Christiane Prifert

High prevalence of antibodies against polyomavirus WU, polyomavirus KI, and human bocavirus in German blood donors

BackgroundDNA of the polyomaviruses WU (WUPyV) and KI (KIPyV) and of human bocavirus (HBoV) has been detected with varying frequency in respiratory tract samples of children. However, only little is known about the humoral immune response against these viruses. Our aim was to establish virus-specific serological assays and to determine the prevalence of immunoglobulin G (IgG) against these three viruses in the general population.MethodsThe capsid proteins VP1 of WUPyV and KIPyV and VP2 of HBoV were cloned into baculovirus vectors and expressed in Sf9 insect cells. IgG antibodies against WUPyV VP1, KIPyV VP1, and HBoV VP2 were determined by immunofluorescence assays in 100 plasma samples of blood donors.ResultsThe median age of the blood donors was 31 years (range 20 - 66 yrs), 52% were male. 89% of the samples were positive for WUPyV IgG (median age 31 yrs, 49.4% male), 67% were positive for KIPyV IgG (median age 32 yrs, 46.3% male), and 76% were positive for HBoV IgG (median age 32 yrs, 51.3% male). For WUPyV and HBoV, there were no significant differences of the seropositivity rates with respect to age groups or gender. For KIPyV, the seropositivity rate increased significantly from 59% in the age group 20 - 29 years to 100% in the age group > 50 years.ConclusionsHigh prevalences of antibodies against WUPyV, KIPyV, and HBoV were found in plasma samples of healthy adults. The results indicate that primary infection with these viruses occurs during childhood or youth. For KIPyV, the seropositivity appears to increase further during adulthood.

Novel Respiratory Syncytial Virus (RSV) Genotype ON1 Predominates in Germany during Winter Season 2012-13

Respiratory syncytial virus (RSV) is the leading cause of hospitalization especially in young children with respiratory tract infections (RTI). Patterns of circulating RSV genotypes can provide a better understanding of the molecular epidemiology of RSV infection. We retrospectively analyzed the genetic diversity of RSV infection in hospitalized children with acute RTI admitted to University Hospital Heidelberg/Germany between October 2012 and April 2013. Nasopharyngeal aspirates (NPA) were routinely obtained in 240 children younger than 2 years of age who presented with clinical symptoms of upper or lower RTI. We analyzed NPAs via PCR and sequence analysis of the second variable region of the RSV G gene coding for the attachment glycoprotein. We obtained medical records reviewing routine clinical data. RSV was detected in 134/240 children. In RSV-positive patients the most common diagnosis was bronchitis/bronchiolitis (75.4%). The mean duration of hospitalization was longer in RSV-positive compared to RSV-negative patients (3.5 vs. 5.1 days; p<0.01). RSV-A was detected in 82.1%, RSV-B in 17.9% of all samples. Phylogenetic analysis of 112 isolates revealed that the majority of RSV-A strains (65%) belonged to the novel ON1 genotype containing a 72-nucleotide duplication. However, genotype ON1 was not associated with a more severe course of illness when taking basic clinical/laboratory parameters into account. Molecular characterization of RSV confirms the co-circulation of multiple genotypes of subtype RSV-A and RSV-B. The duplication in the G gene of genotype ON1 might have an effect on the rapid spread of this emerging RSV strain.

Novel Respiratory Syncytial Virus A Genotype, Germany, 2011–2012

To the Editor: Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and elderly persons. RSV strains have been divided into 2 major antigenic groups (A and B), which are further divided into several genotypes. The main genetic and antigenic differences between genotypes are found within the 2 hypervariable regions of the attachment (G) glycoprotein. In 1999, a novel RSV B genotype, which contained a 60-nt duplication in the second hypervariable region of the G protein, was discovered in Buenos Aires, Argentina, and named BA (1). Since then, genotype BA has almost completely replaced other RSV B strains worldwide and has diversified into several subgenotypes (2). In February 2012, as part of routine RSV surveillance, we identified a novel RSV A genotype with a 72-nt duplication in the second hypervariable region of G, thus representing the first RSV A genotype with nucleotide duplications in the G gene. Shortly thereafter, circulation of this genotype was reported in Ontario, Canada, in 2010–11 and 2011–12, and the genotype was named ON1 (3). To investigate the frequency of genotype ON1 in Germany, we extended the molecular analysis of RSV strains from the previous 2 RSV seasons. The study was approved by the ethics committee of the medical faculty at the University of Wurzburg, Germany. From July 2010 through June 2011 and from July 2011 through June 2012, we identified 271 and 181 RSV-positive patients, respectively. Patients were identified from respiratory specimens sent by hospitals in Bavaria, Germany, for routine testing of respiratory viruses at the Institute of Virology and Immunobiology at the University of Wurzburg. The mean age of all patients was 1.2 years (median 8.2 years; range 0.03–81.4 years), and 259 were male. Of the RSV-positive samples, 183 (67.5%) from season 2010–11 and 171 (94.5%) from season 2011–12 were analyzed by sequencing a fragment of ≈500 nt that encompassed the complete second hypervariable region of the G gene (4). Alignment with reference sequences and phylogenetic analyses were conducted by using MEGA 5.0 (5). Molecular analysis of RSV-positive samples revealed that RSV A and B cocirculated during both seasons (98 A and 85 B during 2010–11; 99 A and 73 B during 2011–12). In accordance with previous reports (2), all RSV B strains from both seasons were identified as genotype BA. The novel RSV A genotype ON1 was not detected during 2010–11. However, 10 (10.1%) of 99 RSV A strains were assigned to genotype ON1 during 2011–12. All other RSV A strains of both seasons belonged to genotype GA2. An amino acid alignment of ON1 sequences is shown in the Figure. The duplication regions of 2 of the 10 ON1 strains contained 2-aa and 3-aa exchanges compared with the ON1 reference sequence (which has no exchanges) (3). Of note, an ON1 sequence from Japan with similar, partially even identical mutations was retrieved from GenBank (Figure). All mutations observed so far did not affect potential O-glycosylation sites. Figure Amino acid sequence alignment of the second hypervariable region of the respiratory syncytial virus (RSV) G gene. RSV ON1 sequences and RSV A reference sequences in the upper part of the alignment are designated by GenBank numbers. ON1 sequences in the ... Of the 99 patients with RSV A infection diagnosed during 2011–12, a total of 91 were hospitalized children. Genotype ON1 was identified in 7 (25.0%) of 28 children in intensive care units (ICUs) and in 2 (3.2%) of 63 children in other wards (p = 0.003, Fisher exact test). Children admitted to an ICU were younger (median 0.2 years) than those not in an ICU (median age 1.2 years; p<0.001, Mann-Whitney U test). An exploratory logistic regression analysis on ICU admittance, adjusted for age, confirmed a strong association between RSV genotype ON1 and ICU admittance (adjusted odds ratio 8.4; 95% CI 1.5%–46.6%; p = 0.015). However, this significant difference should be interpreted with caution for 2 reasons: 1) samples from patients in wards other than an ICU originated mainly in the Wurzburg area, whereas samples from patients in ICUs were received from pediatric hospitals in various regions of Bavaria; 2) clinical information on patients not in ICUs was not available for assessment of whether the difference persisted when taking into account other risk factors for severe RSV disease. In summary, the novel RSV A genotype ON1 containing a 72-nt duplication in the G gene was not found during 2010–11, but it constituted already 10.1% of all RSV A strains in a patient cohort from Bavaria, Germany, in the next season, 2011–12. In the context of the primary report of ON1 in Ontario, Canada (3), and the GenBank entry from Japan, our data suggest worldwide emergence of ON1. The almost complete worldwide replacement of circulating RSV B genotypes with the BA strain containing a comparable 60-nt duplication, which began in 1999, suggests that these duplications provide a selective advantage (2). Thus, molecular analysis of circulating RSV strains should be continued to determine whether ON1 has the potential to replace other RSV A strains in the years to come as did RSV B genotype BA during the past decade.

Molecular Characterization of a Respiratory Syncytial Virus Outbreak in a Hematology Unit in Heidelberg, Germany

ABSTRACT In 2011 and 2012, a large outbreak of respiratory syncytial virus (RSV) infections affecting 57 laboratory-confirmed patients occurred in an adult hematology unit in Heidelberg, Germany. During the outbreak investigation, we performed molecular genotyping of RSV strains to differentiate between single versus multiple introductions of the virus into the unit. Furthermore, we assessed the time of viral shedding of consecutive samples from the patients in order to better understand the possible impact of prolonged shedding for outbreak control management. We used subtype-specific reverse transcription-PCR on nasopharyngeal and bronchoalveolar specimens for routine diagnostics and for measuring the viral shedding period. Samples of 47 RSV-infected patients involved in the outbreak were genotyped by sequence analysis and compared to samples from RSV-infected hospitalized children representing the timing of the annual RSV epidemic in the community. Molecular investigation of the virus strains from clinical samples revealed a unique cluster with identical nucleotide sequences of RSV type A (RSV A outbreak strain) for 41 patients, while 3 patients were infected with different RSV A (nonoutbreak) strains and three other patients with RSV type B. Outbreak strains were identified in samples from November 2011 until January 2012, while nonoutbreak strains were from samples coinciding with the community epidemic in February and March 2012. Median duration of viral shedding time was 24.5 days (range, 1 to 168 days) with no difference between outbreak and nonoutbreak strains (P = 0.45). Our investigation suggests a single introduction of the RSV A outbreak strain into the unit that spread among the immunocompromised patients. Prolonged viral shedding may have contributed to nosocomial transmission and should be taken into account in the infection control management of RSV outbreaks in settings with heavily immunosuppressed patients.

Long-Term Shedding of Influenza Virus, Parainfluenza Virus, Respiratory Syncytial Virus and Nosocomial Epidemiology in Patients with Hematological Disorders.

Respiratory viruses are a cause of upper respiratory tract infections (URTI), but can be associated with severe lower respiratory tract infections (LRTI) in immunocompromised patients. The objective of this study was to investigate the genetic variability of influenza virus, parainfluenza virus and respiratory syncytial virus (RSV) and the duration of viral shedding in hematological patients. Nasopharyngeal swabs from hematological patients were screened for influenza, parainfluenza and RSV on admission as well as on development of respiratory symptoms. Consecutive swabs were collected until viral clearance. Out of 672 tested patients, a total of 111 patients (17%) were infected with one of the investigated viral agents: 40 with influenza, 13 with parainfluenza and 64 with RSV; six patients had influenza/RSV or parainfluenza/RSV co-infections. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic). LRTI was observed in 48 patients, of whom 15 patients developed severe LRTI, and 13 patients with respiratory tract infection died. Phylogenetic analysis revealed a variety of influenza A(H1N1)pdm09, A(H3N2), influenza B, parainfluenza 3 and RSV A, B viruses. RSV A was detected in 54 patients, RSV B in ten patients. The newly emerging RSV A genotype ON1 predominated in the study cohort and was found in 48 (75%) of 64 RSV-infected patients. Furthermore, two distinct clusters were detected for RSV A genotype ON1, identical RSV G gene sequences in these patients are consistent with nosocomial transmission. Long-term viral shedding for more than 30 days was significantly associated with prior allogeneic transplantation (p = 0.01) and was most pronounced in patients with RSV infection (n = 16) with a median duration of viral shedding for 80 days (range 35–334 days). Long-term shedding of respiratory viruses might be a catalyzer of nosocomial transmission and must be considered for efficient infection control in immunocompromised patients.

The innate antiviral factor APOBEC3G targets replication of measles, mumps and respiratory syncytial viruses

The cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme-catalytic polypeptide 3G; A3G) exerts antiviral activity against retroviruses, hepatitis B virus, adeno-associated virus and transposable elements. We assessed whether the negative-strand RNA viruses measles, mumps and respiratory syncytial might be affected by A3G, and found that their infectivity was reduced by 1-2 logs (90-99 %) in A3G overexpressing Vero cells, and in T-cell lines expressing A3G at physiological levels. Viral RNA was co-precipitated with HA-tagged A3G and could be amplified by RT-PCR. Interestingly, A3G reduced viral transcription and protein expression in infected cells by 50-70 %, and caused an increased mutation frequency of 0.95 mutations per 1000 nt in comparison to the background level of 0.22/1000. The observed mutations were not specific for A3G [cytidine to uridine (C→U) or guanine to adenine (G→A) hypermutations], nor specific for ADAR (adenosine deaminase acting on RNA, A→G and U→C transitions, with preference for next neighbour-nucleotides U = A>C>G). In addition, A3G mutants with inactivated catalytic deaminase (H257R and E259Q) were inhibitory, indicating that the deaminase activity is not required for the observed antiviral activity. In combination, impaired transcription and increased mutation frequencies are sufficient to cause the observed reduction in viral infectivity and eliminate virus replication within a few passages in A3G-expressing cells.

Detection of enterovirus D68 in patients hospitalised in three tertiary university hospitals in Germany, 2013 to 2014

Enterovirus D68 (EV-D68) has been recognised as a worldwide emerging pathogen associated with severe respiratory symptoms since 2009. We here report EV-D68 detection in hospitalised patients with acute respiratory infection admitted to three tertiary hospitals in Germany between January 2013 and December 2014. From a total of 14,838 respiratory samples obtained during the study period, 246 (1.7%) tested enterovirus-positive and, among these, 39 (15.9%) were identified as EV-D68. Infection was observed in children and teenagers (0-19 years; n=31), the majority (n=22) being under five years-old, as well as in adults > 50 years of age (n=8). No significant difference in prevalence was observed between the 2013 and 2014 seasons. Phylogenetic analyses based on viral protein 1 (VP1) sequences showed co-circulation of different EV-D68 lineages in Germany. Sequence data encompassing the entire capsid region of the genome were analysed to gain information on amino acid changes possibly relevant for immunogenicity and revealed mutations in two recently described pleconaril binding sites.