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An RGD (arginine-glycine-aspartic acid) motif in coxsackievirus A9 has been implicated in internalization through an interaction with the integrin alpha v beta 3. We have produced a number of virus mutants, lacking the motif, which have a small-plaque phenotype in LLC-Mk2 and A-Vero cells and are phenotypically normal in RD cells. Substitution of flanking amino acids also affected plaque size. The results suggest that interaction between the RGD motif and alpha v beta 3 is not critical for virus viability in the cell lines tested and therefore that alternative regions of the CAV-9 capsid are involved in internalization.

Recent prospective studies have documented serologically an increased frequency of enterovirus infections in prediabetic children, indicating that these infections may initiate and accelerate the beta-cell damaging process several years before the clinical manifestation of type 1 diabetes. The aim of the present study was to establish whether these serological findings would be supported by the detection of enterovirus RNA in a unique prospective series of sera collected from prediabetic children 0-10 years before the manifestation of clinical type 1 diabetes. Reverse transcription followed by polymerase chain reaction employing highly conserved primers among enteroviruses were used to amplify enteroviral sequences. Viral RNA was found in 22% (11/49) of follow-up samples from prediabetic children but in only 2% (2/105) of those from controls (OR 14.9, P0.001). Persisting RNA positivity was not observed in any of these children. The presence of enterovirus RNA was associated with concomitant increases in the levels of autoantibodies against islet cells (OR 21.7, P0.01) and glutamic acid decarboxylase (OR 15.4, P0.05), but not in the levels of antibodies against insulin or the tyrosine phosphatase-like IA-2 protein. In contrast to the prediabetic children, those with newly diagnosed type 1 diabetes were negative for enterovirus RNA. The results thus complement previous serological data, suggesting that enterovirus infections are an important risk factor underlying type 1 diabetes and associated with the induction of beta-cell autoimmunity even years before symptoms appear.

Detection of enteroviruses and rhinoviruses has traditionally been based on laborious and time-consuming virus isolation. Recently, rapid and sensitive assays for detecting enterovirus and rhinovirus genomic sequences by reverse transcription-polymerase chain reaction (RT-PCR) have been introduced. An RT-PCR assay is described that amplifies both enteroviral and rhinoviral sequences, followed by liquid-phase hybridization carried out in a microtiter plate format. In the hybridization assay, amplicons are identified by enterovirus- or rhinovirus-specific probes carrying lanthanide chelate labels, which can be detected simultaneously by time-resolved fluorometry. The sensitivity and specificity of the RT-PCR-hybridization method were evaluated with a representative collection of enteroviruses and rhinoviruses and tested further its applicability to the clinical setting with cerebrospinal fluid samples and nasopharyngeal aspirates. The RT-PCR assay amplified all enteroviruses and rhinoviruses tested, and all but one amplicon gave a positive result in the subsequent hybridization assay. The RT-PCR-hybridization method was more sensitive than virus isolation for the detection of enteroviruses and rhinoviruses in the clinical samples. High sensitivity, rapidity, and easy performance make the assay suitable for the routine diagnosis of enterovirus and rhinovirus infections.

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.

Parainfluenza virus types 1 to 4 (PIV1 to PIV4) are important human pathogens that cause upper and lower respiratory tract infections, especially in infants and children. PIV1, PIV2, and PIV3 are second only to respiratory syncytial virus as a cause of croup in young children. Although some clinical symptoms are typical of PIVs, etiologic diagnosis always requires detection of infectious virus, viral components, or an antibody response. PIVs are typical paramyxoviruses, causing a syncytial cytopathic effect in cell cultures; virus growth can be confirmed either by hemadsorption or by using immunological reagents. Currently, PIV is most often diagnosed by demonstrating viral antigens in clinical specimens by rapid and highly sensitive immunoassays. More recently, PCR has been used for the detection of PIVs. Serological diagnosis is made by detecting a rising titer of immunoglobulin G or by demonstrating immunoglobulin M antibodies. PIVs infect species other than humans, and animal models are used to study the pathogenesis of PIV infections and to test candidate vaccines. Accumulating knowledge on the molecular structure and mechanisms of replication of PIVs has accelerated research on prevention and treatment. Several strategies for vaccine development, such as the use of live attenuated, inactivated, recombinant, and subunit vaccines, have been investigated, and it may become possible to prevent PIV infections in the near future.

Myocarditis is a relatively common complication of viral infections and for example enteroviruses, influenza-viruses and adenoviruses are found in association with this disease. However, in many clinical myocarditis cases the etiology remains unknown when the present routine methods are used for viral diagnosis. The availability of myocardial biopsy samples has now made it possible to analyse the presence of viruses and viral components directly in the heart muscle. In particular the molecular methods, in situ hybridization and polymerase chain reaction (PCR), have recently given promising results. Furthermore, the use of these methods is casting new light in the viral etiology of idiopathic dilated cardiomyopathy.

We have applied a sensitive in situ hybridization method for the detection of coxsackievirus RNA in myocardial tissue. Two radioactive cRNA probes were used: an RNA transcript representing the 5' end of poliovirus 3 which recognizes a highly conserved region among enteroviruses and an RNA transcript of a 1.1 kb fragment from the polymerase gene region of coxsackievirus B3. The reactivity of these probes was tested by dot-blot hybridization against a panel of enteroviruses. Formaldehyde-fixed and paraffin-embedded tissue of experimentally coxsackievirus B3 infected mice was analyzed for the localization of virus RNA. Both the probes gave signals in mouse myocardial cells, disseminated evenly in the heart tissue 7 days postinfection. At this time point, hybridization-positive cells and inflammatory reaction were mainly found in different areas that may be due to the inability of the immune system to recognize the infected cells before cytolysis. The samples were still positive when fixed 52 hours postmortem indicating that virus RNA is in a relatively stable form in the cells.

The presence of mumps virus and enterovirus RNA was studied by in situ hybridization in 15 surgical biopsy specimens from patients with acute pancreatitis. 35S-Labeled cRNA probes, detecting the 5′ end of the poliovirus type 3, a 1.1-kb fragment of the polymerase gene region of coxsackievirus B3, and mumps virus mRNA, encoding the nucleocapsid protein, were used. The controls consisted of mumps virus-infected and uninfected cultured cells, normal human and mouse pancreatic tissue, and mouse tissue from experimental coxsackievirus B3-induced pancreatitis. No specific hybridization signal was observed in any of the acute pancreatitis cases. It is concluded that neither mumps virus nor enteroviruses tested were present in pancreatic tissue of advanced human acute pancreatitis.

Replication of measles virus was restricted in human peripheral blood mononuclear cells (PBMC). However, in in vitro-infected, unstimulated cells, active synthesis of viral RNA and proteins occurred, while the release of infectious virus could not be detected. Stimulation with PHA caused a productive infection cycle comparable to the lytic infection. Replication of viral RNA was demonstrated in both T and B cells, and in both OKT4+- and OKT8+-depleted T cell subsets. The presence of measles virus RNA was detected in PBMC isolated from measles patients, and the production of the immunoreactive hemagglutinin protein was defective in these cells.

We compared healthy blood donors with and without HLA-Dw2 and -Dw3 in immunity assays, the results of which have been found to be abnormal in multiple sclerosis or autoimmune diseases. Tests included lymphocyte blast transformation responses to rubella, mumps and purified tuberculin (PPD), in vitro production of IgG and interferons, natural killer (NK) cell function and measurement of serum antibodies to measles, rubella, mumps and herpes simplex viruses. HLA-Dw2-positive subjects had a lower lymphocyte blast transformation response to rubella virus antigen and a lower NK cell function compared with HLADw2-negative subjects. The presence of HLA-Dw3 was associated with an increased spontaneous and mumps virus-induced immunoglobulin production. No significant differences were found in other assays. These results support the existence of HLA-Dw2- and Dw3associated deviation of immune responsiveness, which may contribute to the susceptibility of multiple sclerosis or other autoimmune type diseases.

Serum and CSF specimens of 30 MS patients and 30 age- and sex-matched control patients with other neurological diseases were tested for autoantibodies. Indirect immunofluorescense was used to detect smooth muscle antibodies and antinuclear antibodies in the serum. Antibodies against DNA and RNA in the serum and CSF were tested by solid phase radioimmunoassays. No significant differences in autoantibody levels were found between MS and control patients.

Purified rubella virus particles contain two glycopolypeptides (62K and 44K to 51K) and one nonglycosylated polypeptide (35K). Glycoproteins can be labeled with tritiated sodiumborohydride after oxidation with galactose oxidase indicating that galactose is the terminal carbohydrate unit. The other carbohydrate components are N-acetyl-D-glucosamine and mannose.

A nucleic acid spot hybridization assay was used to detect Chlamydia trachomatis DNA. The hybridization probes included DNA isolated from elementary bodies of lymphogranuloma venereum (LGV) strains and cloned fragments of both chromosomal and plasmid DNA. The sensitivity of the test was in the range 10 to 100 pg homologous DNA and 10 in vitro infected cells. Cross-reactivity with bacterial DNA was avoided when purified chlamydia-specific DNA fragments were used as probes. C. trachomatis was detectable in most of the clinical specimens with large amounts of infectious particles. Also some isolation-negative specimens gave a positive signal in the test.

The presence of adenovirus DNA in clinical specimens was analyzed by nucleic acid hybridization assays by both radioactive and enzymatic detection systems. The sensitivity of the hybridization tests was in the range of 10 to 100 pg of homologous adenovirus DNA. Minimal background was noticed with unrelated viral and nonviral DNA. Twenty-four nasopharyngeal mucus aspirate specimens, collected from children with acute respiratory infection, were assayed in the hybridization tests and also by an enzyme immunoassay for adenovirus hexon antigen which was used as a reference test. Sixteen specimens positive by the enzyme immunoassay also were positive in the two nucleic acid hybridization tests, and the remaining eight specimens were negative in all of the tests. The results indicate that nucleid acid hybridization tests with both radioactive and nonradioactive probes can be used for diagnosis of microbial infections.

Intercellular adhesion molecule (ICAM)-5 (telencephalin) is an adhesion molecule in telencephalic neurons of the mammalian brain that binds to the leukocyte integrin CD11a/CD18. The authors observed that human cerebral neurons also expressed ICAM-5 and that ICAM-5--mediated neuron--leukocyte binding in cultured hippocampal neurons. This led the authors to examine ICAM-5 expression during clinical CNS inflammation.The authors found, by immunoblotting, a 115-kDa soluble form of ICAM-5 (sICAM-5) cleaved from the membrane-bound (130 kDa) ICAM-5, and established an ELISA assay to measure it. CSF samples of patients with acute encephalitis and MS were studied.sICAM-5 was increased in encephalitis (320 plus minus 107 ng/mL; n = 25), as compared with patients with MS (128 plus minus 10 ng/mL; n = 16) and control subjects without CNS disease (137 plus minus 6 ng/mL; n = 42) (p0.001). The concentration of sICAM-5 correlated with the performance in the immediate recall task (p = 0.013) and with the leukocyte count in the CSF (p = 0.02), especially in cases caused by herpes simplex virus (HSV) (r = 0.94; p = 0.002).sICAM-5 is cleaved from CNS into CSF during acute encephalitis, and it may mediate leukocyte--neuron interactions. sICAM-5 release from cerebral neurons may actively regulate immune responses and leukocyte adhesion during microbial neuroinvasion in humans during encephalitis.