Human herpesvirus 6A and 6B : assay validation, virus-host interaction and clinical relevance

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.

List of scientific papers

I. Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6. Rasmus Gustafsson, Elin Engdahl, Anna Fogdell-Hahn. Virol J. 2012;9:31.
https://doi.org/10.1186/1743-422X-9-311

II. Investigation of reference gene expression during human herpesvirus 6B infection indicates peptidylprolyl isomerase A as a stable reference gene and TATA box binding protein as a gene up-regulated by this virus. Elin Engdahl, Nicky Dunn, Anna Fogdell-Hahn. J Virol Methods. 2016;227:47-9.
https://doi.org/10.1016/j.jviromet.2015.10.011

III. Human herpesvirus 6B induces hypomethylation on chromosome 17p13.3 correlating with increased gene expression and virus integration. Elin Engdahl, Nicky Dunn, Pitt Niehusmann, Sarah Wideman, Peter Wipfler, Albert J Becker, Tomas J Ekström, Malin Almgren, Anna Fogdell-Hahn. [Manuscript]

IV. HLA-A*02, gender and tobacco smoking, but not multiple sclerosis, affects the IgG antibody response against human herpesvirus 6. Elin Engdahl, Rasmus Gustafsson, Ryan Ramanujam, Emilie Sundqvist, Tomas Olsson, Jan Hillert, Lars Alfredsson, Ingrid Kockum, Anna Fogdell-Hahn. Hum Immunol. 2014;75(6):524-30.
https://doi.org/10.1016/j.humimm.2014.03.001

V. Serological response against an immediate-early 1 protein sequence from HHV-6A is positively associated with risk for multiple sclerosis. Elin Engdahl, Rasmus Gustafsson, Jesse Huang, Martin Biström, Izaura Lima Bomfim, Mohsen Khademi, Angelika Michel, Julia Butt, Nicole Brenner, Ingileif Jonsdottir, Kari Stefansson, Daniel Jons, Maria Hortlund, Lucia Alonso-Magdalena, Louis Flamand, Oluf Andersen, Jan Hillert, Lars Alfredsson, Tim Waterboer, Peter Sundström, Tomas Olsson Ingrid Kockum, Anna Fogdell-Hahn. [Manuscript]