Tracing of the molecular remnants of herpes virus infections in necrotic skin tissue

ARTICLE

Auteur(s) : Takenobu Yamamoto, Akiko Yamada, Kazuhide Tsuji, Keiji Iwatsuki

Department of Dermatology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan

accepté le 2 Avril 2008

Herpes virus infection is generally diagnosed with clinical findings and the Tzanck test. However, it is sometimes difficult to discriminate herpes simplex virus (HSV) infection and varicella zoster virus (VZV) infection clinically. Although we can easily detect herpes virus in the Tzanck test, the sensitivity is low. Various microbial DNA can be detected in crusts and scales by PCR amplification [1, 2], but the positive PCR result only indicates the presence of their DNA fragments in the samples, regardless of whether the virus is pathogenic. Furthermore, it is sometimes difficult to exclude the contamination of such viruses. Especially in cases of latently infected viruses, including Epstein-Barr virus (EBV), human herpes virus (HHV)-6, and HHV-7, the detection of viral DNA does not always provide a clue to the pathogenic significance because most people carry the viruses [3-5]. The essential finding required for accurate diagnosis is to detect the involvement of such viruses in the infected sites. For this purpose, detection of herpes virus-derived transcripts, including mRNA and other virus-related RNAs, provide reliable evidence.

We have recently succeeded in detecting EBV-related transcripts in crusts from all patients with hydroa vacciniforme (HV) and EBV-associated NK/T-cell lymphoproliferative disorders [6]. To our surprise, cellular mRNAs as well as EBV-derived RNAs were preserved well enough to be detected by reverse-transcriptase (RT)-PCR in the crust, or in dry necrotic tissue. Based on these findings, we have expanded our study to establish a non-invasive, diagnostic procedure for common herpes virus infections such as herpes simplex (HS), herpes zoster (HZ), and varicella.

Materials and methods

RNA was extracted from the samples with TRIZOL reagent (GIBCO BRL, Gaithersburg, MD), and converted to cDNA for EBV-encoded small nuclear RNA. 1 (EBER1) was generated with an antisense sequence, (5′-AAAACATGCGGACCACCAGC-3′) and a random hexamer (Takara, Kyoto, Japan), in the presence of M-MLV reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA). The cDNA samples were amplified by PCR using specific primer sets for HSV-encoded UL30, VZV-encoded ORF40, and EBV-encoded EBER1 (table 1). To detect a housekeeping gene product, the beta-2-microglobulin (β2-MG) and beta-actin (β-actin) cDNA was amplified by PCR (table 1). The PCR products were subjected to gel electrophoresis using a 2% agarose gel, and positive signals were detected by ethidium bromide staining. Direct sequencing of the PCR-amplified products was carried out to confirm the specific amplification of HSV-encoded UL30 and VZV-encoded ORF40 cDNA.

In order to find an efficient preservative condition, the amounts of β2-MG mRNA determined by quantitative RT-PCR in the same crust samples stored either in a sample left at RT without any treatment, adhesive tape, or saline condition at RT for 5 days, and were compared with those of the freshly obtained samples by real-time RT-PCR with the LightCycler system (Roche Diagnostics, Mannheim, Germany). Three specimens were analyzed and three times were calculated in each sample.
Table 1 RT-PCR primer sequences

Results

HSV-specific, lytic cycle-related transcript, UL30 mRNA was detected in all 15 HS samples (table 2). The sensitivity and specificity of the present assay system were 100% and 88.4%, respectively, and the likelihood ratio was 8.6. Thirteen of 15 HSV samples demonstrated 129 bp of UL30 mRNA encoded by HSV-1, and 2 samples represented 163 bp of UL30 mRNA encoded by HSV-2 (figure 2). One HS case presented not only HSV-1 derived UL30 mRNA, but also slight amounts of VZV-derived mRNA, ORF40.

Of the 23 samples obtained from HZ and varicella, VZV-specific, lytic cycle-related transcript, ORF40 mRNA was detected in 22 samples (table 2, figure 2). The sensitivity and specificity of the present assay system were 95.7% and 97.1%, respectively, and the likelihood ratio was 33.0. Four HZ cases presented not only VZV but HSV-1 or -2. The correct DNA sequence for UL30 and ORF40 was confirmed in the PCR products by a direct sequencing method.

In a control group, UL30 and ORF40 mRNA were not detected in any of 20 samples. EBV-specific, latent cycle-related transcript, EBER-1 was found in all 6 HV samples, and weakly in one case of impetigo.

As compared with the freshly obtained materials, the amount of β2-MG mRNA was reduced to 51% in adhesive tape or 48% in a sample left at RT without any treatment for 5 days, and to 1.2% in the same samples stored in saline (figure 3).
Table 2 Summary of the present assay system in HS, HZ, varicella, and HV

Discussion

Our study has demonstrated that cellular and herpes virus-derived RNAs are preserved in dry crusts, and that such RNAs can be detected by RT-PCR amplification with high sensitivities: 100% for HSV and 95.7% for VZV. The specificities of our assay system using a pair of specific primer sets represented 88.4% for HSV and 97.1% for VZV. In a case of HZ-11 (figure 2), we could detect HSV-2 mRNA without any VZV-related transcripts. Because this patient had a lot of vesicles on the abdomen and no history of herpes simplex, we were given a diagnosis of herpes zoster clinically. But we made a final diagnosis of HSV-2 infection based on molecular evidence. Furthermore, typing of HSV was simultaneously possible by detecting the HSV type-specific transcript, UL30 mRNAs.

The HSV UL30 protein is a late protein and is essential to virus replication. The UL30 protein exhibits polymerase activity and has intrinsic 3’-5’-exonuclease activity [7]. The VZV ORF40 encodes a major capsid protein (gp42) and is a late protein in lytic infection [8]. Therefore, the presence of HSV UL30 and VZV ORF40 mRNA proves not only the infection of HSV and VZV, respectively, but also the presence of lytic cycle infection in the lesions.

One (6.7%) of the 15 crust samples obtained from HS (case HS-1: table 2) exhibited both HSV and VZV mRNA, and 4 (19.0%) of 21 samples from HZ yielded both VZV and HSV mRNA. These observations suggest the possibility that the reactivation of HSV may be induced simultaneously when VZV is led to the lytic cycle, and vice versa [9]. In addition to the detection of both HSV and VZV mRNA, IgM and IgG antibody titers against both HSV and VZV were increased in the case of HZ-21 (figure 1, table 2). These findings suggest the concomitant reactivation of both HSV and VZV, as previously reported in immuno-suppressed patients [10-12]; on the other hand, it is rarely reported in immnuno-competent individuals [9, 13]. The concept of co-infection is supported by the fact that VZV and HSV can co-localize to the same sensory ganglion [11]. Alternatively, we must consider the possibility of contamination of the virus transcripts or cDNA. However, appropriate negative controls revealed negative results in the RT-PCR assay in all investigated cases.

Our assay system is applicable to crust samples that have been stored under dry conditions at R.T. for many days without any pretreatment. We have furthermore confirmed mRNA existence with 2 samples saved for 6 months, as well as a sample stored for 3 months. By contrast, when the samples are stocked in saline, the amounts of detectable mRNA are markedly reduced, probably because of destruction by RNase eluted from the necrotic tissue. In a patient with HZ (case HZ-15: table 2), we could detect VZV ORF40 mRNA both in the crusty lesions obtained at the first visit (HZ-15-a: table 2) and in the scale obtained 1 month after antiviral treatment (HZ-15-b: table 2). This observation indicates that the lytic cycle transcripts of VZV might be preserved in the lesion in an intact fashion, even after antiviral treatment. As such, it appears that both pathogen-derived and cellular RNAs are well-preserved in dry, necrotic tissue, and that crusts, or dry epidermal necrosis with inflammatory cells, may provide beneficial information by means of molecular methods.

Acknowledgments

References

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