Clinical assessment of a generic DNA amplification assay for the identification of respiratory adenovirus infections.

BACKGROUND: respiratory adenoviruses are common, often resulting in serious sporadic and epidemic infections and impaired immunity can dramatically increase their severity. They are now thought capable of establishing latency. Diagnosis by culture is slow while direct antigen detection by immunofluorescence lacks sensitivity. Molecular diagnosis can be both rapid and sensitive but the genetic heterogeneity of adenoviruses poses problems. OBJECTIVES: to design a generic adenovirus nested polymerase chain amplification assay designed to be capable of detecting all respiratory adenoviruses. This was achieved through optimised thermal cycling and the development of a generic degenerate primer set targeting the adenovirus hexon gene. STUDY DESIGN: this was a cross-sectional study on 172 respiratory specimens from hospital-based patients, and one from a general practice, in Northern Ireland. A comparison was made between the amplification assay, virus culture and immunofluorescence. RESULTS: the nested polymerase chain reaction (nPCR) assay had a generic capacity for adenovirus detection and an analytical sensitivity of 6.4x10(2) copies/ml. Using an expanded gold standard (defined as a true positive or a true negative where a specimen was positive or negative by at least two of the study assays, respectively), PCR had a clinical sensitivity and specificity of 46/46 (100%) and 15/126 (91.3%), respectively. Patients with acute respiratory adenovirus infections were more likely to be male (chi(2), p=0.005) and to present with a fever (chi(2), p=0.02) than patients diagnosed with another respiratory virus. Co-infection was identified in 12/172 patients. CONCLUSIONS: the nested amplification assay proved highly sensitive in both the analytical and clinical settings for the detection of respiratory adenovirus infections.

Recombinant measles viruses expressing altered hemagglutinin (H) genes: functional separation of mutations determining H antibody escape from neurovirulence.

Measles virus (MV) strain CAM/RB, which was adapted to growth in the brain of newborn rodents, is highly neurovirulent. It has been reported earlier that experimentally selected virus variants escaping from the monoclonal antibodies (MAbs) Nc32 and L77 to hemagglutinin (H) preserved their neurovirulence, whereas mutants escaping MAbs K71 and K29 were found to be strongly attenuated (U. G. Liebert et al., J. Virol. 68:1486-1493, 1994). To investigate the molecular basis of these findings, we have generated a panel of recombinant MVs expressing the H protein from CAM/RB and introduced the amino acid substitutions thought to be responsible for antibody escape and/or neurovirulence. Using these recombinant viruses, we identified the amino acid changes conferring escape from the MAbs L77 (377R-->Q and 378M-->K), Nc32 (388G-->S), K71 (492E-->K and 550S-->P), and K29 (535E-->G). When the corresponding recombinant viruses were tested in brains of newborn rodents, we found that the mutations mediating antibody escape did not confer differential neurovirulence. In contrast, however, replacement of two different amino acids, at positions 195G-->R and 200S-->N, which had been described for the escape mutant set, caused the change in neurovirulence. Thus, antibody escape and neurovirulence appear not to be associated with the same structural alterations of the MV H protein.

Nitric oxide synthase activity and expression in retinal capillary endothelial cells and pericytes.

The purpose of this study was to examine nitric oxide synthase (NOS) expression in the retinal vasculature in vivo and to study nitric oxide (NO) synthesis in vitro in retinal microvascular endothelial cells and pericytes. Immunoreactivity was examined using a polyclonal antibody raised against porcine cerebellar nitric oxide synthase on frozen sections cut from postmortem human retina and trypsin digests of rat retinal vasculature. The synthesis of nitrite, a stable end product from the interaction of NO with molecular oxygen, was measured in culture supernatants of retinal microvascular cells under basal and stimulated conditions. Expression of constitutive NOS (cNOS) in these cells was examined using the polymerase chain reaction (PCR). Strong NOS immunoreactivity was seen in the endothelium of choroidal and retinal vessels. Nitrite synthesis was documented in supernatants from cultured microvascular endothelial cells which increased significantly following exposure to A23187 and cytokines. Nitrite synthesis by pericytes was not detectable under basal conditions or following stimulation with A23187. Bacterial lipopolysaccharide (LPS), a potent inducer of NOS, caused an increase in nitrite concentrations in pericyte supernatants 24 h after stimulation suggesting the presence of inducible NOS (iNOS). PCR amplification confirmed the presence of the cNOS gene in endothelial cells but not in pericytes. Retinal vascular endothelial cells express significant amounts of NOS constitutively in vivo and in vitro which is activated by Ca++. Also, endothelial cells can be stimulated to synthesize iNOS by cytokines. Retinal pericytes too show iNOS activity following exposure to bacterial LPS. These results suggest that the nitric oxide synthase/nitric oxide pathway may be involved in the regulation of microcirculatory haemodynamics in the retina.