Detectionof reverse transcriptase activity in live attenuated virus vaccines.

BACKGROUND: Safety considerations require that biological products for human use are free from any agent that might pose a potential health hazard. One method to detect the presence of retroviral particles is the reverse transcriptase (RT) assay. This assay is capable of detecting all infectious retrovirus particles, irrespective of genome or protein composition. Recently, a family of ultrasensitive RT tests, named product-enhanced reverse transcriptase (PERT) assays, has been designed with a detection limit that is 10(6) - 10(7) times lower than that of conventional RT tests. OBJECTIVES: To investigate with the PERT assay whether RT activity is detectable in live attenuated virus vaccines and to characterize eventual RT activities. STUDY DESIGN: A total of 12 different monovalent and one trivalent virus vaccines containing live attenuated viruses were tested for RT activity with the PERT assay and a conventional RT test. RT activities were investigated with respect to their susceptibility to RT inhibitors, association with physical particles, and their possible origin. RESULTS: One trivalent and five different monovalent vaccines contained RT activity when tested with the PERT assay, but were negative in a conventional RT assay. All lots tested of these vaccines showed RT activity. The activity in all vaccines was sensitive to AZT-triphosphate and ddTTP and at least part of it was associated with particles. Mg(2+)-dependent RT activity banded at a density of 1.14 g/ml. All positive vaccines were produced using chicken cells. CONCLUSIONS: The data indicate the systematic presence of partially particle-associated retroviral reverse transcriptase in attenuated live virus vaccines that are produced in chicken-derived cells. The identification and further characterization of these particles, as well as the elucidation of possible interactions with the human organism are imperative goals despite the fact that these vaccines have been safely used for many years.

[Diagnosis of hepatitis B: is the additional demonstration of viral genome of a prognostic value?]. / Diagnostik der B-Hepatitis: Ist der zusätzliche Nachweis des Virusgenoms von prognostischer Bedeutung?

HBV markers were tested in sera of patients with various clinical and serological states of HBV infection, including patients with delta-infection, and were compared to the detection of HBV-DNA in the sera by hybridization. The results show that high levels of HBV-DNA (much greater than 100 pg/0.1 ml) are mainly found in sera of patients with chronic hepatitis or in early sera of HBs antigen carriers, while 17 of 20 patients with undetectable or trace amounts of DNA in early sera exhibited a self-limiting hepatitis infection. This test system could be prognostic value.

Isolation of human pathogenic viruses from clinical material on CaCo2 cells.

The use of the epidermal-like cell line CaCo2 and the efficient propagation of viruses from clinical material for diagnostic purposes are reported. Virus replication was observed by cytopathic effects and/or immunofluorescence. The following viruses replicate in CaCo2 cells: enteroviruses (coxsackie B1-B6, poliovirus types 1-3, most echoviruses and coxsackie A viruses), adenoviruses, herpes simplex types 1 and 2, measles, respiratory syncytial, parainfluenza type 2 viruses, and to a lesser extent rubella and mumps viruses. CaCo2 cells were used in neutralization tests for the diagnosis of enteroviral infections.

Cross-reactions of immunoglobulin M and G antibodies with enterovirus-specific viral structural proteins.

We analysed the reactivity of enterovirus-specific human IgM and IgG antibodies with the structural proteins of different enteroviruses by the immunoblot technique. In general, all immunoglobulin G antibodies of the tested sera reacted with capsid polypeptide VP 1 of the viruses tested (echoviruses 9 and 11, coxsackievirus B3 and poliovirus 2). In contrast, enterovirus specific immunoglobulin M antibodies of adults reacted with capsid polypeptides VP 1, VP 2, and/or VP 3 of the viruses mentioned above. The reactions with VP 2 and/or VP 3 were often stronger than with VP 1. IgM antibodies from sera of newborns infected by echovirus 11 reacted with VP 1 and VP 2/3 of echovirus 11 and also with VP 2 and VP 3 of poliovirus 2. Preabsorption experiments indicate that cross-reactive IgG antibodies react with epitopes of VP 1 not present on the surface of intact virus particles. The results from the immunoblot technique were compared to data from microneutralization tests and M-antibody capture radioimmunoassays.

[Possibilities and limits in the detection of nosocomial rotavirus infections]. / Möglichkeiten und Grenzen zum Nachweis nosokomialer Rotavirusinfektionen.

During an epidemiological survey on rotavirus gastroenteritis in the area of Berne (Switzerland) different virus types were analyzed according to their genome segment pattern. Nosocomial rotavirus infections among pediatric patients were carefully investigated. Possible limitations of such studies are discussed in details.

Reaction pattern of immunoglobulin M and G antibodies to echovirus 11 structural proteins.

The immunoglobulin M and G specific immune response of humans to echovirus 11 proteins during an echovirus 11 outbreak was studied by the immunoblot technique. Whereas immunoglobulin G antibodies were directed most exclusively to the VP 1 protein, the immunoglobulin M antibodies were directed against VP 1, VP 2, and VP 3.

Influence of breast milk on nosocomial rotavirus infections in infants.

To prevent nosocomial rotavirus infections in hospitalized children with various non-gastrointestinal diseases, 30 children (mean age five months) received 200 ml of fresh human milk per day in addition to the normal diet for their age. A matched group of children on formula diet served as a control. Fecal samples were routinely screened for rotavirus by a commercial ELISA test. In stools containing rotavirus, the virus RNA segments were analysed by gel electrophoresis to identify the different rotavirus strains. Clinical symptoms were recorded daily and quantified by a score system. Human milk had no effect on the frequency of nosocomial rotavirus infections: ten infected children were fed with human milk and seven were not. However, the severity of the clinical symptoms was clearly reduced: the mean score of clinical symptoms was only half as great and the number of mild or asymptomatic infections was doubled in the group receiving fresh human milk.

Defective viral RNAs in Aedes albopictus C6/36 cells persistently infected with Semliki Forest virus.

A persistent infection of Semliki Forest virus (SFV) has been established in Aedes albopictus C6/36 cells. Only a small number of cells survived the initial infection with this RNA virus and gave rise to a persistently infected culture which produced continuously small amounts of infectious virus. To investigate whether defective viral RNA was analyzed early and late after infection by blot hybridizations. Several defective viral RNAs were detected with a common sequence corresponding to the 3' end of the viral genome during and after the establishment of the persistent infection. These defective viral RNAs resemble the defective interfering RNAs in vertebrate cells generated during serial undiluted passages of standard SFV. The defective viral RNAs are rarely released from cells as virions. The rapid generation of defective viral RNAs may be important for the establishment of a persistent infection in mosquito cells.

[Sensitive virologic evaluation in suspected meningitis: study of a radioimmunotest for the detection of IgM antibodies against ECHO 9 and ECHO 11 viruses]. / Sinnvolle virologische Abklärungen bei Meningitisverdacht: Prüfung eines Radioimmunotests zum Nachweis von IgM-Antikörpern gegen ECHO-9- und ECHO-11-Viren.

The majority of viral meningitis cases is known to be due to ECHO virus infections on one hand, and mumps on the other. While the latter can be diagnosed by IgM antibody detection from one serum sample in the acute stage, diagnosis of enterovirus infections is by virus isolation and typing. An IgM-antibody test for ECHO 9 and 11 viruses is presented to evaluate the possibility of rapid serological diagnosis of ECHO virus meningitis cases. 36 cases from five local outbreaks due to ECHO 6, 9, 11, and 30 viruses were characterized by virus isolation and serum neutralization tests. All sera (88 samples) were assayed by a MACRIA (M-antibody capturing radioimmunoassay) to ECHO 9 and 11 viruses. While sera from all ECHO 9 and 11 cases, when taken at appropriate times, had IgM antibodies to the infecting type, a varying degree of cross-reactivity could be observed. Specificity problems are discussed in comparison with isolated cases of enteroviral infections due to different types, including Coxsackie B viruses.

Selective disappearance of two secreted host proteins in the course of Semliki Forest virus infection of Aedes albopictus cells.

One secreted host protein of molecular weight 54,000 (SP 54) disappeared (from 24 to 48 h after infection) in Semliki Forest virus-infected Aedes albopictus cell clone C6/36 grown in both Mitsuhashi-Maramorosch basal medium and tissue culture medium 199 and reappeared when cells went into the permanently infected state. C6/36 is a high virus producer showing a cytopathic effect. A second secreted host protein of molecular weight 62,000 (SP 62) was prominent if cell clone C6/36 was grown in tissue culture medium 199. After infection in this medium, the protein showed a behavior similar to that described for SP 54. These secreted proteins were not affected in two original Aedes albopictus cell lines. SP 54 and SP 62 are monomeric proteins and structurally not related.