Elina Väisänen

Parvovirus B19 Infection in Fetal Deaths

BACKGROUND Parvovirus B19 infection during pregnancy can lead to nonimmune fetal hydrops, miscarriage, and intrauterine fetal death (IUFD). Some studies have suggested that parvovirus B19 infection may surprisingly often result in nonhydropic fetal death during the third trimester, in the absence of maternal serological evidence of acute infection. This study was conducted to investigate the prevalence of parvovirus B19 DNA among fetuses from miscarriages and IUFDs. METHODS We retrospectively studied 535 unborn fetuses, including 120 fetuses from miscarriages and 169 from IUFDs. The control fetuses were 246 fetuses from induced abortions. All fetuses were autopsied from July 1992 through December 1995 and from January 2003 through December 2005 in Helsinki, Finland. The period included a major epidemic of parvovirus B19 infection in 1993. Formalin-fixed, paraffin-embedded fetal tissues were studied with use of a highly sensitive and specific PCR that was capable of detecting all 3 parvovirus B19 genotypes and by histologic examination. In addition, maternal parvovirus B19 serological status was determined. RESULTS Parvovirus B19 DNA was detected in 5 fetuses with gestational ages of 14, 22, 23, 30, and 39 weeks; these included fetuses from 4 (2.4%) of the 169 IUFDs and 1 (0.8%) of the 120 miscarriages. During the epidemic year 1993, the prevalence of parvovirus B19 DNA-positive fetal deaths was 6 times the prevalence during nonepidemic years. All 5 mothers of the parvovirus B19 DNA-positive fetuses had serological signs of acute parvovirus B19 infection close to the time of fetal death. The only nonhydropic fetus was full-term. CONCLUSIONS Our findings indicate that the prevalence of parvovirus B19 infection among fetuses from IUFDs is low. In particular, our findings did not verify the claimed high prevalence of third-trimester nonhydropic IUFDs associated with parvovirus B19.

Bufavirus in feces of patients with gastroenteritis, Finland.

To the Editor: For nearly 3 decades, human parvovirus B19 (B19V) was considered to be the only pathogenic parvovirus found in humans. Since 2005, several new human parvoviruses have been found, including human bocaviruses (HBoV1–4) and human parvovirus 4 (PARV4) (1–5), and during 2012, metagenomic analysis of fecal samples from children in Burkina Faso with acute diarrhea showed a highly divergent parvovirus, which was named bufavirus (BuV) (6). Its sequence in the coding region showed <31% similarity with known parvoviruses, the closest genera being Protoparvovirus and Amdoparvovirus. Subsequent studies, on the basis of PCR results, showed that 4% of fecal samples from Burkina Faso (n = 98) and 1.6% from Tunisia (n = 63) harbored either of 2 genotypes of this new virus, which belongs to the species Primate protoparvovirus 1 of the genus Protoparvovirus (6,7; http://ictvonline.org). To assess the occurrence of BuV in northern Europe, we analyzed 629 fecal samples from patients of all ages (median 51.5 years, range 0–99) in Finland who had gastroenteritis. To gain a more complete representation of BuV occurrence, we obtained samples retrospectively from routine diagnostics for bacterial and viral gastroenteritis-inducing pathogens (HUSLAB, Helsinki University Central Hospital Laboratory Division, Helsinki, Finland) and analyzed all samples available during the collection periods. The samples originally sent to HUSLAB for bacterial diagnosis (bacterial cohort, n = 243) had been analyzed during October 2012–March 2013 for Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., Vibrio cholerae, and Escherichia coli (subtypes enterohemorraghica, enteropatogena, enterotoxigenic and enteroagregativa) by using culture or PCR (8). In 81 (33.3%) of the samples, >1 bacterial pathogen was found. The samples originally sent for viral diagnosis (viral cohort, n = 386) had been tested in HUSLAB for norovirus during April–May, 2013 by using reverse transcription quantitative PCE (RT-qPCR)(HUSLAB in-house). Further diagnosis for rotavirus and adenovirus had been requested by physicians from 105 (27.2%) of 386 samples (Diarlex MB antigen detection assay, Orion Diagnostica, Espoo, Finland), and for astrovirus from 33 (8.6%) samples (RT-PCR, HUSLAB in-house). A viral pathogen was discovered in 141 (36.5%) samples; in 139, the pathogen was norovirus. The samples had been sent from diverse locations within Finland, and thus were not from a few isolated outbreaks. No further information on patients and samples was available for either cohort, and not enough samples were left for retrospective analysis of additional pathogens. The Ethics Committee of the Hospital District of Helsinki and Uusimaa approved the study. BuV DNA was detected by using a new real-time qPCR with the following primers and probe: BuV forward, 5 ′-ACAGTGTAGACAGTGGATTCAAACTT-3 ′; BuV reverse, 5 ′-GTTGTGGTTGGATTGTGGTTAGTTC-3 ′; BuV qPCR probe, 5 ′-FAM-CGGAAGAGATTTTGACAGTGCYTAGCAA-BHQ1–3 ′. The detailed qPCR protocol is shown in the Technical Appendix. The analytical sensitivity of the RT-qPCR assay was 5–10 copies per reaction. Of the 629 fecal samples, 7 (1.1%) were positive for BuV DNA, of which 4 were from the bacterial cohort and 3 from the viral cohort. BuV DNA quantity was low in all samples, ranging from 1.9 × 103 to 3.2 × 104 copies per milliliter of fecal supernatant (Table). In contrast to the original discovery of the virus in children with diarrhea (6), all positive samples were from adults (median age 53 years, range 21–89 years). All BuV DNA–positive results were confirmed by repeated BuV qPCR, by amplifying and sequencing another area of the virus, or by both methods (Table): all sequenced amplicons were more similar to the BuV genotype 1 (Technical Appendix Figure) (6). Two of the BuV-positive samples were from the same patient, taken 4 days apart, and the latter sample also harbored norovirus. The additional 6 BuV-positive samples were negative for the other viral or bacterial pathogens tested. Table Samples collected for bacterial and viral testing that were subsequently positive for bufavirus DNA* Seven fecal samples collected from adults in Finland contained BuV DNA, indicating that circulation of the virus is restricted neither to children nor to Africa. However, the low DNA loads in all the positive samples suggest that BuV might not be the primary cause of these cases of gastroenteritis. A known gastroenteritis-inducing pathogen (norovirus) was found in 1 of the 7 BuV-positive samples. We did not observe any clustering of the 7 positive samples into a specific period (Table). Although the association with gastroenteritis seems weak, BuV might cause symptoms of other types. We did not include feces from healthy subjects for comparison. The identified BuV DNA in our samples could originate from previous or current infections unrelated to gastroenteritis, or be associated with prolonged virus secretion in the respiratory or digestive tracts, a phenomenon shown, e.g., for (9,10). Acquisition of the virus from a food source cannot be ruled out, although 1 patient harbored the DNA for at least 4 days, during which a 10-fold increase in viral load was observed. Overall, this study shows that BuV circulates in northern Europe and can be found in the feces of patients with gastroenteritis. Despite the absence of known pathogens among 6 of 7 BuVs-shedding patients, the causative role of BuV in gastroenteritis remains uncertain. Serologic studies will help clarify a possible association between BuVs and diarrhea or other diseases. Technical Appendix: Bufavirus quantitative PCR and phylogenetic analylsis. Click here to view.(202K, pdf)

Newly discovered KI, WU, and Merkel cell polyomaviruses: No evidence of mother-to-fetus transmission

BackgroundThree* human polyomaviruses have been discovered recently, KIPyV, WUPyV and MCPyV. These viruses appear to circulate ubiquitously; however, their clinical significance beyond Merkel cell carcinoma is almost completely unknown. In particular, nothing is known about their preponderance in vertical transmission. The aim of this study was to investigate the frequency of fetal infections by these viruses. We sought the three by PCR, and MCPyV also by real-time quantitative PCR (qPCR), from 535 fetal autopsy samples (heart, liver, placenta) from intrauterine fetal deaths (IUFDs) (N = 169), miscarriages (120) or induced abortions (246). We also measured the MCPyV IgG antibodies in the corresponding maternal sera (N = 462) mostly from the first trimester.ResultsNo sample showed KIPyV or WUPyV DNA. Interestingly, one placenta was reproducibly PCR positive for MCPyV. Among the 462 corresponding pregnant women, 212 (45.9%) were MCPyV IgG seropositive.ConclusionsOur data suggest that none of the three emerging polyomaviruses often cause miscarriages or IUFDs, nor are they transmitted to fetuses. Yet, more than half the expectant mothers were susceptible to infection by the MCPyV.

Antigenic diversity and seroprevalences of Torque teno viruses in children and adults by ORF2-based immunoassays.

Torque teno viruses (TTVs) circulate widely among humans, causing persistent viraemia in healthy individuals. Numerous TTV isolates with high genetic variability have been identified and segregated into 29 species of five major phylogenetic groups. To date, the diversity of TTV sequences, challenges in protein expression and the subsequent lack of serological assays have hampered TTV seroprevalence studies. Moreover, the antigenic relationships of different TTVs and their specific seroprevalences in humans remain unknown. For five TTV strains--belonging to different species of four genogroups--we developed, using recombinant glutathione S-transferase (GST)-fused TTV ORF2 proteins, glutathione-GST capture enzyme immunoassays (EIAs) detecting antibodies towards conformational epitopes. We then analysed serum samples from 178 healthy adults and 108 children; IgG reactivities were observed either towards a single strain or towards multiple strains, which pointed to antigenic distinction of TTV species. The overall seroprevalence for the five TTVs peaked at 43 % (18 of 42) in children 2-4 years of age, subsequently declined, and again reached 42 % (74 of 178) among adults. TTV6 species-specific IgG predominated in children, whereas that for TTV13 predominated in adults. During a 3 year follow-up of the same children, both species-specific seroconversions and seroreversions occurred. This is the first EIA-based study of different TTVs, providing a new approach for seroepidemiology and diagnosis of TTV infections. Our data suggest that different TTVs in humans may differ in antiviral antibody profiles, infection patterns and epidemiology.

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.

Absence of human bocavirus from deceased fetuses and their mothers

BACKGROUND The human bocavirus (HBoV), a newly discovered parvovirus, is closely related to the bovine parvovirus and the canine minute virus, which are known to cause adverse pregnancy outcomes. Another human parvovirus, B19, can lead to fetal hydrops, miscarriage and intrauterine fetal death (IUFD). OBJECTIVES To determine the prevalence of HBoV DNA in aborted fetuses and IUFDs. The HBoV serology of the mothers was also studied. STUDY DESIGN We retrospectively studied all available fetuses (N=535) autopsied during 7/1992-12/1995, and 1/2003-12/2005 in Helsinki, Finland. All available formalin-fixed paraffin-embedded fetal tissues - placenta, heart and liver - of 120 miscarriages, 169 IUFDs, and 246 induced abortions were studied by quantitative PCR. We also measured the HBoV IgM and IgG antibodies in the corresponding maternal sera (N=462) mostly of the first trimester. The IgM-positive sera underwent HBoV PCR. RESULTS None of the fetal tissues harbored HBoV DNA. A total of 97% (448/462) of the mothers were positive for IgG antibodies to HBoV, while only 0.9% (4/462) exhibited HBoV-specific IgM antibodies without viremia or respiratory symptoms. One IgM-positive mother had an unexplained fetal loss. CONCLUSIONS We did not find HBoV DNA in any of the deceased fetuses. Almost all pregnant women were HBoV-IgG positive.

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.

A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes.

Epidemiology of two human protoparvoviruses, bufavirus and tusavirus

Two human parvoviruses were recently discovered by metagenomics in Africa, bufavirus (BuV) in 2012 and tusavirus (TuV) in 2014. These viruses have been studied exclusively by PCR in stool and detected only in patients with diarrhoea, although at low prevalence. Three genotypes of BuV have been identified. We detected, by in-house EIA, BuV1-3 IgG antibodies in 7/228 children (3.1%) and 10/180 adults (5.6%), whereas TuV IgG was found in one child (0.4%). All children and 91% of the adults were Finnish, yet interestingly 3/6 adults of Indian origin were BuV-IgG positive. By competition EIA, no cross-reactivity between the BuVs was detected, indicating that the BuV genotypes represent distinct serotypes. Furthermore, we analysed by BuV qPCR stool and nasal swab samples from 955 children with gastroenteritis, respiratory illness, or both, and found BuV DNA in three stools (0.3%) and for the first time in a nasal swab (0.1%). This is the first study documenting the presence of BuV and TuV antibodies in humans. Although the seroprevalences of both viruses were low in Finland, our results indicate that BuV infections might be widespread in Asia. The BuV-specific humoral immune responses appeared to be strong and long-lasting, pointing to systemic infection in humans.

Global Distribution of Human Protoparvoviruses

Development of next-generation sequencing and metagenomics has revolutionized detection of novel viruses. Among these viruses are 3 human protoparvoviruses: bufavirus, tusavirus, and cutavirus. These viruses have been detected in feces of children with diarrhea. In addition, cutavirus has been detected in skin biopsy specimens of cutaneous T-cell lymphoma patients in France and in 1 melanoma patient in Denmark. We studied seroprevalences of IgG against bufavirus, tusavirus, and cutavirus in various populations (n = 840), and found a striking geographic difference in prevalence of bufavirus IgG. Although prevalence was low in adult populations in Finland (1.9%) and the United States (3.6%), bufavirus IgG was highly prevalent in populations in Iraq (84.8%), Iran (56.1%), and Kenya (72.3%). Conversely, cutavirus IgG showed evenly low prevalences (0%–5.6%) in all cohorts, and tusavirus IgG was not detected. These results provide new insights on the global distribution and endemic areas of protoparvoviruses.