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.

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.

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.

Rapid sequence change and geographical spread of human parvovirus B19: comparison of B19 virus evolution in acute and persistent infections.

Parvovirus B19 is a common human pathogen maintained by horizontal transmission between acutely infected individuals. However, B19 virus can also be detected in tissues throughout the life of the host, although little is understood about the nature of such persistence. In the current study, we created large VP1/2 sequence data sets of plasma- and tissue (autopsy)-derived variants of B19 virus with known sample dates to compare the rates of sequence change in exogenous virus populations with those in persistently infected individuals. By using linear regression and likelihood-based methods (such as the BEAST program), we found that plasma-derived B19 virus showed a substitution rate of 4 x 10(-4) and an unconstrained (synonymous)-substitution rate of 18 x 10(-4) per site per year, several times higher than previously estimated and within the range of values for mammalian RNA viruses. The underlying high mutation frequency implied by these substitution rates may enable rapid adaptive changes that are more commonly ascribed to RNA virus populations. These revised estimates predict that the last common ancestor for currently circulating genotype 1 variants of B19 virus existed around 1956 to 1959, fitting well with previous analyses of the B19 virus "bioportfolio" that support a complete cessation of genotype 2 infections and their replacement by genotype 1 infections in the 1960s. In contrast, the evolution of B19 virus amplified from tissue samples was best modeled by using estimated dates of primary infection rather than sample dates, consistent with slow or absent sequence change during persistence. Determining what epidemiological or biological factors led to such a complete and geographically extensive population replacement over this short period is central to further understanding the nature of parvovirus evolution.

Viremic co-infections in children with allogeneic haematopoietic stem cell transplantation are predominated by human polyomaviruses.

BACKGROUND: Viral infections remain the cause of key complications following haematopoietic stem cell transplantation (HSCT). The impact of multiple, concurrent viral reactivations/infections remains to be delineated. METHODS: The clinical correlates of single or multiple viremic infections following HSCT and especially the occurrence of respiratory viruses in the bloodstream were investigated. We retrospectively searched for 23 viruses in a total of 184 sera from 53 paediatric patients. The time-points of interest were pre-HSCT, one, two and three months post-HSCT, and at discharge or death. The viruses were analyzed by quantitative or qualitative PCR. RESULTS: Of the 53 patients, 13 (25%) had viraemias by multiple viruses and 27 (51%) by a single virus. Thirteen patients (25%) had no viruses detected by PCR during the study period. In the children with viremic co-infections, polyomaviruses predominated over herpes viruses. Nearly half the patients, 24/53 (45%) had a polyomavirus in their serum at one or more time-points. At 12/15 time-points and in 11/13 patients with co-infections polyomaviruses were involved, compared with 6/15 time-points and 6/13 patients for cytomegalovirus. Acute graft-versus-host disease (GvHD) and steroid use were significant risk factors for the viraemias caused by more than one virus. CONCLUSIONS: Viral co-detection is a common finding in children undergoing HSCT. With large-scale viral screening also viruses other than CMV could be found as potential pathogens. In this study, BKPyV predominated over CMV as a contributor in viraemias caused by multiple viruses in children receiving HSCT.

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.

Absence of novel human parvovirus (PARV4) in Danish mothers and children.

BACKGROUND: The recently discovered human parvovirus 4 (PARV4) is found most frequently in injection drug users, HIV-positive patients, and in haemophiliacs. Studies from Ghana report the finding of PARV4 in plasma from 2 to 12% of children without acute infection, and in nasal secretions and faecal samples. Studies of PARV4 in children from industrialized countries are few. OBJECTIVES: We aimed to describe the occurrence of PARV4 in a population-based birth cohort of 228 Danish mothers and their healthy children who previously participated in a study of respiratory tract infections in infancy. STUDY DESIGN: Children were included over a whole calendar year and were monitored through monthly home visits through the first year of life. Plasma samples for the present study were available from 228 mothers, 176 newborns, and 202 12-months-old children. All samples were analysed for the presence of PARV4 antibodies by enzyme immunoassay, and samples with detectable antibodies were in addition studied by real-time PCR. RESULTS: One (0.4%) of 228 mothers had PARV4 IgG exceeding the cut-off absorbance level and another had borderline IgG reactivity. No mother among these two had an acute infection, as they were IgM and PARV4 DNA negative. All blood samples from newborns and one-year-old children had IgG and IgM reactivity below cut-off. CONCLUSIONS: PARV4 is rare in Danish mothers and infants. Further studies are needed, in both rural and urban settings, to investigate the epidemiology and clinical significance of this novel human parvovirus.

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.

Reactivation and mutation of newly discovered WU, KI, and Merkel cell carcinoma polyomaviruses in immunosuppressed individuals.

BACKGROUND: Infection with the human polyomaviruses BK (BKV) and JC (JCV) is almost ubiquitous, asymptomatic, and lifelong. However, reactivation during immunosuppression, associated with mutations in the transcriptional control region (TCR) that up-regulates viral replication, can cause life-threatening disease. In this study, we investigated whether the recently discovered WU and KI polyomaviruses (WUPyV and KIPyV) and Merkel cell polyomavirus (MCPyV) could, like BKV and JCV, persist, mutate, and reactivate in immunodeficient subjects. METHODS: Autopsy samples of lymphoid tissue from 42 AIDS-immunosuppressed subjects and 55 control samples were screened by polymerase chain reaction for all 5 polyomaviruses. TCR sequences from KIPyV and WUPyV recovered from both immunosuppressed and nonimmunosuppressed subjects were compared. RESULTS: Combined polyomavirus detection frequencies were much higher for the immunosuppressed group, compared with the nonimmunosuppressed group (35.7% vs. 3.6%), with viral loads in lymphoid tissues ranging from < or = 8.4 x 10(5) to > 1.5 x 10(5) viral genome copies per 10(6) cells. MCPyV was recovered from only 1 HIV-negative study subject. TCR sequences from reactivated WUPyV and KIPyV variants showed a number of point mutations and insertions that were absent in viruses recovered from respiratory tract specimens obtained from nonimmunosuppressed subjects. CONCLUSIONS: KIPyV and WUPyV show reactivation frequencies comparable to those of BKV and JCV during immunosuppression. TCR changes that potentially lead to transcriptional dysregulation may have pathogenic consequences equivalent in severity to those observed for JCV and BKV.

Biological and immunological relations among human parvovirus B19 genotypes 1 to 3.

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 approximately 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.

Bioportfolio: lifelong persistence of variant and prototypic erythrovirus DNA genomes in human tissue.

Human erythrovirus is a minute, single-stranded DNA virus causing many diseases, including erythema infectiosum, arthropathy, and fetal death. After primary infection, the viral genomes persist in solid tissues. Besides the prototype, virus type 1, two major variants (virus types 2 and 3) have been identified recently, the clinical significance and epidemiology of which are mostly unknown. We examined 523 samples of skin, synovium, tonsil, or liver (birth year range, 1913-2000), and 1,640 sera, by qualitative and quantitative molecular assays for the DNA of human erythroviruses. Virus types 1 and 2 were found in 132 (25%) and 58 (11%) tissues, respectively. DNA of virus type 1 was found in all age groups, whereas that of type 2 was strictly confined to those subjects born before 1973 (P < 0.001). Correspondingly, the sera from the past two decades contained DNA of type 1 but not type 2 or 3. Our data suggest strongly that the newly identified human erythrovirus type 2 as well as the prototype 1 circulated in Northern and Central Europe in equal frequency, more than half a century ago, whereafter type 2 disappeared from circulation. Type 3 never attained wide occurrence in this area during the past > or =70 years. The erythrovirus DNA persistence in human tissues is lifelong and represents a source of information about our past, the Bioportfolio, which, at the individual level, provides a registry of one's infectious encounters, and at the population level, a database for epidemiological and phylogenetic analyses.

Detection and differentiation of human parvovirus variants by commercial quantitative real-time PCR tests.

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.