Original Article
Copyright ©2014 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Cardiol. Apr 26, 2014; 6(4): 183-195
Published online Apr 26, 2014. doi: 10.4330/wjc.v6.i4.183
Molecular phenotypes of human parvovirus B19 in patients with myocarditis
C-Thomas Bock, Anja Düchting, Friederike Utta, Eva Brunner, Bui Tien Sy, Karin Klingel, Florian Lang, Meinrad Gawaz, Stephan B Felix, Reinhard Kandolf
C-Thomas Bock, Anja Düchting, Friederike Utta, Eva Brunner, Karin Klingel, Reinhard Kandolf, Department of Molecular Pathology, University Hospital of Tuebingen, 72076 Tuebingen, Germany
C-Thomas Bock, Bui Tien Sy, Robert Koch Institute, 13353 Berlin, Germany
Florian Lang, Department of Physiology, University of Tuebingen, 72070 Tuebingen, Germany
Meinrad Gawaz, Department of Cardiology and Cardiovascular Medicine, University Hospital of Tuebingen, 72076 Tuebingen, Germany
Stephan B Felix, Clinics for Internal Medicine B, Ernst-Moritz-Arndt-University, 17475 Greifswald, Germany
Author contributions: Bock CT, Gawaz M, Felix SB and Kandolf R conceived and designed the research; Bock CT, Düchting A, Utta F, Brunner E, Sy BT, Klingel K, Lang F and Kandolf R performed the experiments; Bock CT, Klingel K, Lang F, Gawaz M, Felix SB and Kandolf R analyzed the data; Klingel K, Gawaz M, Felix SB and Kandolf R contributed to reagents/materials/analysis tools; Bock CT and Kandolf R contributed to drafting of the manuscript; all authors read and approved the final manuscript.
Supported by Grants of the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich-Transregio 19 (project B5)
Correspondence to: C-Thomas Bock, PhD, Director, Professor, Department of Molecular Pathology, University Hospital of Tuebingen, Liebermeisterstr. 8, 72076 Tuebingen, Germany. bockc@rki.de
Telephone: + 49-30-187542379 Fax: + 49-30-187542617
Received: October 11, 2013
Revised: January 16, 2014
Accepted: February 18, 2014
Published online: April 26, 2014

AIM: To investigate molecular phenotypes of myocardial B19V-infection to determine the role of B19V in myocarditis and dilated cardiomyopathy (DCM).

METHODS: Endomyocardial biopsies (EMBs) from 498 B19V-positive patients with myocarditis and DCM were analyzed using molecular methods and functional experiments. EMBs were obtained from the University Hospitals of Greifswald and Tuebingen and additionally from 36 German cardiology centers. Control tissues were obtained at autopsy from 34 victims of accidents, crime or suicide. Identification of mononuclear cell infiltrates in EMBs was performed using immunohistological staining. Anti-B19V-IgM and anti-B19V-IgG were analyzed by enzyme-linked immunosorbent assay (ELISA). B19V viral loads were determined using in-house quantitative real-time polymerase chain reaction (PCR). For B19V-genotyping a new B19V-genotype-specific restriction fragment length polymorphism (RFLP)-PCR was established. B19V-genotyping was verified by direct DNA-sequencing and sequences were aligned using BLAST and BioEdit software. B19V P6-promoter and HHV6-U94-transactivator constructs were generated for cell culture experiments. Transfection experiments were conducted using human endothelial cells 1. Luciferase reporter assays were performed to determine B19V-replication activity. Statistical analysis and graphical representation were calculated using SPSS and Prism5 software.

RESULTS: The prevalence of B19V was significantly more likely to be associated with inflammatory cardiomyopathy (iCMP) compared to uninflamed DCM (59.6% vs 35.3%) (P < 0.0001). The detection of B19V-mRNA replication intermediates proved that replication of B19V was present. RFLP-PCR assays showed that B19V-genotype 1 (57.4%) and B19V-genotype 2 (36.7%) were the most prevalent viral genotypes. B19V-genotype 2 was observed more frequently in EMBs with iCMP (65.0%) compared to DCM (35%) (P = 0.049). Although there was no significant difference in gender-specific B19V-loads, women were more frequently infected with B19V-genotype 2 (44.6%) than men (36.0%) (P = 0.0448). Coinfection with B19V and other cardiotropic viruses was found in 19.2% of tissue samples and was associated with higher B19V viral load compared to B19V-monoinfected tissue (P = 0.0012). The most frequent coinfecting virus was human herpes virus 6 (HHV6, 16.5%). B19V-coinfection with HHV6 showed higher B19V-loads compared to B19V-monoinfected EMBs (P = 0.0033), suggesting that HHV6 had transactivated B19V. In vitro experiments confirmed a 2.4-fold increased B19V P6-promoter activity by the HHV6 U94-transactivator.

CONCLUSION: The finding of significantly increased B19V loads in patients with histologically proven cardiac inflammation suggests a crucial role of B19V-genotypes and reactivation of B19V-infection by HHV6-coinfection in B19V-associated iCMP. Our findings suggest that B19V-infection of the human heart can be a causative event for the development of an endothelial cell-mediated inflammatory disease and that this is related to both viral load and genotype.

Keywords: Myocarditis, Dilated cardiomyopathy, Parvovirus B19, B19V-genotypes, B19V co-infection

Core tip: Human parvovirus B19 (B19V) has recently been shown to be an emerging pathogen for inflammatory cardiomyopathy (iCMP). We showed that B19V replication intermediates could be detected in acute and ongoing myocarditis. B19V-genotypes 1 and 2 were predominant although B19V-genotype 2 was more prevalent in iCMP. Further analyses revealed that B19V-coinfection with other cardiotropic viruses does occur, most frequently with human herpes virus 6 (HHV6). In vitro experiments showed that the HHV6 U94-transactivator element could transactivate the B19V-P6-promoter. We suggest that long-term persistence of B19V DNA in the human heart occurs and that active/reactivated B19V-replication can be associated with iCMP in a viral load and genotype-dependent manner.