Detection of HHV-6-specific mRNA and antigens in PBMCs of individuals with chromosomally integrated HHV-6 (ciHHV-6)

Objective. It is unclear whether chromosomally integrated HHV-6 (ciHHV-6) is able to replicate in vivo. We analysed peripheral blood mononuclear cells (PBMCs) of individuals with ciHHV-6 for HHV-6 specific mRNA (corresponding to proteins U22, U42, and U94): U22 and U42 are expressed only during lytic infection whereas U94 is expressed both during latent and lytic infection. Methods. In 7 specimens of PBMCs from 6 individuals (from 3 families, ciHHV-6A n=4, ciHHV-6B n=2) with FISH proven ciHHV-6, the extracted total RNA was retrotranscribed and the resulting cDNA was analyzed by PCR and nested PCR for U94, U42 and U22 genes. In parallel, cDNA was also analyzed by real time qPCR (rt-qPCR) for the same genes. In addition, nested qPCR was performed using the first round PCR product as the template. Results. First round PCR as well as rt-qPCR was negative for all samples. In nested PCR, 2/7 specimens (out of 2 families, ciHHV-6A both) were positive for U94. In nested rt-qPCR 4/7 (out of 3 families, 2 with ciHHV-6A, 2 with ciHHV-6B) ) were positive for U94. In 1 sample from a healthy 49 year old female U22, U42 as well as U94 transcripts were detected by both nested PCRs and nested rt-qPCR, whereas one 16 year old Hodgkin Disease patient, who was tested twice, had 1 sample positive for U94 and 1 sample negative for all tested transcripts. Conclusions. These preliminary results indicate intermittent in vivo replication of HHV-6 genes in individuals with ciHHV-6. Detection of U22 and U42, which are only expressed during lytic infection, indicates viral reactivation. This might have pathophysiological and possibly clinical implications. Further studies are needed to clarify the frequency and consequences of this condition.

After inheritance of chromosomally integrated HHV-6 (ciHHV-6), viral DNA is found in every nucleated cell. The prevalence of ciHHV-6 is estimated to be 0.2 to 5% of humans. There are conflicting data on the potential of replication possibly leading to clinical implications. We analysed peripheral blood mononuclear cells (PBMCs) from individuals with FISH proven ciHHV-6 for HHV-6 specific mRNA (U94, U42, U22) and antigens by means of reverse transcription PCR and an indirect immunoperoxidase staining. U94 transcripts indicative of latent infection were detected in 6 (54.5%) out of 11 individuals at least once. Transcripts indicative of lytic infection (i.e. U42 and U22) were detected in 4 (36.4%) out of 11 individuals at least once. HHV-6 antigen was detected in 7 (70%) out of 10 individuals at least once. The presence of viral mRNA and proteins supports virus gene expression from ciHHV-6 which may lead to virus replication. Considering the properties of active HHV-6 infection together with obvious replicative activity in individuals with ciHHV-6, pathophysiological effects leading to clinical consequences of chromosomally integrated viral DNA might be considered.