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.
Ivanovsky Institute of Virology, Moscow, Russia.
S
S
Viazov
A
A
Zibert
K
K
Ramakrishnan
A
A
Widell
A
A
Cavicchini
E
E
Schreier
M
M
Roggendorf
D000911:Antibodies, Monoclonal / Q000276:immunology; D001360:Avidin; D001483:Base Sequence; D001710:Biotin; D004247:DNA / Q000276:immunology; D018076:DNA, Complementary / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005858:Germany / Q000453:epidemiology; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006526:Hepatitis C / Q000453:epidemiology / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015995:Prevalence; D012367:RNA, Viral / Q000302:isolation & purification*; D012426:Russia / Q000453:epidemiology; D016415:Sequence Alignment; D014421:Turkey / Q000453:epidemiology
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D012367:RNA, Viral; D001360:Avidin; D001710:Biotin; D004247:DNA