virus neutralization. Reactions with a panel of HRV2 mutant viruses indicated that substitution of some residues could enhance the apparent activity of one of the neutralizing anti-peptide MAbs. For one such substitution, VP2 P164H, there appeared to be a correlation between increased neutralizing activity and enhanced binding. Mapping experiments identified two overlapping neutralization epitopes (amino acids 156-163 and 160-165) and several non-neutralizing epitopes. Although some differences in antibody reactivity were due to epitope specificity alone, the explanation for others was less obvious. Significantly, the majority of MAbs that recognized, and in some cases neutralized, native virus had the same minimum binding sequence and critical residue requirement as others which recognized virus particles only after distortion. This demonstrates that factors other than the linear sequence of the peptide can be crucial in determining the fine specificity, and hence biological relevance, of peptide antigens.

Analysis of NS3-mediated processing of the hepatitis C virus non-structural region in vitro.

The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3-5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.

The prognosis for assisted conception treatment after unexpected failure of fertilization in vitro: a comparative study.

The relative prognosis for further assisted conception treatment (without micro-injection) after initial unexpected failure of fertilization in apparently favourable couples undergoing in-vitro fertilization (IVF) treatment was assessed. After their first cycle of treatment, 481 consecutive couples were grouped according to their fertilization (including cleavage) rate per oocyte into five bands. Proportions of couples proceeding to further cycles of treatment by IVF or gamete intra-Fallopian transfer (GIFT) and resulting fertilization and pregnancy rates were compared. Pregnancy rates in the first cycle of treatment were significantly related to fertilization rate. The fertilization rate was zero in 13 couples (3%) and only 1-24% in 18 (4%). There were no significant differences between these groups in the proportions proceeding to further treatment (31, 50%) compared with others (overall 37%, including some treated by GIFT), or in their median fertilization rates (75, 60% compared with 67%--IVF cycles only), pregnancy rates (20, 38% of cycles compared with 37%--IVF or GIFT) or birth rates (20, 38% of cycles compared with 31%--IVF or GIFT). Amongst couples whose initial fertilization rate was > or = 50% there was no fertilization in 4% of subsequent IVF cycles. We conclude that in couples with well defined favourable conditions, including tests of sperm function for assisted conception treatment, who have unexpected failure of fertilization, the prognosis for further treatment remains favourable without resort to more complex investigations or micro-injection methods. Such failure occurs infrequently and generally as a random event, and should have no appreciable effect on life-table calculation of cumulative pregnancy and birth rates in this group of patients.

A comparison of early mortality and morbidity after single and bilateral internal mammary artery grafting with the free right internal mammary artery.

OBJECTIVE: To compare differences in early mortality and morbidity in patients receiving a single internal mammary artery graft (SIMA) with those receiving bilateral internal mammary artery grafts (BIMA) with a free right internal mammary artery (RIMA). DESIGN: Retrospective analysis of 150 patients undergoing BIMA grafting between 1989-1992 who were carefully matched with 150 patients undergoing SIMA grafting between 1987-1992 for known cardiovascular risk factors, extent of coronary disease, left ventricular function, and number of coronary grafts. Operative variables noted included aortic cross clamp time and bypass time. Postoperative cardiac, respiratory, and wound complications were also noted. RESULTS: Operative mortality was 2% in the SIMA group and 1.3% in the BIMA group (NS). Other than the prevalence of ventricular arrhythmias (P = 0.025), which were more common in the BIMA group, there were no significant differences between the two groups in terms of postoperative morbidity. At median (interquartile range) follow up of 27.94(0.86) and 23.94(0.74) months for the SIMA and BIMA groups respectively there were no deaths. 87% of the SIMA group and 91% of the BIMA group were free of symptoms at follow up. CONCLUSIONS: The earlier fears regarding increased early mortality and morbidity after BIMA surgery were not confirmed by this study. All patients receiving both mammary arteries had a free rather than pedicle right internal mammary graft. The early mortality and morbidity reported here compares favourably with previous reports on the use of a pedicle graft.

Epitope analysis of the T cell response to a complex antigen: proliferative responses to human rhinovirus capsids.

Understanding the factors which regulate the repertoire of a T cell response is important when selecting T helper cell epitopes for inclusion in synthetic viral vaccines. In this study we have examined the T cell response to human rhinovirus (HRV) type 1 A in a mouse model system, using a comprehensive set of synthetic peptides which span all four of the proteins which make up the HRV capsid. This constitutes the first study to use a set of peptides covering the entire sequence of all structural proteins of any virus. This study identifies the major proliferative (CD4) T cell epitopes within the minor receptor group HRV 1 A, and analyzes these epitopes with relation to their location within the three-dimensional structure of the virus. The proliferative response to HRV is highly selective, with strong responses to only a very small number of epitopes, many of which are grouped together within restricted areas of the primary structure of the HRV proteins. The repertoire of the response is almost entirely specific to the major histocompatibility complex haplotype of the host. The major T cell epitopes are spatially distinct from the sites of the major antibody recognition sites, and are buried within the viral capsid. In striking contrast to the antibody responses, the T cell responses are highly cross-reactive against a wide variety of viral serotypes.