Analyses of germline, chromosomally integrated human herpesvirus 6A and B genomes indicate emergent infection and new inflammatory mediators

Abstract

Human herpesvirus-6A (HHV-6A) is rarer than HHV-6B in many infant populations. However, they are similarly prevalent as germline, chromosomally integrated genomes (ciHHV-6A/B). This integrated form affects 0.1-1 % of the human population, where potentially virus gene expression could be in every cell, although virus relationships and health effects are not clear. In a Czech/ German patient cohort ciHHV-6A was more common and diverse than ciHHV-6B. Quantitative PCR, nucleotide sequencing and telomeric integration site amplification characterized ciHHV-6 in 44 German myocarditis/cardiomyopathy and Czech malignancy/inflammatory disease (MI) patients plus donors. Comparisons were made to sequences from global virus reference strains, and blood DNA from childhood-infections from Zambia (HHV-6A mainly) and Japan (HHV-6B). The MI cohort were 86 % (18/21) ciHHV-6A, the cardiac cohort 65 % (13/20) ciHHV-6B, suggesting different disease links. Reactivation was supported by findings of 1) recombination between ciHHV-6A and HHV-6B genes in 20 % (4/21) of the MI cohort; 2) expression in a patient subset, of early/late transcripts from the inflammatory mediator genes chemokine receptor U51 and chemokine U83, both identical to ciHHV-6A DNA sequences; and 3) superinfection shown by deep sequencing identifying minor virus-variants only in ciHHV-6A, which expressed transcripts, indicating virus infection reactivates latent ciHHV-6A. Half the MI cohort had more than two copies per cell, median 5.2, indicative of reactivation. Remarkably, the integrated genomes encoded the secreted-active form of virus chemokines, rare in virus from childhoodinfections. This shows integrated virus genomes can contribute new human genes with links to inflammatory pathology and supports ciHHV-6A reactivation as a source for emergent infection.

Chromosomal integration of HHV-6A during non-productive viral infection

“…Due to the absence of elaborate in vitro studies on the integrated viral genome, it is generally accepted that almost full-length viral genome is integrated into human chromosomes. Several in vivo sequencing studies in over 50 iciHHV-6A/B individuals have demonstrated genome integration, including 5 complete genomes and over a dozen subtelomere integration sites determined 5, 11, 16, 18, 19 . Most of the PCR-based epidemiological studies utilise primer pairs designed against viral ORFs to detect and quantify viral copy numbers.…”

“…Our study is primarily focused on ciHHV-6A. However, it has been shown that chromosomal integration frequency might vary between iciHHV-6A and iciHHV-6B 5 . Hence it will be interesting to see if ciHHV-6B can also show similar characteristic features like ciHHV-6A.…”

“…Chromosomally integrated HHV-6 (ciHHV-6) has been detected only in human sub-telomeric ends suggesting viral integration through homologous recombination between telomeric repeat sequences present at the end of human chromosomes and HHV-6 genome. When integrated into germ cells, HHV-6 can be inherited (iciHHV-6) thereby ending up in 1 or more copies of viral genome in every nucleated cell of the body 4, 5 . Even after several years of research on viral integration, our knowledge is still limited.…”

Chemokines encoded by herpesviruses

“…3). Surprisingly, unlike for the episomal latent virus, fulllength vCCL4 is the predominant form of the chemokine expressed by chromosomally integrated HHV-6 [148]. Although HHVs typically establish latency as episomes in the host cell nucleus, HHV-6 is the HHV with the highest prevalence of chromosomal integration in the germline (1%) [149].…”

Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling

“…pathogenic viral reactivation) or indirect ( e.g. telomeric instability and deletions) mechanisms [5–12]. However, the clinical impact of inherited ciHHV-6 remains poorly understood due to the low prevalence of this condition, lack of routine screening, and the diagnostic challenge of identifying affected patients [13].…”