Barbara Biere

Shedding and Transmission of Novel Influenza Virus A/H1N1 Infection in Households—Germany, 2009

Essential epidemiologic and virologic parameters must be measured to provide evidence for policy/public health recommendations and mathematical modeling concerning novel influenza A/H1N1 virus (NIV) infections. Therefore, from April through August of 2009, the authors collected nasopharyngeal specimens and information on antiviral medication and symptoms from households with NIV infection on a daily basis in Germany. Specimens were analyzed quantitatively by using reverse transcriptase-polymerase chain reaction. In 36 households with 83 household contacts, 15 household contacts became laboratory-confirmed secondary cases of NIV. Among 47 contacts without antiviral prophylaxis, 12 became cases (secondary attack rate of 26%), and 1 (8%) of these was asymptomatic. The mean and median serial interval were 2.6 and 3 days, respectively (range: 1-3 days). On average, the authors detected viral RNA copies for 6.6 illness days (treated in time = 5.7 days, not treated in time = 7.1 days; P = 0.06), but they estimated that most patients cease to excrete viable virus by the fifth illness day. Shedding profiles were consistent with the number and severity of symptoms. Compared with other nasopharyngeal specimen types, nasal wash was the most sensitive. These results support the notion that epidemiologic and virologic characteristics of NIV are in many aspects similar to those of seasonal influenza.

Differentiation of Influenza B Virus Lineages Yamagata and Victoria by Real-Time PCR

ABSTRACT Since the 1970s, influenza B viruses have diverged into two antigenically distinct virus lineages called the Yamagata and Victoria lineages. We present the first real-time PCR assay for virus lineage differentiation to supplement classical antigenic analyses. The assay was successfully applied to 310 primary samples collected in Germany from 2007 to 2009.

Diagnostic Approach for the Differentiation of the Pandemic Influenza A(H1N1)v Virus from Recent Human Influenza Viruses by Real-Time PCR

Background The current spread of pandemic influenza A(H1N1)v virus necessitates an intensified surveillance of influenza virus infections worldwide. So far, in many laboratories routine diagnostics were limited to generic influenza virus detection only. To provide interested laboratories with real-time PCR assays for type and subtype identification, we present a bundle of PCR assays with which any human influenza A and B virus can be easily identified, including assays for the detection of the pandemic A(H1N1)v virus. Principal Findings The assays show optimal performance characteristics in their validation on plasmids containing the respective assay target sequences. All assays have furthermore been applied to several thousand clinical samples since 2007 (assays for seasonal influenza) and April 2009 (pandemic influenza assays), respectively, and showed excellent results also on clinical material. Conclusions We consider the presented assays to be well suited for the detection and subtyping of circulating influenza viruses.

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.

Human adenoviruses in respiratory infections: Sequencing of the hexon hypervariable region reveals high sequence variability

BACKGROUND In respiratory infections, human adenoviruses (hAdV) of species B1 and C are frequently detected, but severe or even fatal disease outbreaks are predominantly caused by only few serotypes. The molecular typing of hAdV hexon sequences can help to speed up the discrimination of serotypes, thus improving on-time epidemiological examinations and patient care. OBJECTIVES We aimed to develop a molecular method for the rapid species B1 and C serotype identification in respiratory samples based on sequence generation of the hexon hypervariable region (HVR). STUDY DESIGN We developed two PCR-based genotyping systems for a generic HVR amplification and sequence determination of species B1 and C viruses. The assays were applied to hAdV prototypes and 106 samples. RESULTS The primer sets proved to be capable of amplifying all B1 and C serotypes. The viruses detected in clinical samples belong to serotypes 1, 2, 3, 5 and 6. The obtained sequences of serotypes 2, 3 and 5 form 2-3 phylogenetic clusters that are based on the characteristic amino acid changes within the variable HVR sites. CONCLUSIONS Our assay can significantly speed up the time-span needed for serotype identification and will improve epidemiological surveillance and patient care. The obtained hexon sequences of field viruses vary significantly and form multiple genetic lineages. The variability is focussed on the HVR sites and can be interpreted as the ongoing evolutionary process. Further research is needed on the hexon sequence variability of other (respiratory) hAdV serotypes.

Growth characteristics of human adenoviruses on porcine cell lines

Human adenoviruses (hAdV) have been recognized as a highly prevalent virus family causing severe disease in immunocompromised patients. In xenotransplantation the xenograft therefore will be exposed to these viruses, which in case of its infection might contribute to posttransplant complications. To evaluate the susceptibility of porcine cells for hAdV, we infected the porcine cell line POEK with seven serotypes representing all six hAdV species. Additionally, a second porcine cell line (ST) was infected with two serotypes. Viral replication of serotypes varied: porcine cells were fully permissive for serotypes 1, 4 and 17, semi-permissive for 11 and 21, and non-permissive for 31 and 40. Furthermore, we demonstrated the interaction of serotype 1 with the porcine homologue of the coxsackie-adenovirus receptor, the receptor used by many hAdV serotypes for cell attachment. Thus, various adenovirus types of different hAdV species may be capable of infecting different porcine tissue types.

Evolution of the hemagglutinin expressed by human influenza A(H1N1)pdm09 and A(H3N2) viruses circulating between 2008–2009 and 2013–2014 in Germany

This report describes the evolution of the influenza A(H1N1)pdm09 and A(H3N2) viruses circulating in Germany between 2008-2009 and 2013-2014. The phylogenetic analysis of the hemagglutinin (HA) genes of both subtypes revealed similar evolution of the HA variants that were also seen worldwide with minor exceptions. The analysis showed seven distinct HA clades for A(H1N1)pdm09 and six HA clades for A(H3N2) viruses. Herald strains of both subtypes appeared sporadically since 2008-2009. Regarding A(H1N1)pdm09, herald strains of HA clade 3 and 4 were detected late in the 2009-2010 season. With respect to A(H3N2), we found herald strains of HA clade 3, 4 and 7 between 2009 and 2012. Those herald strains were predominantly seen for minor and not for major HA clades. Generally, amino acid substitutions were most frequently found in the globular domain, including substitutions near the antigenic sites or the receptor binding site. Differences between both influenza A subtypes were seen with respect to the position of the indicated substitutions in the HA. For A(H1N1)pdm09 viruses, we found more substitutions in the stem region than in the antigenic sites. In contrast, in A(H3N2) viruses most changes were identified in the major antigenic sites and five changes of potential glycosylation sites were identified in the head of the HA monomer. Interestingly, we found in seasons with less influenza activity a relatively high increase of substitutions in the head of the HA in both subtypes. This might be explained by the fact that mutations under negative selection are subsequently compensated by secondary mutations to restore important functions e.g. receptor binding properties. A better knowledge of basic evolution strategies of influenza viruses will contribute to the refinement of predictive mathematical models for identifying novel antigenic drift variants.

Influenza A H1N1 diagnostics: the first, the fastest, and the most reliable

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Molekulare Analyse humaner Influenzaviren

ZusammenfassungDie Evolution der Influenzaviren wird in zunehmendem Umfang außer mittels klassischer Methoden über molekulare Verfahren verfolgt. Im Vordergrund steht dabei die Analyse der beiden Hüllantigene Hämagglutinin (HA) und Neuraminidase (NA), die das Antigenprofil der Viren bestimmen. Die Influenza-A(H3N2)-Viren weisen eine besonders hohe Variabilität auf, sodass in der Regel mindestens 2 Virusvarianten kozirkulieren. Gemeinsam mit den Influenza-B-Viren verursachten sie etwa 90% der in Deutschland in den vergangenen 12 Saisons aufgetretenen Influenzavirus-Infektionen, während die A-(H1N1-)Viren nur eine untergeordnete Rolle spielten. In den Saisons 2001/02 und 2002/03 traten unerwartet reassortierte Viren des Subtyps A(H1N2) auf, die aber nur vereinzelt isoliert wurden und keine epidemiologische Bedeutung erlangten. In der Saison 2001/02 wurden zudem nach 10-jähriger Abwesenheit erstmalig wieder Influenza-B-Viren der Victoria-Linie in Deutschland und in anderen Ländern der Nordhalbkugel nachgewiesen. Auch hier kam es zu Reassortment-Ereignissen mit den kozirkulierenden Yamagata-like-Influenza-B-Viren, die sich am Auftreten von Viren mit einem Victoria-like HA und einem Yamagata-like NA zeigten.AbstractThe evolution of influenza viruses is increasingly pursued by molecular analyses that complement classical methods. The analyses focus on the two surface proteins hemagglutinin (HA) and neuraminidase (NA) which determine the viral antigenic profile. Influenza A(H3N2) viruses are exceptionally variable, so that usually at least two virus variants cocirculate at the same time. Together with influenza B viruses they caused approximately 90% of influenza virus infections in Germany during the last 12 seasons, while influenza A(H1N1) viruses only played a subordinate part. Unexpectedly, reassorted viruses of subtype A(H1N2) appeared during the seasons 2001/02 and 2002/03, but were isolated only rarely and gained no epidemiological significance. Furthermore, during the season 2001/02 influenza B viruses of the Victoria-lineage reappeared in Germany and other countries of the northern hemisphere after 10 years of absence. These viruses reassorted with the cocirculating Yamagata-like influenza B viruses, as could be seen by the appearance of viruses with a Victoria-like HA and a Yamagata-like NA.The evolution of influenza viruses is increasingly pursued by molecular analyses that complement classical methods. The analyses focus on the two surface proteins hemagglutinin (HA) and neuraminidase (NA) which determine the viral antigenic profile. Influenza A(H3N2) viruses are exceptionally variable, so that usually at least two virus variants cocirculate at the same time. Together with influenza B viruses they caused approximately 90% of influenza virus infections in Germany during the last 12 seasons, while influenza A(H1N1) viruses only played a subordinate part. Unexpectedly, reassorted viruses of subtype A(H1N2) appeared during the seasons 2001/02 and 2002/03, but were isolated only rarely and gained no epidemiological significance. Furthermore, during the season 2001/02 influenza B viruses of the Victoria-lineage reappeared in Germany and other countries of the northern hemisphere after 10 years of absence. These viruses reassorted with the cocirculating Yamagata-like influenza B viruses, as could be seen by the appearance of viruses with a Victoria-like HA and a Yamagata-like NA.

The significance of human respiratory syncytial virus (HRSV) in children from Ghana with acute lower respiratory tract infection: A molecular epidemiological analysis, 2006 and 2013-2014

Background Acute lower respiratory tract infection (ALRI) is a leading cause of childhood morbidity and mortality in developing countries. Globally, human respiratory syncytial virus (HRSV) is the most common pathogen of ALRI in infants and children. However, age-stratified HRSV disease burden data are largely absent from Africa, which is a key gap in informing an evidence-based recommendation for the introduction of an HRSV vaccine by the WHO. Methods This study investigated the presence of HRSV in respiratory specimens from 552 children <5 years old with ALRI from Accra, Ghana in 2006 and 2013–2014 by real-time PCR. Of HRSV-positive samples the second hypervariable region of the viral G protein gene was sequenced and analyzed for phylogeny, characteristic amino acid substitutions, and potential glycosylation patterns. Further, HRSV infections have been characterized by age, symptoms and timely occurrence. Results HRSV was observed in 23% (127/552) of the children with ALRI, with the highest incidence in infants younger than one year (33%, 97/295, p = 0.013). Within the observed seasonal circulation time of HRSV from June (mid-wet season) to December (beginning of the dry season) the incidence of ALRI due to HRSV was as high as 46% (125/273). HRSV disease was significantly associated with (broncho-) pneumonia, bronchiolitis, LRTI, and difficulty in breathing. Phylogenetic characterization of HRSV strains from Ghana identified the circulation of the currently worldwide prevailing genotypes ON1 and BA9, and shows evidence of an independent molecular evolution of ON1 and BA9 strains in Ghana resulting in potentially new subgenotypes within ON1 and BA9, provisionally named ON1.5, ON1.6, and BA9-IV. Conclusion This study addresses important knowledge gaps in the forefront of introducing the HRSV vaccine by providing information on the molecular evolution and incidence of HRSV in Accra (Ghana, Africa).