Päivi Norja

Prevalence of Parvovirus B19 and Human Bocavirus DNA in the Heart of Patients with no Evidence of Dilated Cardiomyopathy or Myocarditis

BACKGROUND Although the DNA of parvovirus B19 (B19V) is frequently detected in patients with dilated cardiomyopathy or myocarditis, whether the parvovirus causes disease is questionable, since even in healthy individuals the virus persists in various tissues. The same question applies to human bocavirus (HBoV). We have determined the prevalence and quantity of B19V and HBoV DNA in heart tissue of patients who were not experiencing virus-related heart diseases and analyzed whether the seroprevalence corresponded to DNA prevalence in the heart. METHODS Samples of left-atrium heart tissue and serum were obtained from 100 patients who underwent open-heart surgery. Serum immunoglobulin (Ig) G and IgM against proteins encoded by B19V and HBoV were detected by enzyme-linked immunoabsorption assay and immunoblotting. B19V and HBoV DNA concentrations were determined by quantitative real-time polymerase chain reaction (PCR) in heart tissue and serum samples. Nested PCRs for VP1, K71, and GT3 identified the B19V genotypes. RESULTS The prevalences of serum IgG specific for B19V and HBoV were 85% and 96%, respectively. Of all the patients, 85% had B19V DNA detected in heart tissues, and 4% displayed low-level B19V viremia, whereas only 5% of heart tissue samples and none of the serum samples demonstrated HBoV DNA. The sensitivity of B19V serological testing for B19V DNA in heart samples was 0.96 (95% confidence interval, 0.92-1.0). Specificity was 0.8 (95% confidence interval, 0.6-1.0), and the positive predictive value was 0.96 (95% confidence interval, 0.92-1.0). B19V genotypes 1 and 2 were present in 11% and 89% of heart tissues samples, respectively. B19V genotype 3 was not detected in any of the samples. CONCLUSIONS Our data suggest that B19V but not HBoV demonstrates a lifelong persistence in the heart. The detection of B19V DNA in heart tissue showed no correlation with clinical symptoms. We strongly recommend that serological testing become a standardized procedure for future studies, to obtain representative data concerning the prevalence of B19V in the heart.

Detection and Differentiation of Human Parvovirus Variants by Commercial Quantitative Real-Time PCR Tests

ABSTRACT Parvovirus B19 causes a variety of diseases in humans, with outcomes ranging from asymptomatic to severe, such as chronic anemia in immunocompromised patients or fetal hydrops and death after maternal infection during pregnancy. The virus may be transmitted via plasma-derived products. According to the results of solvent-detergent safety studies, an upper limit of B19 DNA in plasma pools was recently defined. To restrict the input of B19 virus into production pools, a quantitative nucleic acid test is a prerequisite. We examined the suitability of the two commercial quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus) for detection, quantification, and differentiation of the three known B19 genotypes, including the newly described erythrovirus variants (genotypes 2 and 3). The former kit was highly sensitive for genotype 1 but was not suitable for detection of genotype 2 or one of two genotype 3 strains. The latter kit detected and differentiated all three genotypes, albeit with lower sensitivity for one of the genotype-3 strains. We furthermore assessed the prevalence of the three B19 virus genotypes in blood donors, by screening pooled plasma samples derived from 140,160 Finnish blood-donor units. None of the pools contained detectable levels of B19 virus genotypes 2 or 3. The origin, mode of transmission, and clinical significance of these genotypes are unknown and deserve further study. The RealArt Parvo B19 LC PCR is suitable for detection, quantification, and differentiation of all three B19 virus genotypes in molecular and clinical research.

Biological and Immunological Relations among Human Parvovirus B19 Genotypes 1 to 3

ABSTRACT The human parvovirus B19 is now divided into three genotypes: type 1 (prototype), type 2 (A6- and LaLi-like), and type 3 (V9-like). In overall DNA sequence, the three virus types differ by ∼10%. The most striking DNA dissimilarity, of >20%, is observed within the p6 promoter region. Because of the scarcity of data on the biological activities and pathogenetic potentials of virus types 2 and 3, we examined the functional characteristics of these virus types. We found the activities of the three p6 promoters to be of equal strength and to be most active in B19-permissive cells. Virus type 2 capsid protein VP2, alone or together with VP1, was expressed with the baculovirus system and was shown to assemble into icosahedral parvovirus-like particles, which were reactive in the hemagglutination assay. Furthermore, sera containing DNA of any of the three B19 types were shown to hemagglutinate. The infectivities of these sera were examined in two B19-permissive cell lines. Reverse transcription-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS and VP proteins in the infected cells. All three genotypes showed similar functional characteristics in all experiments performed, showing that the three virus types indeed belong to the same species, i.e., human parvovirus B19. Additionally, the antibody activity in sera from B19 type 1- or type 2-infected subjects (long-term immunity) was examined with homo- and heterologous virus-like particles. Cross-reactivity of 100% was observed, indicating that the two B19 genotypes comprise a single serotype.

Parvovirus transmission by blood products – a cause for concern?

The introduction of dual viral inactivation of clotting factor concentrates has practically eliminated infections by viruses associated with significant pathogenicity over the last 20 years. Despite this, theoretical concerns about transmission of infection have remained, as it is known that currently available viral inactivation methods are unable to eliminate parvovirus B19 or prions from these products. Recently, concern has been raised following the identification of the new parvoviruses, human parvovirus 4 (PARV4) and new genotypes of parvovirus B19, in blood products. Parvoviruses do not cause chronic pathogenicity similar to human immunodeficiency virus or hepatitis C virus, but nevertheless may cause clinical manifestations, especially in immunosuppressed patients. Manufacturers should institute measures, such as minipool polymerase chain reaction testing, to ensure that their products contain no known viruses. So far, human bocavirus, another new genus of parvovirus, has not been detected in fractionated blood products, and unless their presence can be demonstrated, routine testing during manufacture is not essential. Continued surveillance of the patients and of the safety of blood products remains an important ongoing issue.

Human parvoviruses B19, PARV4 and bocavirus in pediatric patients with allogeneic hematopoietic SCT

Among the immunocompetent, infections with parvovirus B19 (B19V) and human bocavirus (HBoV) 1 range clinically from asymptomatic to severe, while following allogeneic hematopoietic SCT (HSCT) B19V can cause a persistent severe illness. The epidemiology and clinical impact of HBoV1 and the other emerging parvovirus 4 (PARV4) among immunocompromised patients have not been established. To determine the occurrence and clinical spectrum of B19V, PARV4 and HBoV1 infections, we performed a longitudinal molecular surveillance among 53 allogeneic HSCT recipients for pre- and post-HSCT DNAemias of these parvoviruses. Quantitative real-time PCR showed B19V DNA in sera of 16 (30%) patients, at mean levels of 4.6 × 103, 9.9 × 107, 1.1 × 1010 and 1.6 × 102 B19V DNA copies/mL pre-HSCT (9/53), and at 1 (6/53), 2 (4/53) and 3 months (1/25) post HSCT, respectively. However, no clinical manifestation correlated with the presence of B19V viremia. All B19V sequences were of genotype 1. None of the sera investigated contained PARV4 or HBoV1 DNAs. Our data demonstrate B19V viremia to be frequent among pediatric allogeneic HSCT recipients, yet without apparent clinical correlates. PARV4 or HBoV1 viremias were not seen in these immunocompromised patients.

Occurrence of human bocaviruses and parvovirus 4 in solid tissues

Human bocaviruses 1‐4 (HBoV1‐4) and parvovirus 4 (PARV4) are recently discovered human parvoviruses. HBoV1 is associated with respiratory infections of young children, while HBoV2‐4 are enteric viruses. The clinical manifestations of PARV4 remain unknown. The objective of this study was to determine whether the DNAs of HBoV1‐4 and PARV4 persist in human tissues long after primary infection. Biopsies of tonsillar tissue, skin, and synovia were examined for HBoV1‐4 DNA and PARV4 DNA by PCR. Serum samples from the tissue donors were assayed for HBoV1 and PARV4 IgG and IgM antibodies. To obtain species‐specific seroprevalences for HBoV1 and for HBoV2/3 combined, the sera were analyzed after virus‐like particle (VLP) competition. While HBoV1 DNA was detected exclusively in the tonsillar tissues of 16/438 individuals (3.7%), all of them ≤8 years of age. HBoV2‐4 and PARV4 DNAs were absent from all tissue types. HBoV1 IgG seroprevalence was 94.9%. No subject had HBoV1 or PARV4 IgM, nor did they have PARV4 IgG. The results indicate that HBoV1 DNA occurred in a small proportion of tonsils of young children after recent primary HBoV1 infection, but did not persist long in the other tissue types studied, unlike parvovirus B19 DNA. The results obtained by the PARV4 assays are in line with previous results on PARV4 epidemiology. J. Med. Virol. 84: 1267–1273, 2012.

Expression and serological characterization of polyomavirus WUPyV and KIPyV structural proteins.

The polyomaviruses WUPyV and KIPyV were recently discovered. We expressed their structural proteins VP1, VP2, and VP3, and the corresponding proteins of BKV and JCV, for immunoblotting of IgG antibodies from 115 wheezing young children and 25 asymptomatic adults. Furthermore, nasopharyngeal aspirates (NPA) and sera from the children were examined by PCR for viral DNA. The overlapping minor proteins VP2 and VP3 of WUPyV and KIPyV were more reactive in immunoblots than the major protein VP1; of 100 NPA PCR-negative wheezing children aged < or = 4 y, 31 (31%) and 31 (31%) were positive for WUPyV and KIPyV VP2/VP3, compared to only 3 (3%) and 5 (5%) for VP1, respectively. For comparison, the respective WUPyV and KIPyV IgG seroprevalences as determined by immunofluorescence assay (IFA) with nondenatured VP1 were 80% and 54%, respectively, among 50 NPA PCR-negative children aged < or = 2 y. This difference shows the importance of conformational VP1 antigenicity. Of the 25 adults, 52% and 68% were IgG-positive in immunoblots for VP2/VP3 of WUPyV and KIPyV, and 8% and 12% were for VP1, respectively. Of the 192 NPA samples studied by PCR, 7 (3.6%) were positive for WUPyV, and 3 (1.5%) were positive for KIPyV DNA. Unlike the NPA samples, none of the corresponding 443 sera contained WUPyV or KIPyV DNA. Together with the high VP2/VP3 IgG prevalence, this points to a paucity or brevity of KIPyV and WUPyV viremias among immunocompetent children. Our results indicate the significance of protein conformation in immunoreactivity of VP1, and show the antigenic importance of the WUPyV and KIPyV minor proteins VP2 and VP3. The high and rapidly increasing IgG prevalence rates observed in this study for WUPyV and KIPyV support the notion that these novel polyomaviruses are widespread and are acquired early in childhood.

Tissue persistence and prevalence of B19 virus types 1–3

Human parvovirus B19 is a minute ssDNA virus that causes a wide variety of diseases, including erythema infectiosum, arthropathy, anemias and fetal death. In addition to the B19 prototype, two new variants (B19 types 2 and 3) have been identified. After primary infection, B19 genomic DNA has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. The viral DNA persists as an intact molecule without persistence-specific mutations, and via a storage mechanism with life-long capacity. Thus, the mere presence of B19 DNA in tissue cannot be used as a diagnostic criterion, although a possible role in the pathology of diseases, for example through mRNA or protein production, cannot be excluded. The molecular mechanism, host-cell type and possible clinical significance of tissue persistence are yet to be elucidated.

A new quantitative PCR for human parvovirus B19 genotypes.

Parvovirus B19 (B19V) is a minute ssDNA virus associated with a wide range of diseases from childhood erythema to fetal death. After primary infection, the viral genomes persist lifelong in solid tissues of most types. Quantification of the viral DNA is important in the timing of primary infection, assessment of tissue persistence and screening of blood donor plasma. In this study, we present a new PCR assay for detection and quantification as well as for differentiation of all three B19V genotypes. A new B19V qPCR was designed to target a 154-bp region of the NS1 area. Serum, plasma and solid tissue samples were suitable for testing in the assay. The WHO International Reference Panel for Parvovirus B19 Genotypes was utilized to validate the assay for detection of different genotypes of B19V in clinical material. Each panel member yielded, by the new qPCR, a quantity similar to the one reported by National Institute for Biological Standards and Control (NIBSC). The qPCR was specific for B19V and amplified and quantified all three genotypes with detection sensitivities of ≤10 copies/reaction. The differentiation of B19V genotypes was performed by Sanger sequencing of the amplified products.

Low Prevalence of Parvovirus 4 in HIV-infected Children in Denmark.

Parvovirus 4 (PARV4) has been associated with HIV infection in adults. We examined plasma samples from 46 HIV-infected 0-year-old to 16-year-old children for the presence of PARV4. Four children (8.7%) had detectable PARV4 IgG and 1 had IgM. The result of PARV4 polymerase chain reaction was found to be negative in all patients. PARV4 seropositivity was associated with low CD4 count but not with HIV viral load.