Mohammadreza Sadeghi

Merkel cell polyomavirus DNA in tumor-free tonsillar tissues and upper respiratory tract samples: Implications for respiratory transmission and latency

Abstract Background Merkel cell polyomavirus (MCPyV) was discovered recently. It is considered a potential causative agent of Merkel cell carcinoma, a life-threatening skin cancer. Objectives To study the prevalence of MCPyV in a large number of clinical samples of various types. Most of the samples were examined also for the other newly found polyomaviruses KI (KIPyV) and WU (WUPyV). Study design Altogether 1390 samples from immunocompetent or immunocompromised patients, including (i) tonsillar tissues and sera from tonsillectomy patients; (ii) nasopharyngeal aspirates (NPAs) and sera from wheezing children and (iii) nasal swabs, sera and stools from febrile leukemic children were studied for MCPyV. The tonsils, nasal swabs and stools were also studied for KIPyV and WUPyV. Results MCPyV DNA was detected in 14 samples altogether; 8 of 229 (3.5%) tonsillar tissues, 3 of 140 (2.1%) NPAs, 2 of 106 (1.9%) nasal swabs and 1 of 840 (0.1%) sera. WUPyV and KIPyV were detected in 5 (2.2%) and 0 tonsils, 1 (0.9%) and 4 (3.8%) nasal swabs and 0 and 2 (2.7%) fecal samples, respectively. The patients carrying in tonsils MCPyV were of significantly higher age (median 42years) than those carrying WUPyV (4years, p <0.001). Conclusions MCPyV DNA occurs in tonsils more frequently in adults than in children. By contrast, WUPyV DNA is found preferentially in children. MCPyV occurs also in nasal swabs and NPAs, in a frequency similar to that of KIPyV and WUPyV. The tonsil may be an initial site of WUPyV infection and a site of MCPyV persistence.

Detection of TS polyomavirus DNA in tonsillar tissues of children and adults: evidence for site of viral latency.

BACKGROUND The trichodysplasia spinulosa-associated polyomavirus (TSPyV), a recently discovered species of the family Polyomaviridae, is associated with development of trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised individuals. The viral seroprevalence in the general population is ∼70%, with little known of its route of transmission, latency, or primary infection site. OBJECTIVES We aimed to determine whether the viral DNA is detectable in tonsillar tissue of constitutionally healthy individuals, and what the corresponding antiviral seroreactivities are. STUDY DESIGN We tested 229 matched pairs of tonsillar tissue biopsies and serum samples from asymptomatic donors for TSPyV DNA by real-time quantitative PCR with primer pairs and Taq-Man probes targeting the VP1 and LT genes. The sera were studied by enzyme immunoassay (EIA) for TSPyV-VP1-IgG and the PCR-positive individuals also for -IgM and -IgG-avidity. RESULTS TSPyV DNA was detectable in 8 (3.5%) of 229 tonsillar tissues, and in none of the corresponding sera. TSPyV IgG seroprevalence among children was 39% and among adults 70%. Each of the 8 PCR-positive subjects had antiviral IgG of high avidity but not IgM. CONCLUSIONS TSPyV PCR positivity of tonsillar samples of individuals with long-term immunity provides the first evidence of TSPyV in tonsils and suggests lymphoid tissue as a latency site of this emerging human pathogen.

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.

Possible Involvement of Human Bocavirus 1 in the Death of a Middle-Aged Immunosuppressed Patient

ABSTRACT An immunosuppressed 61-year-old man went to the hospital with fever, nonproductive cough, and increasing shortness of breath. The subject died 8 days later of respiratory complications. PCR of respiratory samples as well as a blood sample revealed exceptionally high DNA levels of the emerging pathogen, human bocavirus 1 (HBoV1), a recently found pathogen associated with respiratory symptoms in young children. We describe the clinical progression of the case and discuss the potential role of HBoV1 in the outcome.

Trichodysplasia spinulosa-Associated Polyomavirus (TSV) and Merkel Cell Polyomavirus: Correlation between Humoral and Cellular Immunity Stronger with TSV

Merkel Cell Polyomavirus (MCV) is a common infectious agent likely to be involved in the pathogenesis of most Merkel cell carcinomas (MCC). Trichodysplasia spinulosa-associated polyomavirus (TSV), which exhibit high seroprevalence in general population, has been detected in trichodysplasia spinulosa (TS) skin lesions suggesting an etiological role for this disease. Previous studies have shown strong MCV-specific T-cell responses, while no data exist on T-cell immunity against TSV. In order to characterize Th-cell immunity against TSV, and to allow comparisons with the MCV-specific Th-cell immunity, we studied TSV-specific proliferation, IFN-γ, IL-10 and IL-13, and MCV-specific IFN-γ and IL-10 responses in 51 healthy volunteers, and in one MCC patient. Recombinant TSV and MCV VP1 virus-like particles (VLPs) were used as antigens. A significant correlation was found between virus-specific Th-cell and antibody responses with TSV; with MCV it proved weaker. Despite significant homology in amino acid sequences, Th-cell crossreactivity was not evident between these viruses. Some subjects seronegative to both TSV and MCV exhibited Th-cell responses to both viruses. The agent initially priming these Th-cells remains an enigma. As CD8+ cells specific to MCV T-Ag oncoprotein clearly provide an important defense against established MCC, the MCV VP1-specific Th-cells may, by suppressing MCV replication with antiviral cytokines such as IFN-γ, significantly contribute to preventing the full process of oncogenesis.

B-Cell Responses to Human Bocaviruses 1-4: New Insights from a Childhood Follow-Up Study.

Human bocaviruses (HBoVs) 1–4 are recently discovered, antigenically similar parvoviruses. We examined the hypothesis that the antigenic similarity of these viruses could give rise to clinically and diagnostically important immunological interactions. IgG and IgM EIAs as well as qPCR were used to study ~2000 sera collected from infancy to early adolescence at 3–6-month intervals from 109 children whose symptoms were recorded. We found that HBoV1-4-specific seroprevalences at age 6 years were 80%, 48%, 10%, and 0%, respectively. HBoV1 infections resulted in significantly weaker IgG responses among children who had pre-existing HBoV2 IgG, and vice versa. Furthermore, we documented a complete absence of virus type-specific immune responses in six viremic children who had pre-existing IgG for another bocavirus, indicating that not all HBoV infections can be diagnosed serologically. Our results strongly indicate that interactions between consecutive HBoV infections affect HBoV immunity via a phenomenon called “original antigenic sin”, cross-protection, or both; however, without evident clinical consequences but with important ramifications for the serodiagnosis of HBoV infections. Serological data is likely to underestimate human exposure to these viruses.

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.

Diagnostic Methods for and Clinical Pictures of Polyomavirus Primary Infections in Children, Finland

We used comprehensive serodiagnostic methods (IgM, IgG, and IgG avidity) and PCR to study Merkel cell polyomavirus and trichodysplasia spinulosa-associated polyomavirus infections in children observed from infancy to adolescence. Comparing seroconversion intervals with previous and subsequent intervals, we found that primary infections with these 2 viruses were asymptomatic in childhood.

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

Abstract 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.

Multiplex detection in tonsillar tissue of all known human polyomaviruses

BackgroundIn the past few years, eleven new human viruses have joined the two previously known members JCPyV and BKPyV of the Polyomaviridae family, by virtue of molecular methods. Serology data suggest that infections with human polyomaviruses (HPyVs) occur since childhood and the viruses are widespread in the general population. However, the viral persistence sites and transmission routes are by and large unknown. Our previous studies demonstrated that the four new HPyVs – KIPyV, WUPyV, MCPyV and TSPyV – were present in the tonsils, and suggested lymphoid tissue as a persistent site of these emerging human viruses.We developed a Luminex-based multiplex assay for simultaneous detection of all 13 HPyVs known, and explored their occurrence in tonsillar tissues of children and adults mostly with tonsillitis or tonsillar hypertrophy.MethodsWe set up and validated a new Luminex-based multiplex assay by using primer pairs and probes targeting the respective HPyV viral protein 1 (VP1) genes. With this assay we tested 78 tonsillar tissues for DNAs of 13 HPyVs.ResultsThe multiplex assay allowed for simultaneous detection of 13 HPyVs with high analytical sensitivity and specificity, with detection limits of 100–102 copies per microliter, and identified correctly all 13 target sequences with no cross reactions. HPyV DNA altogether was found in 14 (17.9%) of 78 tonsils. The most prevalent HPyVs were HPyV6 (7.7%), TSPyV (3.8%) and WUPyV (3.8%). Mixed infection of two HPyVs occurred in one sample.ConclusionsThe Luminex-based HPyV multiplex assay appears highly suitable for clinical diagnostic purposes and large-scale epidemiological studies. Additional evidence was acquired that the lymphoid system plays a role in HPyV infection and persistence. Thereby, shedding from this site during reactivation might take part in transmission of the newly found HPyVs.