Molecular phenotypes of human parvovirus B19 in patients with myocarditis

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.