Study On Association Of Granuloma Fungoides With HSV-1,HSV-2,EBV,HCMV And HHV-8

The aim of this study was to investigate the occurence of human herpesvirus 1 ( herpes simplex virus 1, HSV -1) , 2 ( herpes simplex virus 2, HSV - 2 ) , 4 ( Eptein - Barr virus, EBV ) , 5 ( human cyto-megalovirus, HCMV) and 8 (initially named Kaposi's sarcoma ?associated herpesvirus, KSHV) DNA sequences in the lesional skin biopsies and peripheral blood of granuloma fungoides. The results would allow us to evaluate the possible association of granuloma fungoides with these five types of human herpesviruses.Materials and Methods1. Specimens examinedSkin biopsies and peripheral blood from three groups of individuals from No. 1 hospital of China Medical University were examined.1) The first group was granuloma fungiodes. Thirty - two lesional skin biopsies and fifteen peripheral blood specimens were collected from thirty - three patients, whose diagnosis was confirmed by both clinical and histopathological findings. The lesional skin biopsies were snap - frozen and preserved at - 70C untill they were processed, some of which were embedded with OCT.2) The second group was comprised of nineteen patients affected by chronic eczema and lichen simplex chronicus. Twenty - four le-sional skin biopsies and eleven peripheral blood samples were collected from these patients.3) The third group was referred to as normal controls. Forty peripheral blood specimens from healthy blood donors and twenty - nine normal skin samples were analysed.2. DNA extractionHuman genomic DNA isolation was carried out by using phenol -chloroform procedure. Optical density ( OD) values were measured with spectrophotometry at 260nm and 280nm repectively to determine the concentration and purity of the DNA extracted.3. Polymerase chain reaction (PCR) amplificationAll specimens were tested for amplifiability by PCR with a set of primers ( PC03/PC04) specific for the human (3 - globulin gene, serving as an internal control.PCR was performed to detect specific DNA fragments of the five types of human herpesviruses in samples of lesional skin and peripheral blood leukocytes from patients with granuloma fungoides as well as control subjects. Analysis of HSV - 1, HSV -2, EB virus and HCMV DNA sequences in skin tissue samples and peripheral blood leukocytes were performed using PCR amplification with a set of degenerate primers P - 1/P - 2 as previously described. The amplifier sizes are 518bp, 518bp, 524bp and 589bp respectively. HCMV can be distinguished from the other three types by its largest size. PCR amplification of the 233bp KS330233 fragment specific for human herpesvirus 8 was performed with previously published primer sets KS1/KS2 located in the HHV - 8 open reading frame ( ORF) 26. The 5uL of each PCRproduct were electrophoresed on agarose gel and stained with ethidium bromide. The gels were illuminated with ultraviolet light and analysed. Every sample was tested at least twice for each virus gene.4. Retrieval of the positive productsThe 50uL of PCR mixture containing 520bp or so and 233 bp fragments were separated in agarose gel. The products were retrieved by using a DNA recovery kit as recommended by the manufacturer.5. Digestion of the retrieved products with specific restriction endonu-cleaseThe retrieved products of each virus were analysed by digestion with restriction endonuclease to confirm its origin. HSV - 1 DNA (518bp) can be cleaved into two fragments sized 476bp and 42bp by Sma I , rather than by BamH I ; BamH I , rather than Sma I , can digest HSV -2 DNA (518bp) into 255bp and 293bp. EBV can be cleaved by both BamH I and Sma I , with the digestive fragments of 424bp and lOObp, 247bp and 277bp. The 233bp products can be cleaved into 138bp and 95bp by restriction endonuclease Pst I .6. Control specimensAppropriate positive control specimens were used throughout the study. DNA, extracted from HSV - 1 - infected cell line and BC - 1 cell line co - infected by EB virus and human herpesvirus 8, serve as the positive controls for HSV -1, EBV and human herpesvirus 8.7.