Typing and strain differentiation of clinical herpes simplex virus type 1 and 2 isolates by polymerase chain reaction and subsequent restriction fragment length polymorphism analysis.

In the present study, different combinations of primers were investigated for PCR amplification and typing of clinical herpes simplex virus type 1 (HSV-1) (n = 22) and herpes simplex virus type 2 (HSV-2) (n = 12) isolates. Intratypic strain differentiation was performed by restriction fragment length polymorphism (RFLP) analysis of different PCR amplified HSV genome regions. Enzymatic amplification of HSV DNA from all the clinical isolates could be achieved using primer combinations DNAP5/DNAp3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2). With the primer pair corresponding to the thymidine kinase (TK) genome region, amplification of all the HSV-1 isolates was only possible by nested PCR. With primers HSV UP/DOWN, 10 of 12 HSV-2 isolates could be detected. HSV typing by type-specific primers DNAP5/DNAP3-1 (HSV-1), DNAP5/DNAP3-2 (HSV-2) or restriction enzyme analysis (Ava II) of amplified DNA with HSV-specific UP/DOWN primers showed results which concorded with serotyping by monoclonal antibodies. RFLP analysis of the PCR products using selected restriction enzymes showed sufficient diversity of profiles among strains to differentiate all the HSV-1 isolates and to distinguish four groups of HSV-2 isolates. Amplification of HSV DNA from clinical isolates with subsequent typing and strain differentiation represents a valuable alternative to conventional methods (virus isolation, serotyping and restriction fragment analysis of the entire genomic DNA) and may be suitable for the study of HSV transmission and pathogenesis, especially in labour-intensive clinical samples with low levels of virus replication (i.e. cerebrospinal fluid, vitreous fluid and corneal transplants).