PCR con primer degenerati non evidenzia sequenze virali di HHV-8 ne di una sua variante in cellule stromali midollari di pazienti affetti da gammopatie monoclonali

The association of human herpesvirus-8 (HHV-8) infection with multiple myeloma (MM) is still controversial. The possibility has been raised that either a HHV-8 variant or a novel, as yet unidentified, γ-herpesvirus related to HHV-8, are frequently associated with MM, which could explain the lack of antibodies to HHV-8 antigens and the discordant results from polymerase chain reaction studies (PCR) of HHV-8 specific genomic sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the long term-cultures (LTC) of bone marrow stromal cells (BMSCs) from 21 MM, 3 monoclonal gammopathy of undetermined significance (MGUS) and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and non adherent fractions. Direct sequencing and alignment of the nucleotide and amino acidic sequences showed that the DNA sequences were 100% identical to Epstein-Barr virus DNA. PCR with specific primers targeting the latent membrane protein-1 (LMP-1) gene confirmed the presence of EBV. The PCR positivity was due to the presence of EBV infected lymphoblastoid cells (LCLs) with plasmacytoid features, expressing the LMP-1 protein and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV-8 or a novel herpesviral variant related to HHV-8 in MM, not even in the presence of EBV co-infection.