Molecular phenotypes of human parvovirus B19 in patients with myocarditis

Abstract

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: B19V co-infection; B19V-genotypes; Dilated cardiomyopathy; Myocarditis; Parvovirus B19.

Figure 1

Generation of B19V-genotype 1 to 3 specific restriction fragment length polymorphism-polymerase chain reaction. A: Schematic representation of the B19V genome showing localization of the B19V-genotype-specific restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) in the B19V NS1-VP1u region. Sequences of B19V-1, B19V-2 and B19V-3 showing the RFLP-PCR fragment and HpaI and TaqI restriction enzyme sites (lower panel). Primer positions for 1^st and 2^nd RFLP-PCR are indicated (1. PCR and 2. PCR, see also Table 2); B: Expected fragment size and digestion pattern after HpaI and TaqI digestions (right panel). Agarose gel electrophoresis showing respective PCR-fragments after HpaI and TaqI digestion for each B19V-genotype (left panel); C: Representative agarose gel electrophoresis of patient-specific B19V RFLP-PCRs. B19V-1: B19V-genotype 1.

Figure 2

Typical histopathological and immunohistological findings in acute myocarditis (A and B), chronic myocarditis/inflammatory myocarditis (C and D), chronic dilated cardiomyopathy without inflammation (E and F), and non-failure control hearts (G and H). Masson trichrome staining (A, C, E and G) and immunohistological detection of CD3⁺ T-lymphocytes (B, D, F and H).

Figure 3

Representative B19V-specific reverse transcription-polymerase chain reaction showing B19V mRNA replication intermediates isolated from endomyocardial biopsies of patients with acute myocarditis (lane 2) and chronic myocarditis/iCMP (lane 3). iCMP: Inflammatory cardiomyopathy.

Figure 4

Genotype specific myocardial B19V loads of patients with chronic myocarditis. A: Prevalence of B19V-genotype 1 (B19V-1) and B19V-2 in endomyocardial biopsies of patients with myocarditis [inflammatory cardiomyopathy (iCMP), grey columns] and dilated cardiomyopathy (DCM, white columns). Patient number is given in %; B: qPCR of myocardial of B19V-1 and B19V-2 loads in endomyocardial biopsies (EMBs); C: B19V genotype-specific myocardial viral loads in EMBs of patients with chronic myocarditis (iCMP, white columns) and DCM (grey columns) determined by qPCR. One-way Anova was highly significant (P < 0.0001). P < 0.05 is statistically significant (two-tailed T-test). qPCR: Quantitative real-time polymerase chain reaction.

Figure 5

Age and gender dependent distribution of B19V-genotypes in endomyocardial biopsies of patients with myocarditis. A: Distribution of B19V-genotype 1 (B19V-1) and B19V-2 according to year of birth; B: Gender-specific mean age of our patient cohort; C: Gender-specific distribution of B19V-1 (white columns) and B19V-2 (grey columns). P < 0.05 is statistically significant (two-tailed T-test).

Figure 6

Distribution of B19V-coinfection with cardiotropic viruses. A: In endomyocardial biopsies determined by virus-specific nPCR; B: Frequency of B19V-coinfection with cardiotropic viruses; C: qPCR of B19V loads in B19V mono- and co-infection; D: Distribution of B19V-genotype 1 and 2 in B19V mono- and co-infection in endomyocardial biopsies (EMBs); E: B19V loads of B19V mono- and co-infection in EMBs of patients with iCMP and DCM. One-way Anova was highly significant (P < 0.0001); F: B19V loads of B19V mono- and co-infection with cardiotropic viruses. One-way Anova was highly significant (P = 0.0091); G: Luciferase reporter assay to determine transactivation capacity of the HHV6-U94 transactivator on the B19V P6-promoter activity. P < 0.05 is statistically significant (two-tailed T-test). HHV6: Human herpesvirus 6; EV: Enterovirus; HCMV: Human cytomegalovirus; EBV: Epstein-Barr virus; DCM: Dilated cardiomyopathy; iCMP: Inflammatory cardiomyopathy; qPCR: Quantitative real-time polymerase chain reaction.