Petri Auvinen

Pathogenetic differences between coxsackie A and B virus infections in newborn mice

Coxsackieviruses are divided into A and B subgroups on the basis of their pathogenicity in newborn mice. Although used in the classification of these viruses, our understanding of the details of the infection is incomplete due to the lack of sensitive and specific techniques to localize the viruses in affected tissue. We have used in situ hybridization to detect coxsackievirus genomes in tissues of newborn mice after infection by five serotypes (A2, A9, A21, B3 and B4) through different administration routes. Our results indicate that coxsackie A viruses are able to affect both skeletal and heart muscle while the coxsackievirus B subgroup infects a wide range of tissues. In addition to striated muscle these include central nervous system, liver, exocrine pancreas and brown fat. This model will make it possible to analyze molecular factors determining tissue tropism.

Genetic diversity of enterovirus subgroups

SummaryEnterovirus serotypes were studied using nucleic acid hybridization and nucleotide sequence analysis. A great majority of enteroviruses could be roughly divided into two larger subgroups the first consisting of poliovirus and certain coxsackievirus A serotypes. The second subgroup included coxsackie B viruses, most ECHO viruses, enterovirus 71 and representatives of coxsackie A viruses. Enterovirus 70 showed low homology to the viruses in both groups. Interestingly, ECHO virus 22 failed to react with any of the hybridization probes indicating a relatively distant relationship. The close relationship between coxsackie B and ECHO viruses as well as between polio and certain coxsackie A viruses was also evident when nucleotide sequences of the 3′ end noncoding parts were compared.

Mapping of antigenic sites of coxsackievirus B3 by synthetic peptides

Peptides presenting predicted antigenic sites of CBV3 capsid proteins and peptide sequences from conserved regions of the nonstructural proteins were synthesized, and rabbit antipeptide sera were tested for their immunoreactivity. Peptides derived from different capsid regions were able to induce production of neutralizing antibodies in rabbits. As measured by EIA, all peptides representing four different proposed antigenic sites were immunogenic, inducing an antibody response against the homologous peptide and purified CBV3 as measured by EIA. Immunization with inactivated CBV3 induced a secondary response especially in rabbits primed with peptides representing polypeptide VP2. Antisera against the nonstructural protein sequences were highly cross‐reactive with other enteroviruses, while the capsid peptide antisera were mainly type‐specific when tested by immuno‐blotting against a panel of enteroviruses. Four of the capsid region peptides also exhibited distinct T‐cell reactivity in a mouse T‐cell proliferation assay.

Typing of herpes simplex virus isolates with monoclonal antibodies and by nucleic acid spot hybridization

Fifty-one clinical isolates of herpes simplex virus (HSV) were typed by an enzyme immunoassay (EIA) using mouse monoclonal antibodies, by DNA spot hybridization, and by restriction enzyme analysis using restriction endonuclease Eco RI. Extracts of VERO cells infected with the isolates were used for coating microtitre plates or denatured and spotted onto nitrocellulose filters. Viral antigens passively adsorbed to microtitre plates were detected by an indirect EIA using mouse monoclonal antibodies specific for HSV type 1 (HSV-1) or HSV type 2 (HSV-2). Spotted DNA was hybridized with 32P-labeled probes containing Hind III/Sal I-fragments of either HSV-1 or HSV-2 DNA and bound radioactivity was detected by autoradiography and counted in a liquid scintillation counter. All the three methods gave identical results for the 51 isolates studied. Twenty-six isolates were identified as HSV-1 and 25 as HSV-2. An additional 30 specimens were tested only by EIA and hybridization. Results by both techniques were in complete agreement.

Use of enzyme immunoassay and nucleic acid hybridization for detecting Sindbis virus in infected mosquitoes

Aedes aegypti mosquitoes were inoculated intrathoracically with prototype Sindbis virus, held at 26.7 degrees C for from 0-95 h and placed at -70 degrees C. Individual mosquitoes were tested for virus by plaque assay in Vero cells, for viral RNA by nucleic acid hybridization using a cloned cDNA probe, and for viral protein by enzyme-linked immunosorbent assay. Virus was detected by plaque assay as early as 8 h after infection. Sindbis virus RNA was detected by nucleic acid hybridization 18 h after infection and by enzyme-linked immunosorbent assay 10 h after infection. The results of these comparisons suggest that both nucleic acid hybridization and enzyme-linked immunosorbent assay are applicable to direct detection of Sindbis virus in mosquitoes containing virus at levels usually found during arbovirus epidemics.

Detection and Typing of Enteroviruses by Nucleic Acid Probes and Monoclonal Antibodies

Enteroviruses are currently detected using virus isolation followed by a neutralization test with a panel of antisera for specific typing of the virus strain. One of the problems in this method is the long time period needed for final diagnosis. In addition some coxsackievirus (CV) type A (CVA) strains can only be isolated in newborn mice. Gene technology and hybridoma methods offer new possibilities for development of reagents which can be used in enterovirus detection. The increasing knowledge on the genetic and antigenic structure, and mutual relatedness of enteroviruses further facilitates the applicability of these techniques.

Genomic variation of herpes simplex virus type 2 isolates analysed by hybridization after electroblotting from polyacrylamide gels

Genomic variation of herpes simplex virus type 2 (HSV-2) strains was analysed by polyacrylamide gradient gel electrophoresis and subsequent hybridization to cloned HSV-2 sequences. Two probes were used, one from the L-segment and one from the S-segment of the HSV-2 genome. The probes did not contain a-repeat sequences. Hybridization to the specific sequences in individual DNA fragments obtained by use of the frequently cleaving restriction endonuclease Alu I revealed variations in the genome not detectable by analysing the fragment size only. The use of 35S-labelled deoxynucleotide in the radioactive labelling of the probe further improved the resolution of the method.