Development and evaluation of quantitative-competitive PCR for quantitation of coxsackievirus B3 RNA in experimentally infected murine tissues.

A method is described for quantitation of enterovirus RNA in experimentally infected murine tissues. Viral RNA was extracted from tissue samples and amplified by reverse transcriptase PCR in the presence of an internal standard RNA. The ratio of PCR product derived from viral RNA and internal standard RNA was then determined using specific probes in a post-PCR electrochemiluminescent hybridization assay. This provided an estimate of the viral RNA copy number in the original sample, and detection of PCR product derived from internal standard RNA validated sample processing and amplification procedures. RNA copy number correlated with viral infectivity of cell culture-derived virus, and one tissue culture infective dose was found to contain approximately 10(3) genome equivalents. The ratio of RNA copy number to infectivity in myocardial tissue taken from mice during the acute phase of coxsackievirus B3 myocarditis was more variable ranging from 10(4)-10(7), and was dependent on the stage of infection, reflecting differential rates of clearance for viral RNA and viral infectivity. The assay is rapid, and could facilitate investigations which currently rely upon enterovirus quantitation by titration in cell culture. This would be useful for experimental studies of viral pathogenesis, prophylaxis and antiviral therapy.

Serological and molecular evidence of enterovirus infection in patients with end-stage dilated cardiomyopathy.

OBJECTIVE: To study the relative diagnostic value of enterovirus-specific molecular biological and serological assays in patients with end-stage dilated cardiomyopathy, and to investigate the possible role of other cardiotropic viruses in dilated cardiomyopathy. DESIGN: Analysis of recipient myocardial tissue and serum from patients with dilated cardiomyopathy and controls undergoing cardiac transplantation for end-stage cardiac disease. SETTING: University virology department and transplantation unit. METHODS: Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis of myocardial RNA and DNA; enterovirus-specific in situ hybridization; enterovirus-specific immunoglobulin M detection. RESULTS: Enterovirus RNA was detected in myocardial tissue from only a small proportion of (five of 75) hearts. However, although enterovirus-specific immunoglobulin M responses were detected in 22 (28%) of 39 controls patients, a significantly higher prevalence was observed among patients with dilated cardiomyopathy (22 (56%) of 39 patients; P < 0.005). All enteroviruses detected in myocardium showed greatest nucleotide sequence homology with coxsackievirus type B3. Detection of enterovirus RNA in myocardium by the polymerase chain reaction and by in situ hybridisation gave comparable results. Other potentially cardiotropic virus genomes, including human cytomegalovirus, influenzaviruses, and coronaviruses were not detected in myocardium. CONCLUSION: This study found that enterovirus-specific immunoglobulin M responses provided the strongest evidence of enterovirus involvement in patients with end-stage dilated cardiomyopathy. However, the high background prevalence of these responses limits their diagnostic value. The finding that enteroviruses detected in myocardium were coxsackievirus type B3 accords with recent findings in patients with acute myocarditis, and indicates that this serotype is the major cardiotropic human enterovirus.

Molecular detection and serotypic analysis of enterovirus RNA in archival specimens from patients with acute myocarditis.

OBJECTIVE: To determine whether enterovirus RNA can be demonstrated in archival necropsy material in acute myocarditis. DESIGN: Analysis of paraffin embedded myocardial tissue from cases of acute myocarditis. SETTING: University virology department. METHODS: Extraction of RNA from tissue followed by polymerase chain reaction (PCR) and DNA sequence analysis. PATIENTS: Six patients with histologically proven myocarditis and eight controls. RESULTS: Enterovirus RNA was identified in 5 of 6 patients with myocarditis and in none of the controls. The nucleotide sequences of the PCR products showed greatest similarity to group B coxsackieviruses, particularly coxsackievirus B3. CONCLUSION: This study indicates that archival tissue samples, even histologically stained tissue sections, can be used to study the role of enteroviruses in myocardial disease using molecular detection techniques. If a predominant role for coxsackievirus B3 in myocarditis is confirmed by further study, this may have implications for the development of a specific vaccine.

Immunoelectron microscopical labelling of a glycolipid in the envelopes of brain cell-derived budding viruses, Semliki Forest, influenza and measles, using a monoclonal antibody directed chiefly against galactocerebroside resulting from Semliki Forest virus infection.

Neurotropic RNA budding viruses such as Semliki Forest virus (SFV), influenza and measles were each grown in identical mouse brain cell cultures. Positive immunoelectron microscopical labelling with gold was seen in the envelope of these viruses using an anti-SFV derived glycolipid monoclonal antibody (MAb), 373 shown to be directed chiefly against galactocerebroside. The results indicate that each enveloped virus grown from the same cell type contains the same glycolipid in its envelope. The presence of common glycolipids derived from the host cell in the envelopes of various enveloped budding viruses may play a significant role in the pathogenesis of virus induced, immune mediated CNS autoimmunity and demyelination, particularly in multiple sclerosis (MS).

Enhancement of demyelination in the central nervous system of Semliki Forest virus infected mice after pretreatment with muramyl dipeptide.

Semliki Forest virus infections in mice produce an encephalitis with demyelination. If before giving the virus the mice are treated with muramyl dipeptide in Freund's incomplete adjuvant, there is a significant increase in demyelination. If ovalbumin is added to the above and then followed after an interval by a second dose of ovalbumin and finally by the virus, the demyelination is further, but only marginally increased. The addition of ovalbumin without muramyl dipeptide in the schedule appears to increase the number of infiltrating cells and to a lesser extent the perivascular cuffing, but does not increase the demyelination as compared to that obtained when Semliki Forest virus is given on its own.

The pathological effect on the central nervous system of mice following single and repeated infections of the demyelinating A7(74) strain of Semliki Forest virus.

The avirulent strain A7(74) of Semliki Forest virus was inoculated intraperitoneally into mice at weekly intervals for 7 weeks. Pathological, virological and serological studies were carried out twice weekly, after each infecting dose. Similar studies were performed on mice that had been given repeated inoculations at 2, 3 or 4 weekly intervals as were a group of control mice given a single dose of SFV. Results showed grossly enhanced central nervous system lesions, in particular the perivascular cuffing and demyelination, after the 2nd and 3rd weekly inoculation. With further injections there was no increase in severity of the lesions and by the eighth inoculation the pathological changes in the brain appeared to have recovered. The maximum and most persistent damage to the brain was seen after the 2nd and 3rd weekly inoculations. As the interval between two SFV inoculations was increased, the lesions in the central nervous system were reduced and protection increased. Virus in the blood was only detectable after the first inoculation and brain virus after the first and second inoculations. Peak IgG antibody levels were seen on day 46 after a single inoculation and day 35 in the multiple inoculations. It was concluded that repeated inoculations of SFV do not produce a relapsing demyelinating disease, but the 7th and 14th day inoculations do enhance the lesions which are seen to persist after the inoculation on the 21st day. In spite of the gross pathological changes inflicted, the brain damage appears to recover.

Demyelination induced in mice by avirulent Semliki Forest virus. I. Virology and effects on optic nerve.

Swiss A2G mice were infected intraperitoneally with an avirulent strain of Semliki Forest virus. The virus titres in the optic nerves, retina and brain were estimated on post-inoculation days 3 to 8. High titres of virus were obtained in the optic nerve, retina and brain. The optic nerves, retina and brain were studied by light microscopy up to post-inoculation day 67. Electron microscope studies wee carried out on the optic nerve on post-inoculation days 11 and 14. Inflammatory, microcystic changes and demyelination seen in the optic nerve were similar to those found in he central nervous system.

The effect of mouse strain on the pathogenesis of the encephalitis and demyelination induced by avirulent Semliki Forest virus infections.

Mice from the following strains--Simpson, SWR/J, TO, CBA/Ca, CW (outbred), LAC/G (outbred), SJL/J and Swiss A2G (outbred)--were infected i.p. with avirulent Semliki Forest virus. Clinical signs of disease were noted, histopathological changes assessed and the blood and brain virus infectivities and serum immunoglobulin levels were measured. Only the CW, TO and Swiss/A2G animals showed convincing evidence of demyelination at the light microscopy level but all strains developed encephalitis and microcystic (spongiform) lesions.

Maintenance of encephalitogenic viruses by non-neuronal cerebral cells.

Mouse and human brain glial elements have been successfully tissu-cultured and inoculated with two highly encephalitogenic viruses without apparent cytopathogenic effect but with long-term continued production of virus