Dengue virus infection induces upregulation of GRP78, which acts to chaperone viral antigen production.

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

Troponin-I positivity in patients referred to Rapid Access Chest Pain Clinic.

BACKGROUND: Rapid Access Chest Pain Clinics (RACPCs) are set up to access patients with new onset chest pain (within the preceding three weeks), of possible cardiac origin. These patients are seen in the clinic within two weeks of referral and the attending physician takes a history, performs a routine clinical examination, and if clinically justified, a treadmill exercise test is performed according to Bruce Protocol. Within the group of patients referred to the RACPC with new onset but otherwise stable angina, there is a potential overlap with patients who in fact may have an evolving acute coronary syndrome, i.e., unstable angina. The aim of this study was to assess the prevalence of Troponin-I positivity as an indicator of acute coronary syndrome. METHODS: This cross-sectional descriptive study included 60 consecutive patients referred to the RACPC with history of recent onset chest pain (within the last three weeks) of possible cardiac origin and positive ETT or confirmed abnormal ischemic ECG at baseline. Troponin-L was measured in these patients. RESULTS: Out of the total 60 patients, 8.33% of the patients referred to RACPC with new onset angina had positive cTnI. CONCLUSION: Point of care test (POCT) for cTnI can help to identify the high risk patient referred to RACPC.

Rapid detection of influenza A virus in clinical samples using an ion channel switch biosensor.

This report describes the fabrication and successful use of the ion channel switch biosensor (ICSB) for rapid point-of-care detection of influenza A in different types of respiratory specimens. Virus culture -- regarded as the "gold standard" -- and an immunochromatographic rapid point-of-care test for influenza A virus were compared with the biosensor. The ICSB rapid test provided an objective readout within 10 min of specimen inoculation into the ICSB chamber wells, without the need for chemical or other pretreatments. Construction of the ICSB with specific antibodies also enables rapid detection and identification of appropriate influenza A subtypes.

Disease progression and adverse events in patients listed for elective percutaneous coronary intervention.

OBJECTIVE: To record disease progression and the timing of adverse events in patients on a waiting list for elective percutaneous coronary intervention (PCI). DESIGN: Observational prospective study. SETTINGS: A UK tertiary cardiothoracic centre, at a time when waiting lists for PCI were up to 18 months. PATIENTS: 145 patients (116 men, median age 59.5 years) placed on an elective waiting list for PCI between October 1998 and September 1999. MAIN OUTCOME MEASURES: Adverse events recorded were death, myocardial infarction, need for urgent hospital admission because of unstable angina, and need for emergency revascularisation while waiting for PCI. RESULTS: During a median follow up of 10 months (range 1-18 months), nine (6.2%) patients experienced an adverse event. Eight (5.52%) patients were admitted with unstable angina as emergencies. One was admitted with a myocardial infarction. Twenty nine (20.0%) patients had significant disease progression at the time of the repeat angiogram before PCI. In 10 (7%), disease had progressed so that PCI was no longer feasible and patients were referred for coronary artery bypass graft. Sixteen (11%) were removed from the PCI waiting list because of almost complete resolution of their anginal symptoms. CONCLUSION: Adverse coronary events and clinically significant disease progression occur commonly in patients waiting for PCI. Despite the presence of severe coronary lesions, myocardial infarction was rare and no patients died while on the waiting list. Resolution of anginal symptoms was also comparatively common. The pathophysiology of disease progression frequently necessitates a change in the treatment of patients waiting for PCI.

Enhancement or inhibition of HIV-1 replication by intracellular expression of sense or antisense RNA targeted at different intermediates of reverse transcription.

OBJECTIVES: To construct retroviral vectors expressing sense or antisense RNA targeted at HIV reverse transcription intermediates, and to test the anti-HIV properties of these constructs in transduced T cells. DESIGN: Five double-copy retroviral vectors were constructed, in which the expression of the sense or antisense RNA corresponding to HIV minus- or plus-strand strong-stop DNA was driven by the human tRNA(met) promoter. METHOD: The templates for the sense or antisense RNA were polymerase chain reaction-cloned from HIV pNL43 into a murine leukaemia virus-based vector and corresponding defective virions were packaged in PA317 cells. Human Jurkat T cells transduced with these vectors were challenged with HIV and monitored for viral RNA, viral DNA and p24 production for 23 weeks. RESULTS: Intracellular expression of HIV sense RU5 sequences (RNA complementary to minus-strand strong-stop DNA) enhanced HIV replication in T cells. Expression of HIV sense or antisense U3RU5 sequences (identical or complementary to plus-strand strong-stop DNA) conferred long-term inhibition of HIV replication, despite continuous presence of viral challenge in the transduced cell cultures. CONCLUSION: Plus-strand strong-stop DNA as an intermediate in the early process of viral reverse transcription can be explored as an additional target for anti-HIV gene therapy.

Interference between effector RNAs expressed from conventional dual-function anti-HIV retroviral vectors can be circumvented using dual-effector-cassette retroviral vectors.

Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.

Serological analysis of duck hepatitis B virus infection.

A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen and antibody; viraemia (DHBV DNA or DHBsAg) was detected in all ducks inoculated within 3 weeks post-hatch, and persistent infection developed in 93% of birds in this group. In contrast, only 80% and 60% of ducks inoculated 4- and 6-weeks post-hatch respectively developed viraemia, and approximately 70% of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated 8 weeks post-hatch and in uninfected controls. A typical anti-DHBs seroconversion developed subsequently in 2 of 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection develops with increasing age that may be related to several factors including host immunity. This model may help elucidate similar age-related features of human hepatitis B virus infections.

Synthesis of human immunodeficiency virus DNA in a cell-to-cell transmission model.

Cell-to-cell transmission of human immunodeficiency virus type 1 (HIV-1) was modelled by coculturing virus-infected cells with uninfected target cells at a ratio of 1:4. While H9 cells persistently infected with HTLV-IIIB did not contain unintegrated viral DNA detectable by Southern blotting, when cocultured with uninfected HUT-78 cells the mixed culture effectively underwent a new round one-step virus replication which began de novo synthesis of free viral DNA within 4 hours. Linear DNA was synthesized before the accumulation of circular DNA, and two seemingly distinct phases of viral DNA synthesis were involved. When both virus donor cells and recipient cells were arrested in the G0/G1 phase of the cell cycle, accumulation of circular viral DNA was inhibited. In contrast to cell-free virus infection of resting human peripheral blood mononuclear cells (PBMC), where no free viral DNA of discrete sizes could be detected by Southern blot, cell-to-cell transmission infection of resting PBMC resulted in the synthesis of full-length linear as well as circular viral DNA. The efficiency with which cell-to-cell transmission of HIV initiates virus replication underlines the importance of this mode of transmission in virus dissemination in vivo.

A survey of nosocomial respiratory viral infections in a children's hospital: occult respiratory infection in patients admitted during an epidemic season.

OBJECTIVE: To define the extent of shedding of respiratory viruses and Mycoplasma pneumoniae among a population of pediatric patients admitted to the hospital during a winter epidemic period and to identify nosocomial infections within this population. DESIGN: An open, prospective survey of patients admitted to three wards (General Medical, Respiratory Infectious, and Infectious Diseases) of a pediatric hospital during a defined three-month period. PATIENTS: All patients with medical, respiratory, and infectious conditions admitted to three wards of the Adelaide Children's Hospital had nasopharyngeal aspirations performed at the time of admission with the purpose of documenting viral and M pneumoniae shedding. Patients were monitored daily for the development of symptoms of respiratory infection or new symptoms of respiratory disease. Such patients underwent a further nasopharyngeal aspiration for the purpose of diagnosing hospital-acquired infection. RESULTS: Nasopharyngeal aspirations were obtained from 601 patients. Forty-seven percent of asymptomatic patients were positive for a respiratory virus or M pneumoniae, and 61% of patients with respiratory symptoms were also positive. Gastroenteritis patients shed viruses in 66% of cases. Respiratory symptoms were initially overlooked by admitting physicians but subsequently identified in 110 cases, and 46% of these were found to be positive for a respiratory virus or M pneumoniae. There were 18 possible hospital acquired infections among the 293 initially virus-negative patients. Multiple isolates were obtained from a substantial number of patients, especially those with respiratory symptoms. CONCLUSIONS: A substantial proportion of all patients admitted to a pediatric hospital during winter represent a potential source of infection, and strict infection control measures should be enacted to limit the spread of these infections.

Isolated clinic hypertension is not an innocent phenomenon: effect on the carotid artery structure.

This study examines the common carotid intimal-medial wall thickness (CCA-IMT) in untreated patients with elevated clinic blood pressure (BP) but normal ambulatory BP (isolated clinic hypertension, n = 22), in comparison with a group with elevated clinic and ambulatory BP (hypertensives, n = 41) and a group with normal clinic and ambulatory BP (normotensives, n = 17) readings. The three groups did not differ in age, male/female ratio, lipid profile, glucose tolerance test, or smoking habits. No difference existed in CCA-IMT values between the groups with hypertension (0.67 +/- 0.18 mm) and isolated clinic hypertension (0.68 +/- 0.14 mm), but the values in these two groups were significantly higher (one-way ANOVA; F = 8.09, P < .001) than in the group of normotensives (0.50 +/- 0.09 mm). The CCA-IMT did not correlate with clinic systolic or diastolic BP readings or with BP derivatives of 24-h ambulatory monitoring. Mean 24-h BP in the isolated clinic hypertensives did not differ from that in the normotensives, whereas both were lower than in the hypertensives. We conclude that changes in the CCA-IMT occuring in subjects with isolated clinic hypertension are equal to the changes in sustained hypertension, indicating that isolated clinic hypertension may not be a benign condition.

Differing prevalence of human papillomavirus RNA in penile dysplasias and carcinomas may reflect differing etiologies.

Penile intraepithelial neoplasias grade 3 (PIN 3) and penile carcinomas were examined for the presence of human papillomavirus (HPV) RNA transcripts by in situ hybridization using 125I-labeled RNA probes. Human papillomavirus transcripts were detected in all 10 PIN 3 lesions not associated with invasive malignant conditions but were present in only 29% of penile carcinomas (9 of 26 squamous cell carcinomas and none of 5 verrucous carcinomas). Human papillomavirus RNA-positive penile cancers were significantly more likely to exhibit adjacent PIN 3 lesions than were HPV-negative tumors, and PIN 3 lesions adjacent to tumors always contained the same HPV-RNA type as was present in the invasive tumor. The development of most penile cancers may be unrelated to HPV infection. Future epidemiologic studies of the role of sexually transmitted factors in the development of penile carcinoma should distinguish between HPV-positive and HPV-negative penile cancers.

Widespread presence of cytoplasmic HBcAg in hepatitis B infected liver detected by improved immunochemical methods.

Cytoplasmic and cell membrane associated hepatitis B core antigen (HBcAg) were found to be more widespread within infected liver using indirect immunofluorescence on frozen sections than with the widely used direct immunofluorescence method. Fixation of frozen sections with carbon tetrachloride improved tissue histology without reducing the sensitivity of antigen detection. In tissue blocks fixed with formalin or ethanol-acetic acid, detection of HBcAg was reduced in comparison with frozen sections, and many cells containing low concentrations of (usually cytoplasmic and membranous) HBcAg could not be identified even using indirect immunofluorescence or peroxidase-antiperoxidase reactions. In contrast, intracellular hepatitis B surface antigen (HBsAg) was well detected in fixed sections, but membrane associated HBsAg was not detectable after fixation.

Antibody to hepatitis B antigen in haemophiliacs and their household contacts.

The prevalence of antibody to hepatitis B antigen, detectable by radioimmunoassay, was found to be no higher among 58 long-term household contacts of multiply transfused haemophiliacs than among 100 randomly chosen blood donors. This suggested that such contacts do not have greater exposure to serum hepatitis virus than that occurring through natural means. Among those persons possessing antibody, the multiply transfused haemophiliacs showed a marked tendency for higher antibody titres than their contacts, implying differences in pathogenesis between infection acquired through multiple transfusion and infection acquired naturally.

Antibody to hepatitis B surface antigen in haemophiliacs on long-term therapy with Scottish factor VIII.

Thirty-five patients with haemophilia A were studied clinically and serologically between 1971-2 and 1975-6 for evidence of hepatitis B infection. One patient suffered from clinical hepatitis B, and a further eight patients showed antibody responses to hepatitis B surface antigen (HBsAg) consistent with exposure to HBsAg during this period. No evidence for HBsAg exposure was found in 14 patients, while the remaining 12 patients had high titres of antibody to HBsAg at both times and no inferences could be drawn about HBsAg exposure. All patients had received exclusively replacement factor VIII material prepared locally from HBsAg-screened voluntary Scottish blood donations. From the details of the therapy given we calculated that the rate of HBsAg seroconversion in these patients represented about 0.3 HBsAg-containing donations/1000 donations.

Hepatitis Be antigen and antibody in hepatitis B surface antigen positive blood donors.

In a study of 105 asymptomatic HBsAg positive blood donors, 9 (8.6%) were found to have HBeAg, 38 (36.2%) anti-HBe, and the remaining 58 (55.2%) neither marker detectable by gel diffusion. There was no correlation between HBeAg/anti-HBe status and HBsAg sub-types, Glm allotypes, the presence of anti-Gm, red cell antibodies, or rheumatoid factor. Rheumatoid factor activity could be removed from anti-HBe positive sera without removing anti-HBe activity, indicating that separate entities were involved. HBeAg was found only in donors under the age of 30 (P less than 0.005), while anti-HBe did not show an age-related trend. HBeAg was also found less commonly in donors of blood group A than in the total carrier population (P less than 0.05), indicating an apparent protection in carriers of group A. The blood group distribution for the 105 HBsAg positive donors was similar to that of the general population.

Routine diagnosis of seven respiratory viruses and Mycoplasma pneumoniae by enzyme immunoassay.

A composite EIA, using 8-well microstrips, was used for the rapid detection of seven respiratory viruses and M. pneumoniae. The viruses included influenza A and B, parainfluenza 1, 2 and 3, adenovirus and respiratory syncytial virus. During the 61 month period--June 1988 to June 1993--17326 respiratory specimens, submitted from three states, were tested by this EIA. The specimens were mainly from a paediatric population (hospitals and private physicians). RSV was the predominant virus detected, followed by adenovirus, parainfluenza 3, M. pneumoniae, influenza A, parainfluenza 2, influenza B and parainfluenza 1. The use of blocking antibodies confirmed the identification of the agents, in particular with samples showing absorbance values greater than the cutoff with more than one infectious agent. Different methods for processing specimens in order to obtain a uniform suspension, and interpretation of non-specific reactions, are discussed. The assays showed an average sensitivity of 85% and specificity of 99%, compared to virus culture. This EIA system provided an efficient method for the rapid diagnosis of viral and mycoplasmal infections in a busy diagnostic laboratory.

Use of recombinant hepatitis delta antigen in diagnostic assays for HDV antibody.

The gene encoding the hepatitis delta virus (HDV) structural antigen (HD Ag) was inserted into a Rous sarcoma virus expression vector and the recombinant plasmid used to direct the synthesis of recombinant HD Ag (rHD Ag) in a continuous hepatoma cell line. A competitive radioimmunoassay for serum antibody to HDV using rHD Ag was developed and was found to be equally suitable for diagnostic purposes to a radioimmunoassay using infected liver-derived HD Ag. Similarly, rHD Ag was shown to be serologically equivalent to liver-derived HD Ag within the limit of the blocking titrations performed. The rHD Ag-positive cell line was also used in an indirect immunofluorescence assay to detect anti-HD. Similar titres of anti-HD were detected by both radioimmunoassay and immunofluorescence and identical samples were positive for anti-HD by either assay. In a sample of prison inmates with high prevalence of both HBV and HDV, anti-HD was confined almost exclusively to those with persistent HBV infection and not to those in whom HBV infection had cleared. The availability of rHD Ag will permit wider development of diagnostic anti-HD assays, and the use of two such assays is presented in this study.

Anomalous origin of the left coronary artery from the pulmonary artery: natural history and normal pregnancies.

Two female patients are described with anomalous origin of the left coronary artery arising from the pulmonary artery who sustained an anterolateral myocardial infarction in infancy. Neither patient received surgical treatment although both have lived to middle age with minimal cardiovascular problems and have had uncomplicated pregnancies. Good exercise tolerance and long term survival may be possible even without surgery for patients with this anomaly.

Diagnostic cardiac catheterisation in a hospital without on-site cardiac surgery.

OBJECTIVE: To assess the feasibility, safety, and clinical impact of diagnostic cardiac catheterisation in a multipurpose laboratory in a district general hospital without cardiac surgery. METHODS: A prospective audit of the first 2000 consecutive cases between September 1992 and March 1997. Unstable patients were referred to a surgical centre for investigation, in line with subsequently published British Cardiac Society (BCS) guidelines, but all other patients requiring cardiac catheterisation were investigated locally and are included in this report. The function of the laboratory was also compatible with the BCS guidelines regarding staffing, operators, equipment, number of cases, and locally available vascular surgery. RESULTS: Of the 2000 cases, 1988 studies were completed (99%), 1985 (99%) included coronary angiography, and 1798 (90%) were performed as day cases. Left main stem disease was present in 157 (8%), three vessel disease in 683 (34%), two vessel disease in 387 (19%), single vessel disease in 424 (21%), and normal coronary arteries in 494 (25%). Of the latter, 284 (14% of the total) had another cardiac diagnosis for which they were investigated (for example, valvar heart disease). Referral for cardiac intervention following catheterisation was made in 1172 of the 2000 cases (intervention rate 59%; catheter:intervention ratio 1. 7:1). The interventions performed were coronary artery bypass grafting (CABG) in 736 of the 1172 cases (63%), other types of cardiac surgery in 122 (10%), combined CABG and other cardiac surgery in 71 (6%), and percutaneous transluminal coronary angioplasty in 243 (21%). There were two catheter related deaths (0. 1%), both of which occurred within 24 hours of the procedure, and a further nine major cardiovascular complications with residual morbidity (0.45%). These were myocardial infarction in two (0.1%), cerebrovascular events in two (0.1%), and surgical vascular complications in five (0.25%). In addition, there were eight successfully treated, life threatening arrhythmias (0.4%). CONCLUSIONS: Diagnostic cardiac catheterisation can be performed safely and successfully in a local hospital. When BCS guidelines are followed, the mortality is similar to published pooled data from regional centres (0.1% v 0.12%). The high intervention rate indicates a persistent unmet demand in the districts, which will continue to affect surgical and interventional services.

Successful external cardioversion for ventricular tachycardia using 2 Joules.

We present a patient with recurrent sustained monomorphic ventricular tachycardia following myocardial infarction, refractory to drug therapy. He has been reluctant to have an implantable cardioverter-defibrillator, but responds to very low energy transthoracic direct current cardioversion administered via external pacing electrodes under sedation. The use of very low energies may avoid shock-induced myocardial damage in patients with recurrent ventricular tachycardia.