Detection and sequence diversity of Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) DNA

ARTICLE

Kaposi's sarcoma (KS) was originally described as a rare vascular neoplasm of elderly persons in certain areas of the Mediterranean or eastern Europe. However, in the 1980s, the incidence of KS increased dramatically in patients with acquired immunodeficiency syndrome (AIDS), and therefore, the etiology of KS in human immunodeficiency virus (HIV)-positive patients was speculated to be distinct from other forms of KS [1]. Because of its epidemiological features, HIV-associated KS is considered to be a sexually transmitted disease, and it has been suggested that viral infection may play a role in its development [2].

In 1994, a partial DNA sequence of a new human herpesvirus termed KS-associated herpesvirus (KSHV) was isolated from HIV-positive patients with KS [3]. Subsequently, KSHV was detected in various forms of KS [4-8], and it was also designated human herpesvirus type 8 (HHV-8) [9]. The KSHV DNA sequence in KS has been detected not only in HIV-positive, but also in HIV-negative patients [10-13], and more recently in some patients with diseases other than KS, such as lymphoproliferative disorders [14-18], vascular neoplasms [19-21], skin tumors [22], immunosuppression after organ transplantation [23, 24], and even autoimmune diseases [25]. In order to clarify the relationship between KSHV detection and HIV infection or disease specificity, we examined the presence of amplification of the KSHV DNA sequence in patients with several diseases, with or without HIV infection, and carried out sequence analysis of the detected DNA fragments.

Materials and methods

Patients

The patients comprised 5 who were HIV-positive with KS, 2 who were HIV-positive with syphilis, one who was HIV-positive with prurigo, and 2 who were HIV-negative with angiosarcoma (Table I). All had been definitively diagnosed on the basis of clinical appearance, and pathological and laboratory findings. Biopsy specimens from skin lesions, peripheral blood and regional lymph nodes were examined. The patients were all Japanese except for one African patient with KS who was HIV-positive (case 1).

DNA extraction

DNA isolated from the skin and lymph nodes was extracted from formalin-fixed, paraffin-embedded tissue. Ten 5-µm-thick sections of paraffin-embedded biopsy specimens were deparaffinized in xylene, and washed with 100% ethanol. After drying, the sample was incubated with Proteinase K and lysis buffer (10 mM Tris-Hcl, 150 mM NaCl, 10 mM EDTA, 2% SDS, pH 8.0) and extracted with phenol and chloroform. DNA was precipitated with ethanol and resuspended in 50 µl TE buffer (10 mM Tris-Hcl, 1 mM EDTA, pH 8.0). One mililiter of peripheral blood was also treated for DNA extraction in lysate with the same final concentration as that described above, and 20 µl of DNA solution was prepared for polymerase chain reaction (PCR) analysis.

PCR and DNA sequencing

A set of KS330₂₃₃ primers (5'-AGCCGAAAGGATTCCACCAT-3', 5'-TCCGTGTTGTCTACGTCCAG-3') [3] was used, and 1 µl of each prepared DNA solution was applied for amplification of the 233-bp KSHV DNA fragment. The PCR conditions consisted of denaturation for 1 min at 94° C, annealing for 1.5 min at 58° C, and extension 1 min at 72° C. Amplifications were carried out in a DNA thermal cycler (Perkin-Elmer Cetus, CT, USA) for 40 cycles, and PCR products were visualized using electrophoresis on 3.5% Nusieve agarose gel. Primers corresponding to the beta-globin gene were used in control reactions to verify the quality of the extracted DNA. Amplified DNA fragments were then subjected to sequence analysis using the direct sequencing method.

Results

DNA extracted from 14 skin, 2 peripheral blood, one lymph node and one oral mucosa sample was subjected to PCR amplification of KSHV sequences. Specific amplification of the 233-bp DNA fragment was observed with 8 samples obtained from 4 KS patients (Table I), while all samples were positive for beta-globin gene amplification. KSHV DNA sequences were detected only in HIV-positive KS patients. An eczematous skin lesion diagnosed as toxicodermia in a KS patient whose KS skin lesion was positive for KSHV, did not generate DNA amplification. DNA amplification was not observed in other samples with HIV infection or HIV negative angiosarcoma.

Discussion

Because KSHV sequences have been frequently detected in various forms of KS and angiosarcoma, a relationship between KSHV and HIV, and the tumorigenicity of KSHV on endothelial cells has been discussed [26, 27]. In this study, KSHV was detected in 4 out of 5 HIV-positive patients with KS, which is in agreement with many previous studies showing a high rate of KSHV detection in HIV-positive patients with KS. However, our results did not show a strict correlation with either HIV positivity or HIV-negative angiosarcoma. Positive KSHV detection in the peripheral blood in case 1 and in lymph nodes in case 4 suggested a systemic distribution of KSHV in KS patients, whereas a skin biopsy specimen in case 2 from a site unaffected by KS did not show KSHV amplification. Similar discrepant KSHV expression as a function of sampling sites has been occasionally reported [8, 13, 28], suggesting the dominant presence of KSHV in the development of skin lesions in KS. The nucleotide sequences of the amplified DNA fragments were determined in this study, and alignment of the 4 sequences with the prototypic sequence of KS330₂₃₃ revealed 5 point mutations (Fig. 1). According to 20 reports [4-7, 11, 12, 14, 19, 21, 22, 25, 28-36] presenting 81 sequences of KS330₂₃₃ fragments, 16 site changes were evident upon comparison with the original sequence (Fig. 1). Moreover, 6 mutation sites among 16 were frequently detected (Table II), and 5 point mutations revealed in this study were also included in frequent mutations. An identical sequence with 5 substitutions was observed in cases 2 and 4. One base change in case 3, and four base changes in case 1 were also observed in cases 2 and 4. Five of the substituted positions demonstrated in our cases had already been identified in other reports. In previous reports, the mutations were identical base substitutions at each mutation site, and neither addition nor deletion of bases was observed (Table II). Similar substitutions were frequently observed in each of the sequences detected worldwide, indicating that the sequence diversity was highly conserved. These changes in nucleotide sequences may include normal polymorphisms, and the pathogenic role of amino acid changes induced by base substitutions is not clear. Although the functional role of the sequence in KSHV has not been fully clarified, epidemiological analysis of the route of KSHV, and the correlation between amino acid substitutions and disease specificity should be examined in many additional cases.

Although several investigators have discussed the involvement of KSHV in immunosuppression or immune dysfunction, the precise role of KSHV in the pathogenesis of KS or other diseases has still not been clarified. Since our results reconfirmed the relationship between KSHV and HIV-positive KS, KSHV infection must be one of the most important factors in the development of KS. Elucidation of the pathogenic role of KSHV in KS may also provide clues for clarifying the tumorigenic effects of other viruses such as Epstein-Barr virus, and papilloma virus, which are thought to be involved in several neoplastic disorders. As the molecular investigation of KSHV has been reported recently [37, 38], functional analyses for investigating the mechanisms of gene regulation or factors influencing the host cell are expected. Because KSHV is thought to be sexually transmitted [1, 34], elucidation of its pathogenic role in tumorigenicity will be important for future increase in the number of neoplasms associated with KSHV.

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