Description of a Nonlethal Herpes Simplex Virus Type 1 Glycoprotein D Deletion Mutant Affecting a Site Frequently Used for PCR

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) infections are among the most commonly clinically encountered infections in medical practice and, worldwide, are major causes of mucocutaneous ulceration (2, 6, 8, 11); in Northern Ireland, HSV-1 is the most commonly recognized cause of genital ulceration in women (4). The virus attaches to cells and gains entry through the action of a number of surface glycoproteins. Aurelius et al. (1) described a nested primer set targeting the nucleic acid sequence of HSV-1 glycoprotein D (gpD), which has been widely used and cited in over 220 publications. We have used this set in a multiplex nested PCR for HSV-1 and HSV-2 (5) and have identified an HSV-1 mutant which yielded the expected first-round product but repeatedly failed second-round amplification; the outer and inner primer pairs were labeled 5A-5B and 5C-5D, respectively. The mutant virus was demonstrated in a specimen (16253/99) of dermal scrapings, submitted on a glass slide, from an 84-yearold woman who presented with a discrete vesicular rash on the left cheek which healed without specific antiviral therapy. The presentation of the vesicular lesions was preceded by a prodromal sensation and was clinically diagnosed as an HSV eruption. The diagnosis was made at a dermatology clinic which the patient attended routinely for the skin condition bullous pemphigoid, which was controlled by the application of dermovate ointment as required. To investigate the failure of second-round amplification, we sequenced through the internal primer sites of the mutant’s first-round product, plus those of a second HSV-1 isolate (20209/99) and the HSV-1 laboratory control. The first- and second-round PCRs were undertaken as previously described (5). Briefly, the master mixes were made in nuclease-free water (NFW) with Promega Taq DNA polymerase (in storage buffer B), magnesium-free reaction buffer (103) supplied with the Taq enzyme, MgCl2 (Promega), and primers. The amounts of final working concentrations of the components of each 25-ml test mixture were 5 pmol for each primer, 10 mM Tris-HCl (pH 9.0), 3.5 mM MgCl2, 50 mM KCl, 0.2 mM for each deoxynucleoside triphosphate, 0.1% Triton X-100, and 0.625 U of Taq enzyme. Hot-start PCR was carried out in a Perkin-Elmer GeneAmp 2400 thermal cycler with the cycling conditions shown in Table 1. First- and second-round products were visualized together on an ethidium bromidestained 4% MetaPhor high-resolution agarose gel (FMC