Typing of hepatitis C virus isolates by DNA enzyme immunoassay.

Recently, at least six types of hepatitis C viruses (HCV) have been identified. Different types of HCV appear to possess different pathogenic properties and a different sensitivity to interferon treatment. Typing of HCV isolates may therefore be an important diagnostic procedure. We report on a new method for identification of HCV types 1a, 1b, 2a, 2b and 3a which are most prevalent in Europe, North America and Japan. The assay is based on a combination of two well established techniques, the polymerase chain reaction (PCR) and DNA enzyme immunoassay (DEIA). In the first step of the method a cDNA of about 250 bp corresponding to the HCV core-region is amplified by nested PCR. The target cDNA is then hybridized to type-specific oligonucleotides fixed to a solid phase through an avidin-biotin bridge. The formed hybrids are detected by a standard ELISA using monoclonal antibodies reacting with double-stranded DNA. Typically, signal-to-noise (S/N) ratios between 18.2 and 48.6 could be observed when different HCV types/subtypes were analyzed by this method. The test was evaluated using cloned HCV cDNAs of known types and by sequence determination of some of the typed cDNAs. Typing of 115 isolates from Germany, Russia and Turkey revealed that subtype 1b (59-100%) and 1a (24-32%) are most prevalent in these countries.

Influence of twenty potentially antiviral substances on in vitro multiplication of hepatitis A virus.

A multiwell tissue culture system was developed to study the influence of various substances on hepatitis A virus (HAV) propagation. A panel of 20 substances of different structure types, each with known effect against at least some viruses, was studied at a concentration of 100 microM. Three substances showed reproducible inhibition. The strongest inhibitor, arabinosylcytosine, also produced cytotoxic changes in cells down to a concentration of 1 microM, and its effect was considered as nonspecific. Amantadine and ribavirin showed a moderate effect at 100 microM. A stronger inhibition was seen at 250 and 500 microM, doses that are toxic and impractical for clinical use. Although no promising candidates for antiviral treatment of hepatitis A have emerged from the present study, the assay model described here would seem useful in the screening of substances with inhibitory effects on HAV.

Sensitivity of HSV strains isolated before and after treatment with acyclovir.

Sequential isolates of herpes simplex virus (HSV) from 5 patients, treated for six episodes with orally administered acyclovir, were tested for in vitro sensitivity to the drug. In addition, isolates from an acyclovir treated immunocompromised patient with oral and genital herpes infections were examined for sensitivity to acyclovir. All isolates were tested in a production (24 h-yield) inhibition assay and defined as sensitive or resistant by the parallel examination of thymidine kinase (TK) positive and negative reference strains of HSV. No evidence for decreased or altered sensitivity to acyclovir was recorded of the genital HSV strains, isolated during seven episodes of recurrences. However, in agreement with the clinical response to therapy, genital isolates obtained from the immunocompromised patient pre- and post-treatment with acyclovir displayed in vitro sensitivity values indicating a rapid and complete development of resistance.