Molecular phenotypes of human parvovirus B19 in patients with myocarditis

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 DCMwere 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 DNAsequencing 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 B19Vreplication 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 tissuesamples 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 incr