Clin Res Cardiol (2021)
DOI DOI https://doi.org/10.1007/s00392-021-01843-w
Parvovirus B19 NS1 and VP1/2 mRNA expression indicates viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure
H. Pietsch1, F. Escher2, G. Aleshcheva1, C. Baumeier1, C.-T. Bock3, H.-P. Schultheiss1
1IKDT - Institut Kardiale Diagnostik und Therapie GmbH, Berlin; 2CC11: Med. Klinik m.S. Kardiologie, Charité - Universitätsmedizin Berlin, Berlin; 3Abteilung für Infektionskrankheiten, Robert Koch-Institut, Berlin;
Background
Human erythroparvovirus B19 (B19V) is the most frequently found virus in endomyocardial biopsies (EMBs). Recent studies have detected B19V genomes in various organs of symptomatic and asymptomatic individuals including endothelial cells of the heart muscle. Accordingly, B19V as a causative pathogen of myocardial damage has been questioned. To date, the majority of studies focused on the presence of viral DNA in the myocardium, but lack the analysis of viral RNAs as markers for viral activity. In contrast to latent infection, the expression of viral mRNAs is of clinical importance as we could demonstrate in previous studies. Only recently, novel antiviral treatment strategies have become available. A sensitive and specific method to identify and monitor patients that will profit from antiviral treatment is a prerequisite for successful therapy. During this study, we established a new PCR-based diagnostic method to characterize the expression of B19V RNAs coding for the non-structural (NS1) and capsid protein (VP1/2) in endomyocardial biopsies of patients with unexplained heart failure and demonstrated its feasibility in a clinical setting of molecular diagnostics.

Methods
To quantify B19V RNA expression, we developed real-time (reverse transcription) polymerase chain reactions (qPCRs) targeting the NS1 and VP1/2 regions of B19V genome. 576 randomly selected EMBs of patients with unexplained heart failure were tested for B19V genomes and transcription intermediates using qPCR and nested PCR.  

Results
403 of 576 (69.9%) EMBs were positive for B19V genomes (B19V DNA). Viral loads ranged from 15 to 51,118 genome equivalents/µg isolated DNA. Of the 403 B19V DNA-positive samples, B19V mRNAs NS1 and/or VP1/2, indicating viral activity, could be detected in 155/403 (38.4%) EMBs using the newly established B19V qPCRs. 89 of 403 samples were characterized by only NS1 mRNA detection while 24 of 403 revealed only VP1/2 mRNA expression. Detection of both intermediates was successful in 42 of 403 samples. Patients characterized by active B19V infection presented with a significantly reduced left ventricular ejection fraction (30.7±13.1%) in contrast to virus free patients (35.5±15.9%; p=0.025) or latently infected patients (34.7±15.7; p=0.043).

Conclusion
Our newly established molecular testing will improve detection and quantification of B19V. As we could demonstrate here, it is essential to screen for both viral transcription intermediates, the NS1 and VP1/2 viral mRNAs, in order to precisely differentiate between latent infection or transcriptional active infection of the heart muscle. Whereas merely viral genomes are detectable during latent infection, a clinically relevant B19V-infection with transcriptional activity of the heart muscle is characterized by B19V-NS1 and VP1/2 viral mRNA expression. The results of our study revealed that a remarkable high proportion of 26.9% (155 of 576) of patients with unexplained heart failure is affected by transcriptional active B19V infection of the myocardium. Prospective studies must be conducted to evaluate our findings and furthermore, effective antiviral treatment strategies need to be adapted.

 
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