Human herpesvirus 6A and 6B : assay validation, virus-host interaction and clinical relevance
Engdahl, Elin
2016-12-16
09.00
sal Eken, Norrbacka S2:02, Karolinska Universitetssjukhuset, Solna
Inst för klinisk neurovetenskap / Dept of Clinical Neuroscience
Human herpesvirus (HHV)-6A and HHV-6B are two ubiquitous herpesviruses that after primary
infection can stay latent in the host. The two viruses can reactivate from latency and cause
secondary complications. Also, diseases like multiple sclerosis and epilepsy have been associated
with HHV-6A and HHV-6B. However, these associations need to be further examined in order to
prove relation and to present evidence for how virus-host interactions can play a role in disease
pathology. In order to investigate the effect of virusesin disease it can, at least in some stages, be
necessary to perform in vitro experiments. When conducting in vitro experiments, optimal
conditions are needed in order to obtain accurate and reliable results.
The first part of the thesis focuses on improvement of in vitro experiential setups. In Study I, we
sought to develop a correct and robust measurement of virus titers aiming for increased accuracy
of experiments and better harmonization within the HHV-6A/6B research field. This was done by
optimizing the classical TCID50 method with a new qPCR readout. This qPCR readout was found to
be more robust compared to other readouts and this method can be used for HHV-6A titer
determination. In order to analyze relative gene expression during virus infections, a gene with
stable expression is needed for normalization. In Study II we searched for a gene with stable
expression during HHV-6B infection of Molt-3 cells. Comparison of eight different commonly used
reference genes revealed that PPIA is a gene suitable to use as reference gene when working with
HHV-6B.
The second part of the thesis focuses on how HHV-6B affects host cell DNA methylation and if this
could be one mechanism by which this virus has been associated to epilepsy. Study III reveals, for
the first time, that HHV-6B induces locus specific host cell DNA hypomethylation close to the
telomeres. This hypomethylation was observed already 2 days after infection and correlated with
increased gene expression and integration of the virus genome into the host cell genome. No
difference in methylation could be observed in epileptic brain tissue which could be due to a
transient hypomethylation present only in the acute phase of infection or because of underpowered
study design.
The third and last part of the thesis investigates the association between IgG antibody responses
against HHV-6A and HHV-6B in MS in order to elucidate if these viruses may play a role in this
disease. In addition, the influence of environmental factors and host genetics was investigated.
These studies revealed that there was no difference in response against HHV-6B viral lysate
between MS cases and controls (Study IV) but that established MS patients and pre-symptomatic
MS patients had an increased response against the HHV-6A protein IE1A and, to a lesser extent,
the HHV-6B protein 101K (Study V). The epitope specific IgG responses were highly dependent on
the host HLA status. Antibody responses can be interpreted in different ways, but the results of
Study V indicate that HHV-6A and HHV-6B may play a role in MS disease etiology. This virus-host
interaction in relation to MS needs further investigation.