WO2007096199A2

WO2007096199A2 - Differential diagnostics and therapy of virally induced heart muscle disease

Info

Publication number: WO2007096199A2
Application number: PCT/EP2007/001691
Authority: WO
Inventors: Dirk Lassner; Heinz-Peter Schultheiss; Uwe Kühl
Original assignee: Ikdt Institut Kardiale Diagnostik Und Therapie Gmbh; Charite-Universitätsmedizin Berlin
Priority date: 2006-02-22
Filing date: 2007-02-22
Publication date: 2007-08-30
Also published as: WO2007096199A3

Abstract

Novel polynucleotide sequences and amino acid sequences of parvovirus B19, methods for differential diagnosis of viral infection in myocardial disease and pharmaceutical compositions for the treatment thereof are disclosed. A method for the detection of a viral infection of a patient comprises the detection in a blood vessel or heart muscle tissue sample, outside of the human body, the presence or absence of Parvovirus B19 genotype 1 or Parvovirus B19 genotype 2 or Human Herpesvirus 6 or both. Similarly provided is the use of Interferon alpha in the preparation of a pharmaceutical composition for the prevention or treatment of myocardial infection by Parvovirus B19 or Human Herpesvirus 6, optionally with the addition of ribavirin.

Description

Differential diagnostics and therapy of virally induced heart muscle disease

Description

This invention relates to the diagnosis and treatment of viral infection in association with heart and coronary disease. Specifically, the invention relates to the detection and treatment of erythrovirus and human herpesvirus 6 genotypes in heart tissue of patients.

Myocarditis is defined as a focal or diffuse, acute or chronic recurrent inflammatory process in the myocardium which may be induced by a number of physical, chemical, and infectious agents, including viruses. Molecular biology techniques demonstrate that a number of viruses, e.g. Adeno- and Enteroviruses (Pauschinger et al.1999, Circulation 99:889-895, and ibid. 99:1348-1354), are detectable in endomyocardial biopsies of patients with acute and chronic myocarditis. Viral infection may damage the myocardium by e.g. inflammatory processes and virus replication, making evaluation of virus persistence and inflammatory state in endomyocardial biopsies essential for the development of specific therapeutic strategies. The indication "chronic viral cardiomyopathy" as used herein includes viral infections with and without inflammation. Clinically, viral myocarditis may appear in a wide variety of forms, ranging from a total lack of clinical manifestations to progressive myocardial failure and sudden death. Chronic viral infection leads to progressive myocardial damage and is considered to be an essential cause for the development of dilated cardiomyopathy. Dilated cardiomyopathy may lead to a deterioration in clinical symptoms up to heart failure which in the final stage requires a ventricular assist device or heart transplantation. In The Myocarditis Treatment Trial for example, the mortality rate was 20% at one year and 56% at 4.3 years for the entire group (Mason et al.1995 N Engl J Med 333:269-275).

At present, patients are treated symptomatically and surgically for heart failure. The standard therapy consists of e.g. Angiotensin converting enzyme (ACE) inhibitors, diuretics, digitalis, beta-blockers, aldosterone antagonists, anticoagulants, and/or antiarrhythmic agents. Although there have been several attempts to introduce more specific therapies, there are no recommendations on the basis of randomized trials so far.

Interferons (IFN^'s) are a family of small proteins. Four types of interferons have been identified which differ in both structure and antigenic properties: IFN alpha, beta, gamma and omega derived from different cell types. The important role played by IFN as a natural defence against viruses is documented by three types of experimental and clinical observations: in many viral infections a strong correlation has been established between IFN production and natural recovery; inhibition of IFN production or action enhances the severity of infection and treatment with IFN protects against viral infection. The antiviral effect is independent from virus type and results in an intracellular block of the viral replication cycle. Immunomodulatory effects include activation of macrophages and natural killer cells as well as enhancement of major histocompatibility complex (MHC) antigen expression.

WO2005/120540 A2 (Schering AG) shows the use of Interferon beta in the treatment of cardiomyopathy. The document does not show differential treatment or diagnosis in relation to different genotypes of viruses.

Parvovirus B19 (PVB19) is a member of the human erythrovirus family frequently detected in endomyocardial biopsies (EMB) from chronic dilated cardiomyopathy (DCM) patients. Based on nucleotide sequence comparisons, human erythroviruses cluster into three genotypes 1 to 3 (Servant et al., J Virol 2002;76:9124-34), which share a high degree of homology between major structural proteins and may cause clinically and serologically indistinguishable infections. These 3 genotypes share a high degree of homology between major structural proteins and cause infections which are clinically and serologically indistinguishable (Schalasta et al., J Med Virol 2004;73:54-9, Heegaard et al., J Med Virol 2002;66:246-52). Recently, several different viral genomes have been detected in EMBs from patients presenting with DCM (Baboonian et al., Heart 1997;78:539-43; Bowles et al., J Am Coll Cardiol 2003;42:466-72.; Kϋhl et al., Circulation 2005;111 :887-93). The prevalence of genotypes 2 and 3 in patients with heart failure has not yet been characterized.

In human cardiac tissue erythrovirus genotypes other than PVB19 have not yet been reported.

The clinical course of DCM has been linked to the spontaneous course of the viral infections. Spontaneous and treatment- dependent virus clearance was associated with spontaneous clinical improvement while myocardial dysfunction progressed over time in those patients who developed virus persistence (Frustaci et al., Circulation 2003; 107:857- 63; Kϋhl et al., ibid. 2003; 107:2793-8; Kϋhl et al., ibid. 2005;112:1965-70). US2003/0170612A1 shows diagnostic assays for parvovirus. WO2006/031608 shows methods and compositions for detecting erythrovirus genotypes.

In individual patients, significant discrepancies have been recognized with respect to the spontaneous viral course and the resolution or progression of heart failure. This holds specifically true for patients with detected genomes of parvovirus B19, which were identified in a high frequency of individuals undergoing biopsy for evaluation of heart failure of unknown cause.

Human herpesvirus 6 (HHV-6) is a lymphotropic virus with lifelong persistence after childhood infection and primarily associated with non-cardiac diseases. Recent studies have identified HHV-6 as a possible pathogenetic cause of myocarditis and idiopathic cardiomyopathy. Its prevalence, subtype involvement and cardiac infection site are unknown.

In the course of the present invention, the prevalence and course of a HHV-6 reactivation in 1622 baseline and 458 follow-up endomyocardial biopsies (EMB) of consecutive patients with clinically suspected acquired cardiomyopathies or persisting symptomatic non-ischemic heart failure was investigated. The patients were clinically grouped into suspected myocarditis (870), dilated cardiomyopathy (531 ) and other diseases known to influence cardiac contractility (221 ). Subgroup analyses identified the prevalence of HHV-6 variants in EMB and isolated blood cells, and followed the spontaneous or treatment dependent course of the HHV-6 infection. HHV-6 variant specific infection sites within the myocardium were identified by immunohistochemistry and electron microscopy. Systemic HHV-6 reactivation was analyzed by nested PCR in blood samples of patients with proven viral genomes in EMB tissues (764).

231 patients (14.2%) with cardiac HHV-6 reactivation, including 144 tissue samples in which multiple viral infections (62.3%) were identified. Sequence analysis confirmed HHV- 6 genotype B as the major pathogen (90.0%). A systemic HHV-6 reactivation was seen in 24 patients (4.4%) and primarily associated with HHV-6 genotype A reactivation (85.7%). 0.3 % of the HHV-6 positive patients presented systemic HHV-6 infection/reactivation, cardiac involvement and high virus loads of >50000 virion copies/μg isolated DNA indicating genomic suggesting chromosomal integration of viral DNA in these minor group of patients. While persistent or new HHV-6 reactivation was associated with progression of left ventricular dysfunction (p<0.001 ), LV-EF improved in association with resolved HHV-6 reactivation. It can be concluded that reactivation of HHV-6 genotypes A and B is frequently detected in the myocardium of patients with symptomatic heart failure. The course of the viral disease is associated with the symptomatic and hemodynamic presentation of the patients. Given the frequency of HHV-6 infections or reactivation in the whole population, HHV-6 cardiac involvement should be considered as possible pathogen in unexplained heart failure of patients with cardiac and non-cardiac diseases.

Surprisingly, it was found that within a group of human patients with symptomatic dilated cardiomyopathy, different previously unknown genotypes of erythrovirus and human herpesvirus 6 could be isolated. Furthermore, the presence or absence of different viruses correlates with age and clinical symptoms, as well as with the response and likelihood of virus elimination upon IFN beta therapy.

Interestingly, the differences between the VP2 sequence of erythrovirus type 1 and 2 isolated from myocardial tissue correspond to the exposed structural amino acid sequence loop in a surface protein of erythrovirus, providing a binding region for the cellular receptor of erythrovirus alpha-5-beta-1 Integrin. Sequence variations on the level of the protein sequence may lead to differential antigenicity and recognition of the protein by antibodies or cells of the immune system. This in turn may provide the opportunity for both targeting diagnostic recognition and preventive or therapeutic vaccination with these genotype- specific sequences.

According to one embodiment of the invention, polynucleotide and amino acid sequences are provided that allow for the diagnosis, therapy and prevention of infection by the previously unknown genotypes of erythrovirus. These include siRNA, mRNA, peptides or proteins for preventive or therapeutic vaccination, other known vectors for genetic vaccination such as naked DNA or viruses.

According to another embodiment of the invention, polynucleotide sequences of the invention can be used to provide diagnostic tools, for example surfaces to which polynucleotide sequences are attached that allow for the distinction of different erythrovirus genotype amplified nucleotide sequences. Amplification may be effected, by example, by polymerase chain reaction, reverse, nested, multiplex or quantitative polymerase chain reaction.

Nested polymerase chain reaction is a modification of the Polymerase chain reaction (PCR) intended to reduce the contaminations in products due to the amplification of unexpected primer binding sites. Nested polymerase chain reaction involves two sets of primers, used in two successive runs of polymerase chain reaction, the second set intended to amplify a secondary target within the first run product.

Quantitative PCR allows for the determination of the quantity of nucleic acid samples of the detected sequence, for example by activation of fluorescently active molecules attached to labelled probes by the 5'-3' exonuclease activity of the PCR processing enzyme. Frequently used technologies are known to the person skilled in the art as the "TaqMan" and "lightCycler" technologies.

As is shown in example 2, cardiomyopathy can also be associated to human herpesvirus 6 (HHV6) infection. It was previously unknown that 2 different variants A and B of HHV6 were involved in infection myocardial tissue. Hence, the diagnosis of the status of a patient with respect to HHV6 can be very useful in order to determine pathways of further diagnosis or intervention. According to yet another embodiment of the invention, diagnostic tools are provided that allow for the detection of HHV6 in patients suffering from or suspected to suffer from myocardial disease.

One example for such diagnostic tool is a set of primer molecules for multiplex PCR, which enables the concomitant analysis of a heart tissue or blood sample for the presence of genotypes of erythrovirus, HHV6, or both. Another example is a diagnostic method that allows for the detection of the presence or absence of erythrovirus genotypes or HHV6 genotypes, or both, in a sample of heart tissue or vein tissue, the sample being analysed outside of the body of a patient.

Another embodiment of the invention is a device such as a chip or other surface-bound technology that allows for the detection of sequence variants or the presence or absence of a genotype of erythrovirus or HHV6 or both.

Yet another embodiment of the invention is a plurality of PCR primer molecules that enable the simultaneous performance of PCR reactions on different templates, so-called "multiplex PCR". This term refers to the use of multiple, unique primer sets within a single PCR reaction to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple sequences at once, additional information may be elicited from a single reaction that otherwise would require several times the reagents and technician time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and amplicon sizes should be separated by sufficient difference in final base pair length to form distinct bands via gel electrophoresis.

According to embodiments of the present invention, a multiplex PCR reaction for the detection of Parvovirus B19 or HHV6 or both can be performed using at least two primer pairs (the term "pair" referring to two distinct primer oligodeoxynucleotides, hybridizing to the 5' and 3'-end of the sequence that is the target of the specific primer pair, on opposite strands). Similarly, a multiplex PCR reaction employing three, four, five or ten, fifteen, twenty, 25, 30 or more primer pairs for the detection of Parvovirus B19 or HHV6 or both can be employed.

In the context of the present invention, the term "a kind" in reference to a polynucleotide primer is to be understood as a plurality of primer molecules of the same sequence.

According to yet another aspect of the invention, a pharmaceutical composition is provided that allows for the treatment of a patient suffering from heart disease. In the course of the present invention, it was found -to the surprise of the clinical investigator- that patients suffering from cardiomyopathy will benefit substantially from a treatment with interferon alpha, possibly enhanced by concomitant treatment with ribavirin (i-(β-D-Ribofuranosyl) - 1 H-1 ,2,4-triazole-3-carboxamide).

Interferon can be used in pegylated or non-pegylated form as interferon alpha 2a or alpha 2b or alphacon1 ,9. The person skilled in the art is able to determine the adequate schedule of application from the schedule of treatment given in example 4.

Fig. 1 shows a schematic position of PCR primers and TaqMan probes used for the detection of PVB19 as described in example 1.

Fig. 2 shows positions of amino acid exchange sites within the 536 bp nucleotide sequences of Myocard 1 and 2. Marked areas correspond to the coding regions of the phospholipase A (nucleotides 1 to 220) and the co-receptor binding site (nucleotides 411 to 511 ), respectively.

Fig. 3 shows age-dependant distribution of erythrovirus genotypes Fig. 4 shows a significant reduction of LV-EF in the genotype 1 positive cohort of patients (PVB19) as compared to genotype 2-positive patients.

Fig 5 shows the echogram examination of heart wall oedema (increase of wall thickness) in a patient treated with interferon alpha (a) and three patients treated with interferon beta. Y-axis: wall thickness in mm. X-axis: time of treatment (weeks). Circled "+" signifies biopsies positive for virus; circled "-" biopsies negative for virus.

Examples

Example 1 : Detection of Parvovirus B19 in cardiac tissue

317 patients with symptomatic dilated cardiomyopathy (DCM) (median LV-EF=28.6%, range 5%-45%) who underwent endomyocardial biopsy (EMB) for the elucidation of the etiology, were analyzed using a new consensus PCR assay designed for the detection of the three erythrovirus genotype sequences.

Among 317 consecutive DCM patients (mean age 54.3±11.6 years) who underwent first diagnostic EMB for the elucidation of heart failure of unknown origin, we identified 151 patients (47.6%) with erythrovirus-positive PCR and 161 virus-negative patients (52.6%) who complained for symptoms of moderate to severe heart failure including fatigue, reduced physical capacity, angina, or dyspnea on exertion. Global systolic left ventricular ejection fraction (LV-EF) was reduced below 45% (LV-EF=28.6±9.8%, range 5% to 45%). Mean history of symptoms was above 3 months in all patients. Patients with clinically suspected recent myocarditis and patients with multiple or other viral infections (e.g. enterovirus, adenovirus, human Herpesvirus, or Epstein-Barr-virus) were excluded from this analysis. Coronary heart disease, hypertension, diabetes mellitus, obesity, valvular disease, lung disease, renal dysfunction, or other severe concomitant diseases known to be associated with ventricular dysfunction were excluded in all patients before biopsy obtainment. The basic characteristics of the patients are listed in Table 1.

Table 1

Number of patients 317

Age (years ± SD) 54.3 ± 11.6

Male 242 (76.8%)

NYHA II/III/IV (%) 50.7.0/44.0/5.3

EF (%) 28.6 ± 9.8 LVEDP (mmHg) 15.5 ± 8.2

LV-EDD (mm) 65.3 ± 8.9

LV-ESD (mm) 52.7 ± 11.5

Medication

Glycosides 46.0%

Diuretics 91.8%

Spironolactone 88.1 %

ACE-lnhibitor 90.3% β-blocker 83.8%

Cumarin 52.5%

Antiarrhythmics 11.5%

Example 1 a: Detection of Erythrovirus Genomes in EMBs by nested-PCR

Genomic DNA from endomyocardial biopsies was extracted by Puregene Mousetail Kit (Gentra, Minneapolis, USA). For exclusion of systemic virus infection peripheral blood cells were isolated from 0.5 ml peripheral blood after lysis of erythrocytes. Detection of erythrovirus DNA by nested PCR (nPCR) was performed with primers specific for the VP1/VP2 coding sequence. PVB19-specific primers used for PCR resulted in a PCR amplicon of 290 bp and, in the second round of PCR, in a 173-bp amplicon. Specificity of PCR products was confirmed by automated DNA sequencing. Detected sequences were matched to the NCBI GenBank and compared with a recently described PVB19 genome (GenBank accession No. AY386330). As a control for successful extraction of nucleic acids the sequences of house-keeping gene beta actine (ACTB) were simultaneously amplified from each DNA sample. Primer sequences were chosen from the sequence of the beta actine (ACTB) gene (Table 2). Except the TaqMan-PCR probe SPV all primers were obtained from TIB MOLBIOL (Berlin, Germany). Amplification of 664bp long-PCR fragment for following sequencing of 553bp PCR product was performed by nested-PCR using two new primer pairs (Table 2). 1. PCR was performed by 35 three-step cycles (45 s at 95 °C, 45 s at 57°C, 45s at 72⁰C) after initial denaturation for 7 min at 95°C. Nested PCR was performed by 40 cycles with same thermal profile as first PCR round. Table 2 PCR Primer sequences

Table 3: Sequence variation in erythrovirus subtypes:

Table 3 shows Identification of nucleotide exchanges in comparison with Genebank isolates: The 536 bp PCR products reveal 25 positions which allow the discrimination of genotype 1 from genotype 2. In 26 additional positions genotype 1 is identical to genotype 2 but differs from genotype 3 (Accession number AJ249437.1 , for virus V9). Erythrovirus genotype 3 sequence were not detected in any EMB, although single base exchanges at some wobble bases corresponded to pattern of genotype 3. Example 1 b: TaqMan QPCR for Ervthrovirus Genomes

For comparison of qualitative nested-PCR and quantitative values, primers and probes for erythrovirus TaqMan QPCR were designed in the nested-PCR sequence fragment. Primers and the minor groove binding site (MGB) probe SPV were selected according to general rules of primer and probe design for real-time PCR by PrimerExpress software (Applied Biosystems, Darmstadt, Germany). The chosen primer set amplifies a 70 bp long fragment in the VP1/VP2 gene region of PVB19. Sequences of used primers and probe are listed in Table 2. The amplicon-specific probe SPV was labeled with the fluorescent reporter dye FAM (6-carboxy-fluorescein) at the 5 end and the blackhole MGB quencher dye at the 3^' end (Applied Biosystems, Darmstadt, Germany).

The TaqMan-PCR was carried out in a 96-well microtiter plate format (Perkin-Elmer, USA). The PCR mix was made up to a volume of 25 μl using ready-to use Universal Mastermix containing AmpliTaq DNA polymerase, uracil-N-glycosylase (UNG), dNTPs, KCI, MgCI2 and ROX as a passive reference all in optimized concentrations (Applied Biosystems, USA). Applied concentrations per assay of forward and reverse primers and the fluorescence-labelled probe were 0.2 μM and 4μl patient sample DNA. After UNG treatment at 50 °C for 2 min and initial denaturation at 95 °C for 10 min, the DNA was amplified by 38 two-step cycles (15 s at 95 ⁰C, 1 min at 57 ⁰C). The amplification was followed on the ABI Prism HT7900 Sequence Detection System (Applied Biosystems,

USA). Reactions and cycling were performed as recommended by the manufacturer' s instructions (ABI Prism 7900HT, Applied Biosystems). Serial dilutions of PVB19 plasmid DNA standard (3.5 to 3.5x10⁴ genomes per assay, Genexpress, Berlin, Germany) were simultaneously coamplified to generate standard curve. Subsequent calculation of viral load was performed by ratio of estimated viral genome copy number in TaqMan assay to amount of incorporated human DNA amount measured by UV spectroscopy

Example 1 c: DNA Sequence analysis

DNA fragments overlapping the VP1/VP2 region nucleotide sequences (290 or 664 bp) of erythrovirus genome were amplified by nested PCR using primer pairs indicated in Table 2. Nested-PCR products were digested for primer and dNTPs elimination by ExoSAP (USB, Cleveland, USA). Sequencing reaction fragments were generated by 30 three-step cycles (20 s at 96 ⁰C, 20 sec 55°C, 2 min at 60⁰C) using CEQ Quick kit (Beckman-Coulter, Germany) for cycle sequencing and PVB4 or PVB3 as corresponding sequencing primer. For long-nested-PCR sequencing fragments last incubation step was 4 min at 60⁰C using primers PVB4 or PVB6, respectively.

Single stranded sequencing products were purified by CleanSeq magnetic beads purification kit (Agentcourt, Beverly, USA). Nucleotide sequences were obtained with a 8- capillary sequencing system CEQ 8000 (Beckman-Coulter, Krefeld) All obtained sequences were aligned individually against NCBI database BLAST. Alignment for determination of the erythrovirus genotype was performed by manual editing using Phylogenetic Data Editor (PHYDE) Software (Institute for Botanies, Dresden/Bonn, Germany).

Example 1 d: Histological and immunohistoloqical Assessment of EMBs

Histological evaluations were performed on paraffin sections according to the Dallas classification, lmmunohistological assessment of myocardial inflammation was carried out as described in Noutsias et al., Circulation 1999;99:2124-31.

Example 1 e: Statistics

Results for quantitative features are given as means ± SD. Student's t-test, one-way analysis of variance, chi-square or Fisher's exact test were used as appropriate. All P values were two-tailed; P values below 0.05 were considered to indicate statistical significance. The independent prognostic effect of the significant variables identified by univariate analysis was tested by forward stepwise regression analysis. All statistical analyses were performed using the JMP software version 5.1 (SAS Institute Inc., USA).

Example 1 : Results

Biopsy-based detection of different erythrovirus genotypes

DNA sequence analysis: By first using standard nested-PCR primers for erythrovirus screening, 151 DCM patients were identified whose EMBs yielded an 173 bp nested-PCR product. We then amplified a longer PCR fragment encompassing a total of 553 bp from the VP1/VP2 region of human erythroviruses by using a second primer set (Figure 1 and Table 2). Characterization of these PCR products by cycle sequencing revealed two distinct 536 bp nucleotide sequences (Myocard 1 and 2) only one of which corresponded to genotype 1 , i.e. PVB19 in the strict sense, whereas the other corresponded to genotype 2 (prototype virus LaLi). Genotype 1-like sequences were identified in 28.5% (n=43), and genotype 2-like sequences in 71.5% (n=108) of the analyzed tissues, respectively. Genotype 3 (prototype virus V9) was not identified in any of the EMBs. The 536 bp sequence genotype 1 showed 99% nucleotide identity to the J35 virus of genotype 1 containing 14 wobble-base positions (2 K, 2 M, 5 R, 1 S, 5 Y) (Figure 2, Table 3). The second 536 bp sequence 2 showed 100% nucleotide identity to the LaLi virus and only 1 mismatch to the HaAM virus of genotype 2 containing 25 wobble base positions (4 M, 3 R, 1 S, 1 V, 3 W, 13 Y). Both sequences differed in 18 of 536 nucleotide positions (3,2%).

Sequence alignment of the two nucleotide sequences Myocard 1 and 2 with previously published isolates showed highest similarity with Genebank accession numbers AY386330 (J35) ¹³ (genotype 1 ) and AY044266.1 (LaLi) and AY044268.1 (HaAM ¹⁴) (genotype 2). The 536 bp PCR products contained 25 nucleotide positions which allow discrimination of genotype 1 from genotype 2 (Table 3). In 26 additional nucleotide positions genotype 1 is identical to genotype 2 but differs from genotype 3 (Accession number AJ249437.1 , for virus V9).

The low genetic diversity of less than 2% over the whole genome sequence among the human erythroviruses allows distinction of different genotypes by analyzing only about 10% (536bp) of the genome. Moreover, the VP1/VP2 region shows lowest genetic variability (0-0.6%) within the whole genome. Thus, detection of Myocard 1 vs. Myocard 2 sequences corresponds to presence of genotypes 1 and 2, respectively in the endomyocardial tissues.

Comparison of amino acid sequences of capsid proteins VP1 and VP2

The capsid of human erythroviruses consists of 60 structural subunits 95% of which are the major viral protein VP2 (58 kDa). The other structural protein (VP1 ) differs from VP2 only in a N-terminal "unique region" composed of 227 additional amino acids predominantly located at the surface of the virion (Kaufmann et al., PNAS 2004;101 :11628-1 1633; Cotmore et al., J. Virol. 1986;60:548-557; Kawase et al., Virology 1995;211 :359-366). The 536 bp Moycard 1 and 2 sequences encode 89 amino acids residues of the VP1 and 69 amino acids of the VP2 protein. Within this sequence of 178 amino acids we found the two published amino acid exchanges between genotypes 1 and 2 at positions V192A and S259T (Table 4). In individual isolates of Myocard 1 and 2 an additional amino acid substitution was identified within the phospholipase A protein domain at position K18ON.

Table 4: Positions of amino acid exchanges: Analysis of wobble base combinations in the sequenced of the 536 bp long primer sequence (178 amino acids) identifies 8 sites for possible amino acid sequence exchanges for the myocard sequences in comparison with J35 and LaLi which were preferentially present in Myocard 2.

Quantification of virus load and exclusion of systemic erythrovirus infection

To assess whether the virus loads of the two erythroviruses genotypes differed in cardiac tissues, quantification of the erythroviral loads was performed by using TaqMan PCR assay. Mean viral load of tissue infected by both genotype 1 (451 ± 514 copies/μg genomic DNA, range: 35 - 5702) and genotype 2 (614 ± 2316 copies/μg genomic DNA, range: 46 -22.000) in cardiac tissue did not differ significantly (p=0.52).

In order to exclude a contamination of cardiac tissue by virus infected blood cells and to exclude recent systemic infection, isolated peripheral blood cells of all patients were analyzed by PCR. Erythrovirus-specific DNA was undetectable in all analyzed blood samples. In the serological typing all patients with identified myocardial erythrovirus genotypes 1 and 2 (PCR) were PVB19-lgG positive. Positive IgM was not detected in any of our patients with chronic disease, excluding recent erythrovirus infection.

Myocardial inflammation

By conventional histological analysis active myocarditis according to the Dallas classification was detected in EMB of virus-negativ and Erythrovirus -positive patients (Table 5). Borderline myocarditis was detected in EMB of virus-negativ and genotype 2 - positive but not in EMB of genotype 7-positive patients. These differences were not statistically significant (p=0.68). The three patient cohorts also presented with similar frequencies of myocardial fibrosis and myocyte hypertrophy in conventional stained histology, lmmunohistochemical analysis of myocardial inflammatory cells showed that numbers of infiltrating CD3-positive lymphocytes (11.6±8.1 vs. 15.1±27.3 cells/mm², p=0,41 ) and macrophages (37.9±26.9 vs. 34.7±24.6 cells/mm², 0,50) were comparable in genotype 1 and 2 positive patients. The subtype of perforin-positive effector T cells (2.1±3.2 vs. 1.1± 1.6, p<0.05) were, however, significantly increased in genotype 1 postitive individuals.

Age distribution and clinical presentation of patients with different erythrovirus genotypes:

Although erythroviral genomes were detected in patients of all age groups, genotype 1 was preferentially found in the younger patients below 45 years of age (n=28/40, 70%) (Figure 3). Above 45 years of age, genotype 2 became predominant (n=96/111 , 86.5%). In heart tissues of patients above 65 years of age, only virus genotype 2 was present. The mean age of patients with genotype 1 vs genotype 2 infections was 44.6 ± 8.7 years and 57.8 ± 10.1 years, respectively (p<0.0001 ). The mean age of genotype 1 (p<0.0001 ) and genotype 2 (p=0.02) differed from that of virus-negative patients (54.6 ± 11.8 years).

In spite of the younger age, LV-EF was reduced in erythrovirus genotype 1 -postitive patients as compared to erythrovirus genotype 2-positive patients (LV-EF: 24.4±10.4% vs. 31.0±9.5%, p=0.01 ), at least at the time point of our initial diagnostic analysis (Figure 4). Mean LV-EF of virus negative patients was 28.1±9.3%. In summary, EMBs of 151 (47.6%) patients were erythrovirus-positive. By using genotype- specific PCR primers encompassing a total of 553 bp from the VP1/VP2 region of the human erythrovirus family we identified two different genotype-specific sequences. Genotype 1 -specific sequences (prototype virus PVB19) were detected in 43/151 (28.5%) of positive biopsy samples, whereas genotype 2-specific sequences (prototype virus LaLi) so far considered rare in human diseases and not yet been described in human heart tissue was identified in 108/151 (71.5%) of virus-positive EMBs with a preference in patients above 50 years of age. Sequence analysis showed a mutation of possible clinical relevance in the phospholipase A2 like domaine of VP1. In spite of younger age, systolic left ventricular dysfunction of genotype 1 -positive patients was significantly reduced as compared to genotype 2-positive patients (LVEF: 24.4±10.4% vs 31.0±9.5%, p=0.0001 ) at the initial presentation.

Two genetically distinct erythrovirus-variants with a different age-distribution were detected in EMBs of DCM patients. The erythrovirus-genotype 2, previously not described in human heart tissue, was highly prevalent in the hearts but the less prevalent genotype 1 was associated with more severely disturbed cardiac function.

Example 2: Detection of Human Herpesvirus 6 in cardiac tissue

Selection of Patients

We prospectively studied 1622 patients who underwent a first EMB at Charite Berlin between June 2002 and June 2006. The patients presented with unexplained cardiac symptoms including fatigue, reduced physical capacity, angina, or dyspnea on exertion, arrythmias, abnormal electrocardiograms, enlargement of cardiac ventricles on echocardiographic examination, or regional or global left ventricular dysfunction of unknown cause. After angiographic exclusion of coronary artery disease requiring cardiovascular intervention and after exclusion of other severe concomitant diseases known to be associated with ventricular dysfunction such as hypertension, diabetes mellitus, obesity, valvular diseases, lung disease, renal dysfunction, all patients gave written informed consent for the biopsy-based analysis of the underlying cause of the disease.

Endomyocardial biopsy:

All patients underwent endomyocardial biopsy and right heart catheterization in a standardized manner.

At least three specimens were used for the evaluation of RNA or DNA viruses by nested PCR, respectively. DNA and RNA were extracted separately from frozen heart muscle tissue probes. Polymerase chain reaction (PCR)/reverse transcription PCR (RT-PCR) was performed for the detection of enteroviruses, adenoviruses, erythroviruses, human herpes virus type 6, human cytomegalovirus, Epstein-Barr virus, influenca Virus A and B, Chlamydia pneumonia, and herpes simplex virus 1 and 2. For exclusion of a systemic infection/reactivation with erythroviruses, EBV, and HHV6, DNA was extracted from peripheral blood cells (PBLs). Subtype specificity of HHV-6 PCR products was confirmed by automated DNA sequencing. Detected sequences were matched to the NCBI GenBank and compared with a recently described HHV-6 variant A and B genomes (GenBank accession No. X83413.1 and AF157706.1).

For comparison of qualitative nested-PCR and quantitative values, primers and probes for HHV-6 TaqMan QPCR were designed in the nested-PCR sequence fragment.

Detection of HHV6 Genomes in EMBs and blood cells by nested-PCR:

Genomic DNA from endomyocardial biopsies was extracted by Puregene Mousetail Kit (Gentra, Minneapolis, USA). For exclusion of systemic virus infection peripheral blood cells were isolated from 0.5 ml peripheral blood after lysis of erythrocytes.

Total RNA was extracted from endomyocardial biopsies or peripheral blood cells by Trizol B method. The whole extracted RNAs (20μl) were reverse transcribed in cDNA by application of High Capacity cDNA Archive kit (Applied Biosystems, Foster City, USA)

Detection of HHV6 DNA or cDNA by nested PCR (nPCR) was performed with primers specific for the U94 coding sequence. HHV6-specific primers used for PCR resulted in a PCR amplicon of 431 bp and, in the second round of PCR, in a 367-bp amplicon (Table 6). Specificity of PCR products was confirmed by automated DNA sequencing. Detected sequences were matched to the NCBI GenBank and compared with a recently described HHV6 variant A and B genomes (GenBank accession No. X83413.1 and AF157706.1 ). Concentrations of isolated genomic human DNA. amount were quantitatively measured by Quantifiler Human DNA quantification kit (Applied Biosystems, Foster City, USA) according to manufacturers instructions. As a control for successful extraction of RNA and for normalization of gene expression (de Kok) the sequences of house-keeping gene hypoxanthine ribosyltransferase (HPRT) were simultaneously amplified from each cDNA sample by use of corresponding, pre-designed TaqMan gene expression assay (Applied Biosystems, Foster City, USA). All primers and the TaqMan fluorescent probe TU94S for detection of HHV6 DNA were obtained from TIB MOLBIOL (Berlin, Germany).

Table 6: PCR Primers

TaqMan QPCR for HHV6 Genomes

For comparison of qualitative nested-PCR and quantitative values, primers and probes for HHV6 TaqMan QPCR were designed in the nested-PCR sequence fragment. Primers and the probe TU94S were selected according to general rules of primer and probe design for real-time PCR by PrimerExpress software (Applied Biosystems, Darmstadt, Germany). The primer set chosen amplifies a 95 bp long fragment in the U94 gene region of HHV6 (). Sequences of used primers and probe are listed in Table 6. The amplicon-specific probe TU94S was labeled with the fluorescent reporter dye FAM (6-carboxy-fluorescein) at the 5 end and the blackhole DABcyl ((4-(4'-dimethylaminophenylazo)benzoic acid) dye at the 3^' end.

The TaqMan-PCR was carried out in a 96-well microtiter plate format (Perkin-Elmer, USA). The PCR mix was made up to a volume of 25 μl using ready-to use Universal Mastermix containing AmpliTaq DNA polymerase, uracil-N-glycosylase (UNG), dNTPs, KCI, MgCI2 and ROX as a passive reference all in optimized concentrations (Applied Biosystems, USA). Applied concentrations per assay of forward and reverse primers and the fluorescence-labelled probe were 0.2 μM and 4μl patient sample DNA or cDNA. After UNG treatment at 50 °C for 2 min and initial denaturation at 95 °C for 10 min, the DNA was amplified by 38 two-step cycles (15 s at 95 ⁰C, 1 min at 57 ⁰C). The amplification was followed on the ABI Prism HT7900 Sequence Detection System (Applied Biosystems,

USA). Reactions and cycling were performed as recommended by the manufacturer' s instructions (ABI Prism 7900HT, Applied Biosystems). Serial dilutions of HHV6 plasmid DNA standard (3.28 to 3.28x10⁴ genomes copies per assay, Genexpress, Berlin, Germany) were simultaneously coamplified to generate standard curve. Subsequent calculation of viral load was performed by ratio of estimated viral genome copy number in TaqMan assay to amount of incorporated human DNA.

DNA Sequence analysis

DNA fragments overlapping the U94 region nucleotide sequences (367 or 431 bp) of HHV6 genome were amplified by nested PCR using primer pairs indicated in Table 6. Nested-PCR products were digested for primer and dNTPs elimination by ExoSAP (USB, Cleveland, USA). Sequencing reaction fragments were generated by 30 three-step cycles (20 s at 96 ⁰C, 20 sec 55°C, 2 min at 60⁰C) using CEQ Quick kit (Beckman-Coulter, Germany) for cycle sequencing and U94C or U94D as corresponding sequencing primer.

Single stranded sequencing products were purified by CleanSeq magnetic beads purification kit (Agentcourt, Beverly, USA). Nucleotide sequences were obtained with a 8- capillary sequencing system CEQ 8000 (Beckman-Coulter, Krefeld) All obtained sequences were aligned individually against NCBI database BLAST. Alignment for determination of the HHV6 genotyp was performed by manual editing using Phylogenetic Data Editor (PHYDE) Software (Institute for Botanies, Dresden/Bonn, Germany). Results:

A total of 1622 consecutive baseline and 458 follow-up EMB were analyzed by histological, immunohistological and molecular biological methods for the presence of signs of myocardial inflammation and/or viral genomes. In the baseline biopsies we identified 231 patients (14.2%) (age: 48.6±13.7 years, 130 men (54.4%), LV-EF: 52.3±18.8%, history 23.6±45.2 months) with positive nested PCR (nPCR) results indicating HHV-6 infection or reactivation. In EMBs of 87 patients (37.7%) HHV-6 specific genomes were detected, exclusively. Multiple infections with additional RNA or DNA sequences specific for enteroviruses (EV, n=13), adenoviruses (ADV, n=3), Epstein Barr virus (EBV, n=5), Chlamydia pneumonia (n=1), hepatitis virus C (HCV, n=1 ), or Parvovirus B19 (PVB19, n=139), respectively were detected in the remaining 144 EMBs.

Systemic HHV-6 infection or reactivation:

Isolated peripheral blood cells (PBL) were available from 223 HHV6-positive patients (96.5%) and 115 blood samples of patients without myocardial virus infection. Isolated peripheral blood cells (PBL) were analyzed for systemic viral infection or reactivation. 162 of the 223 analyzed blood samples Of HHV-6 positive patients (72.7%) did not contain any virus-specific RNA or DNA. Virus-specific DNA was detected in PBL of 64 of these patients (28.7%). Simultaneous detection of both systemic and myocardial HHV-6 reactivity by nPCR was found in 39 samples (17.5%). In these patients a contamination of EMB tissues by HHV6-positive blood cells cannot be excluded. In 8 of the HHV6-positive blood samples other viral DNA was present (EBV: 6 patients, PVB19: 2 patients). Single systemic infection with EBV and PVB19 were found in 19 (8.5%) and 2 (0.9%) analyzed samples. Systemic HHV-6 and EBV reactivations were found in 6% and 15.7% of patients without detected viral genomes in EMB, respectively. PBL of other virus positive patients were routinely analyzed only for those viruses which were detected in the EMB specimens and are therefore not reported here.

Prevalence of HHV-6 variants A and B:

Sequence analysis of 77 consecutive HHV-6-positive patients was routinely carried out since January 2005. The prevalence of HHV6-positive patients within these 18 months was 15.7% and thereby comparable to the general prevalence of HHV-6 reactivation (14.7%) in the whole cohort of 1622 patients. Sequence analysis identified HHV-6 variant A in 7 EMB tissues (9.1%) while HHV-6 type B was present in the remaining 70 EMB specimens (90.9%). Systemic HHV-6 genomes were found in 5 patients (71.4%) with HHV-6A infection/reactivation and 12 patients (19%) in whom HHV-6B was identified. The median copy numbers of HHV-6 B in EMB was below the detection limit of 10 copies/μg isolated myocardial DNA (range <10 to 171357) and 28717 copies/μg isolated myocardial (range <10 to 100677) DNA for type A (p=0.002). Corresponding values of isolated peripheral blood cells were <10 copies/μg isolated myocardial DNA (range <10 to 166575 copies) for HHV-6 B and 118806 copies/μg isolated myocardial DNA (range <10 to 177200) for type A (p=0.245, respectively.

Spontaneous course of HHV-6 variants:

92 patients underwent a follow-up biopsy after a mean interval of 7 months including 14 patients who initially had been HHV-6 negative at the time of the baseline analysis. These patients underwent a follow-up biopsy for follow-up of other viral infections (e.g. PVB19 or EV) or evaluation of the cause for progression of unexplained heart failure. 35 other patients with a history longer than 12 months were included in different treatment trials and therefore were lost for the analysis of the spontaneous course of HHV-6 reactivation. HHV- 6 reactivation persisted in 35 patients up to several months and resolved in 57 individuals. During this short follow-up period both newly acquired and persisting HHV-6 reactivation were associated with progression of left ventricular dysfunction, in spite of constant heart failure medication. LV-EF decreased from 58.2±16.0% to 51.8±17.2% (p<0.001 ) while left ventricular diameters mildly increased. In contrast, LV-EF improved from 54.9±15.4% to 60.7±13.1% in those 57 patients in whom HHV-6 genomes were undetectable at the follow-up biopsy (p<0.001 ). This hemodynamic improvement was associated with a significant decrease of left ventricular endsystolic but not enddiastolic diameters. Progression or improvement of LV-EF dysfunction did also occur in patients with multiple viral infections. The HHV-6 associated hemodynamic changes in patients with PVB19/HHV6 double infections in which the second virus persists indicates that the HHV-6 reactivation rather than other viruses, e.g. PVB19, contributes to the spontaneous course of the disease of these patients, and that HHV-6 influences myocardial function.

Example 3: Multiplex-PCR and Fragment analysis

Analyzed viral genomes: PVB19, HHV6 Material required:

The specific primer sets for fragment analysis was calculated by eXpress Profiler software (Beckman-Coulter). Multiplex primer sets were designed for the following viruses:

For PVB19 (15 Plex) 15 forward and reverse primer each (15 fragments) For HHV6 (36 Plex) 36 forward und reverse primer each (36 fragments) The final concentration of each primer in the multiplex assay is 200 nmol/l.

The PCR reaction was conducted with the selected primer combination according to standard procedure.

Thermal Cycler Programme for PCR multiplex reaction:

Pre-Dilution for analysis on Gene Sequenzer CEQ8000 (Beckman-Coulter) was performed, if required, by diluting the PCR reaction 1 :5 with TE-buffer pH 8,0 (2μl Template + 8μl TE) before preparation of the master mix for analysis on gene sequencer Sample- buffer plate Setup and fragment analysis of fluorescent dye labelled PCR product were performed on a CEQ 8000 8-capillary sequencer by fragment analysis tool of the corresponding genetic analysis software (Beckman-Coulter).

Example 4: treatment of patients with Ribavirin and Interferon alpha

A female patient testing positive for PVB19 genotype 1 was treated with 180 microgram PEGINF alpha-2a (pegylated interferon alpha) plus 400 - 600 mg Ribavirin (combination therapy) daily over the course of several weeks. The patient developed clinical signs of a myocarditis with angina pectoris, wall oedema and tissue loosening in the echogram. The examination by magnetic resonance tomography (MRT) showed a significant improvement with a decrease of the extent and multifocal distribution of contrasting agent accumulations. The pericardial effusion was no longer detectable. The data obtained in MRT and echocardiogram reveal that the patient responded positively to interferon-alpha and Ribavirin treatment. The triggering of an inflammation response in PVB19-positive patients is not achieved by interferon beta treatment, resulting in insufficient elimination of the virus. Interferon alpha treatment, however, does achieve such elimination of the virus. See Figure 5 for comparison.

Example 5: Differential cytokine analysis as prognostic parameters for spontaneous course of different viral infections

Differential cytokine analysis by ELISA/Multiplex ELISA recognizes virus-specific cytokine profiles (see Figures) which demonstrate a close correlation with the spontaneous course of the virus infections. According to one embodiment of the present invention, increased endogenous interferon-β levels are associated with spontaneous enterovirus clearance while low interferon-β levels were associated with enterovirus persistence. Similar prognostic differential cytokine profiles exist for PVB19 and/or HHV6 (example Figure xx) and for the different genotypes of these viruses. The combination of cytokine profiles together with the molecular biological virus analyses (as described above) allows a more sensitive and specific decision for patient management and the outlined specific antiviral treatment options.

Further embodiments are described in the appendices, which are part of the description of the present invention.

_{* * * * *}

Claims

1. Polynucleotide sequence comprising at least one of the nucleotide sequences listed consecutively from SEQ ID NO 147 to SEQ ID NO 157.

2. Amino acid sequence comprising at least one of the amino acid sequences listed consecutively from SEQ ID NO 001 to SEQ ID NO 032.

3. Antibody binding to an amino acid sequence comprising at least one of the amino acid sequences according to claim 2.

4. Method for the detection of a viral infection of a patient, comprising the detection in a tissue sample or blood sample, outside of the human or animal body, the presence or absence of

- a nucleic acid sequence comprising at least one polynucleotide sequence according to claim 1 , and / or

- an amino acid sequence comprising at least one amino acid sequence according to claim 2, and / or

- an antibody according to claim 3.

5. Method according to claim 4, characterized in that the patient is a human patient.

6. Method according to claim 4 or 5, characterized in that the sample is heart tissue.

7. Method for the detection of a viral infection of a patient comprising the detection in a blood vessel or heart tissue sample, outside of the human body, the presence or absence of

- Parvovirus B19 genotype 1. Parvovirus B19 genotype 2, or

- Human Herpesvirus 6 or

- Parvovirus B19 and Human Herpesvirus 6.

8. Method according to claim 7, characterized in that the Human Herpesvirus is Human Herpesvirus variant A or B.

9. Method according to at least one of claim 4 to 8, characterized in that the detection is performed by polymerase chain reaction (PCR).

10. Method according to at least one of claim 4 to 9, characterized in that the detection is performed by nested polymerase chain reaction.

11. Method according to at least one of claim 4 to 10, characterized in that the detection is performed by quantitative polymerase chain reaction.

12. Method according to claim 11 , characterized in that the reaction comprises at least one kind of polynucleotide primer molecule comprising a sequence identical to one of the sequences listed consecutively from SEQ ID NO 036 to SEQ ID NO 041.

13. Method according to claim 12, characterized in that the reaction additionally comprises at least one kind of polynucleotide primer molecule comprising a sequence identical to one of the sequences listed consecutively from SEQ ID NO 033 to SEQ ID NO 035 or from SEQ ID NO 042 to SEQ ID NO 044.

14. Method according to at least one of claim 4 to 11 , characterized in that the detection is performed by multiplex polymerase chain reaction.

15. Method according to at least one of claim 4 to 14, characterized in that the detection is performed employing at least 4 kinds of primer molecules comprising each a different sequence, said sequence being identical to one of the sequences listed consecutively from SEQ ID NO 045 to SEQ ID NO 146.

16. Method according to at least one of claim 4 to 8, characterized in that the detection is performed by Enzyme-linked-immunosorbent Assay (ELISA).

17. Method according to at least one of claim 4 to 16, characterized in that the cytokine level in the sample of the cytokine interferon alpha, interferon beta, interferon gamma, interleukine 2, interleukine 4, interleukine 6, interleukine 8, interleukinelO, interleukine 12, interleukine 13, interleukine 5, interleukine 7, interleukine 17, GM- CSF, MCP-1 or MIP-I b or any combination of these cytokines is determined.

18. Device for the detection of a nucleic acid in a sample, comprising two or more isolated polynucleotide sequences attached to a surface, which under stringent conditions hybridize to one or more polynucleotides according to claim 1 or to a Human Herpesvirus 6 polynucleotide sequence or to both a Parvovirus B19 and to a Human Herpesvirus 6 polynucleotide sequence.

19. Device for the detection of an antibody in a sample, comprising at least one of the isolated amino acid sequences of claim 2 attached to a surface.

20. Device according to claim 19, characterized in that the device further comprises an isolated amino acid sequence of Human Herpesvirus 6 or an isolated amino acid sequence of Parvovirus B19 or isolated amino acid sequences of both Human Herpesvirus 6 and Parvovirus B19.

21. The use of an isolated polynucleotide sequence according to claim 1 in the preparation of a pharmaceutical composition for the prevention or treatment of a disease.

22. The use of an isolated amino acid sequence according to claim 2 in the preparation of a pharmaceutical composition for the prevention or treatment of a disease.

23. The use according to claim 21 or 22 for the prevention or treatment of myocardial infection.

24. The use of Interferon alpha in the preparation of a pharmaceutical composition for the prevention or treatment of myocardial infection by Parvovirus B19 or Human Herpesvirus 6.

25. The use of Interferon alpha in the preparation of a pharmaceutical composition according to claim 24, characterized in that the interferon alpha is pegylated interferon alpha 2a.

26. The use of Interferon alpha in the preparation of a pharmaceutical composition according to claim 24 or 25, characterized in that the pharmaceutical composition contains ribavirin.

27. The use of Interferon alpha in the preparation of a pharmaceutical composition according to claim 24 or 26, characterized in that the applied dose of interferon alpha is 400 microgram to 1200 microgram per 75 kg patient body weight.