High Incidence of Genotypic Variance between Sequential Herpes Simplex Virus Type 2 Isolates from HIV-1–Seropositive Patients with Recurrent Genital Herpes

Genital herpes caused by herpes simplex virus type 2 (HSV-2) remains a common cause of genital ulceration worldwide. One of the hallmarks of the disease is the appearance of recurrent genital herpes (RGH) lesions, which cause high morbidity and are postulated to increase the efficiency of HIV-1 transmission [1]. Generally, recurrences result from reactivation of latent virus and subsequent anterograde axonal transport to the site of primary infection [2]. Alternatively, patients may have been reinfected with an exogenous HSV-2 strain at the same anatomical site, referred to as “HSV-2 superinfection.” However, at present, the incidence and the risk factors of HSV-2 superinfection are incompletely understood, and further investigation is necessary. In the past, intratypic differences between HSV-2 strains were demonstrated by morphological analysis of plaque, serological analysis, and restriction fragment–length polymorphism (RFLP) analysis [3–7]. The objective of the present study was 2-fold. First, we sought to develop and validate a polymerase chain reaction (PCR) approach, based on our previous work on HSV-1 genotyping relying on strain-to-strain variation in tandemly repeated DNA sequences (called “reiterated sequences” [Re]) in the viral genome [8, 9], to enable a rapid and accurate distinction between unrelated HSV-2 strains. Second, we sought to determine the incidence of HSV-2 genotype variation, which may indicate an HSV-2 superinfection, between sequential genital HSV-2 isolates obtained from HIV-1–seronegative and HIV-1–seropositive patients with RGH

Patients, materials, and methodsGenital HSV-2 isolates were obtained from 51 patients with symptomatic genital herpes visiting the sexually transmitted disease unit of the Department of Dermatology and Venereology at the Erasmus Medical Center. Additionally, serial swabs (2/patient) from genital lesions, from the same anatomical site, were obtained from 11 HIV-1–seropositive patients (6 women and 5 men; mean age, 33 years) at a mean time interval of 14 months (range, 1–58 months) and 8 HIV-1–seronegative patients (4 women and 4 men; mean age, 32 years) at a mean time interval of 17 months (range, 1–49 months). The genital swabs were inoculated onto Vero cell monolayers. The virus was harvested when ∼75% of the monolayer showed viral cytopathic effect and was subsequently typed for HSV-1 or HIV-2 by immunocytological analysis and PCR, as described elsewhere [10]. The 11 HIV-1–seropositive patients were classified according to Centers for Disease Control and Prevention classifications (available at: http://wonder.cdc.gov/wonder/help/AIDS/MMWR-12-18-1992.html) (table 1). Twelve subclones were generated from the HSV-2 reference strain MS (ATCC VR-540), obtained after subcloning twice by limiting dilution as described elsewhere [9]. All patients provided written, informed consent, and the ethics committee of the Erasmus Medical Center approved the protocol

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Table 1

Genetic characterization of sequential herpes simplex virus type 2 isolates (collected at time points a and b) from patients with recurrent genital herpes (RGH)

Guanidine isothiocyanate–Celite binding (Janssen Chemika) was used to extract DNA from 100 μL of culture medium of Vero cells exhibiting overt cytopathic effect on inoculation with the clinical HSV-2 isolates [9, 10]. Extracted DNA was resuspended in 100 μL of water, and 5 μL of DNA suspension was used for each PCR. Primers were designed to amplify Re IV–containing regions in the introns of the HSV-2 genes US1 and US12 [8]. The sequences of the forward and reverse primers for US1 were 5′-ACTGCAGCCTTATCGCAGGTACGG-3′ (HSV-2 genome sequence nucleotide positions 133199–133222; GenBank reference no. Z86099) and 5′-GCCTTGTCGCCAGTTACCACACCG-3′ (nucleotide positions 133706–133729), respectively. In the case of US12 the forward and reverse primers were 5′-CCAAGACATGGTGTCCCGTCCACG-3′ (nucleotide positions 148027–148050) and 5′-ACTGCAGCCTTATCGCAGGTACGG-3′ (nucleotide positions 148520–148543), respectively. The PCRs were performed in 50-μL volumes containing 1.25 U of cloned Pfu DNA polymerase and were optimized using the Opti-Prime PCR Optimization kit (both from Stratagene). The defined optimum PCR contained 100 mmol/L Tris-HCl (pH 8.3), 15 mmol/L MgCl₂, and 250 mmol/L KCl, supplemented with 5% (vol/vol) dimethyl sulfoxide (DMSO); primers at a concentration of 1 μmol/L; and each deoxynucleoside triphosphate, including an equimolar amount of dGTP and 7-deaza-2′-dGTP (Boehringer Mannheim) at a concentration of 200 μmol/L. PCR amplification was, except for annealing at 60°C for both primer sets, performed as described elsewhere [9, 10]

Amplicons were size fractionated on 2% agarose gels and were visualized by ethidium bromide staining. The length of the PCR products (i.e., amplicons) were estimated by comparison with 50- and 100-bp DNA molecular weight markers (Life Technologies). The specificities of the amplicons were confirmed by Southern blotting using an HSV-2 Re IV–specific oligonucleotide (5′-GTCCCCCCGTCCCCCCGTCCCCCC-3′), as described elsewhere [9, 10]

ResultsWe previously developed a PCR approach to discriminate HSV-1 strains. The method is based on strain-to-strain variation in the number of reiterated sequences located in the HSV-1 genes US10 and US11 (Re VII) and US1 and US12 (Re IV and Re VIII) [8–10]. Given the presence of an analogous Re IV in the noncoding regions of the HSV-2 genes US1 and US12 [8], the option to use these regions for a similar PCR-mediated HSV-2 genotyping approach was inventoried. First, PCR primers and conditions were optimized to amplify and determine the variability of the Re IV–containing regions on unrelated genital HSV-2 isolates obtained from 51 patients with RGH. Combining the results of the 2 amplified regions showed that 47 (92%) of the 51 isolates had a unique combination of amplicons (table 2). Results were reproducible in subsequent experiments, and sequencing of representative amplicons demonstrated that the amplicon-length variation observed was indeed the result of differences in Re IV numbers (data not shown). Occasionally, no PCR product could be visualized by ethidium bromide staining, or multiple amplicons appeared. Subsequent Southern blot analyses with the HSV-2 Re IV–specific probe did, however, reveal 1 specific amplicon in these samples (data not shown). Second, because DNA repeats may have the tendency to change profiles because of multiple replication rounds in time, the stability of the target sequence was determined. The amplicons from all 12 subclones tested were identical in size to those of the parental strain, indicating the stability of Re IV pending 2 cycles of limiting dilution (data not shown)

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Table 2

Estimated length of reiterated sequence IV–specific amplicons of genital herpes simplex virus type 2 (HSV-2) isolates obtained from unrelated patients with genital herpes

Sequential genital HSV-2 isolates from patients with RGH were genotyped. The results of the PCR analyses are summarized in table 1. The sequential genital HSV-2 isolates from all 11 HIV-1–seropositive, but from only 1 of the 8 HIV-1–seronegative patients with RGH, showed different genotypes (P = .03; Fisher’s exact test)

DiscussionThe results of an investigation into the value of a PCR method for rapidly and accurately distinquishing between HSV-2 strains are reported here. The method commonly used to differentiate HSV-2 strains is RFLP analysis, a time-consuming and highly labor-intensive method requiring infectious virus that is not always easy to obtain from clinical samples [5–7]. Thus, we developed a PCR-based HSV-2 genotyping assay that overcame these shortcomings. The method is based on strain-to-strain differences in the number of DNA repeats located in the introns of the HSV-2 genes US1 and US12 [8]. The principle of using the variation in length of reiterated sequences to study transmission routes and molecular epidemiological aspects has been proven useful for HSV-1 [9–12]. As for HSV-1, the high G:C ratio of the target sequences necessitated the inclusion of various additives such as DMSO and 7-deaza-2′-dGTP, to limit formation of secondary structures in the PCR buffer for consistent and reproducible amplification of the Re IV–containing regions of HSV-2 US1 and US12 [9, 13]. The hypervariability of these regions, crucial for the general applicability of the PCR method developed, was demonstrated by genotyping 51 unrelated HSV-2 isolates. Comparison of the alleles from both regions showed unique combinations for 47 (92%) of the 51 HSV-2 isolates, which is similar to the results of the HSV-1 PCR genotyping method described elsewhere [9, 10]. Subsequently, we demonstrated that the target sequences were stable during in vitro culture, similar to the analogous regions in the HSV-1 genome [9]

Previous studies have reported that prior infection with HSV-2 is an important risk factor for acquiring HIV-1 infection and that HSV-2 shedding occurs relatively more frequently in individuals coinfected with HSV-2 and HIV-1 [14, 15]. It is generally assumed that shedding is caused by reactivation of the latent HSV strain acquired previously [7]. A recent study reported on HSV shedding in immunocompetent patients treated with acyclovir [16]. The authors suggested that the observed day-to-day and side-to-side variability in HSV-2 sensitivity to acyclovir might represent local variants that possibly emerged at mucosal sites rather than within the innervating ganglia [16]. In the present study, 11 HIV-1–seropositive and 8 HIV-1–seronegative patients were investigated to determine the incidence of genetic variability of HSV-2 in patients with RGH. In previous RFLP studies of sequential HSV-2 isolates from patients with RGH (who were not tested for HIV-1), the incidence of HSV-2 variability ranged from 3% to 25% [5, 6]. The genotypic variation in 13% of the HIV-1–seronegative patients with RGH described here was within the same range, as were the results of our previous study of HSV-1 RGH [10]. Noticeably, genetic variation between the sequential HSV-2 isolates obtained from the same anatomic location was observed in all 11 HIV-1–seropositive patients with RGH (table 1). The low rate of genetic recombination in DNA viruses, exemplified by the stability of the Re IV–containing target sequences and the proofreading activity of the Pfu DNA polymerase used, implies that the intraindividual HSV-2 genotype variations are most likely not attributable to genetic alteration of the initial strain or to errors during PCR amplification, respectively. The genotypic differences between the sequential HSV-2 isolates, obtained from the same anatomic site of the patients with RGH, may be due to reactivation of different strains that have established latency in the innervating sensory ganglia. We are currently testing this hypothesis by genotyping HSV strains in human sensory ganglia latently infected with HSV. Alternatively, HSV-2 superinfection may have occurred during the sampling interval. Compared with the HIV-1–seronegative patients with RGH, the HIV-1–seropositive patients in our study reported persistent high-risk sexual behavior (table 1). This, together with the high level of immune suppression because of underlying AIDS, probably reflects the ability of an exogenous virus to superinfect this affected mucosal tissue

The clinical and therapeutic implications as well as the epidemiological significance of transient mucosal shedding of HSV-2 strains or variants certainly warrants further research. This may involve a comparative investigation into HIV-1–seropositive and HIV-1–seronegative patients with RGH who are persistently engaged in high-risk sexual behavior

• Received May 5, 2006.
• Accepted June 6, 2006.

• © 2006 by the Infectious Diseases Society of America