Virologische Analysen im Nationalen Referenzzentrum für Influenzaviren während der Influenzasaison 2023/2024

Im Nationalen Referenzzentrum für Influenzaviren unterschied sich die Erfassung und Untersuchung der Influenzasaison 2023/2024 von den vorhergehenden Saisons durch den Aufbau eines Sentinels im stationären Bereich. Zusätzlich zu den 134 Arztpraxen, die am ambulanten Sentinel teilnehmen, sandten 15 Kliniken Abstrichproben aus dem stationären Bereich ein. Dies ermöglichte zum ersten Mal einen Vergleich der Zirkulationsmuster der respiratorischen Viren im ambulanten und stationären Bereich. Es werden die vergleichende genetische Analyse der Influenzaviren im ambulanten und stationären Sentinel in der Saison 2023/2024 sowie die antigenen Charak-terisierungen in Bezug auf die Passgenauigkeit der Impfstämme und Resistenzprüfungen beschrieben

Todesfall eines Kindes im Rahmen eines Influenza-Ausbruchs in einem Kindergarten

Im Epidemiologischen Bulletin 22/2018 wird ein Ausbruchsgeschehen durch Influenza-A(H1N1)pdm09-Virus in einer Kita und deren sozialer Umgebung analysiert. Die Mehrheit der involvierten Personen war nicht gegen Influenza immunisiert. Die Variation der Symptome bei den Erkrankten von leichtem Husten bis hin zu respiratorischem Versagen infolge Pneumonie bei diesem Influenza-Ausbruch mit genetisch, innerhalb des Kerngeschehens, unverändertem Virus zeigt, dass die Influenza auf individueller Ebene unterschiedliche Verläufe nehmen kann, die nicht vorhersagbar sind.Peer Reviewe

Synopse virologischer Analysen im Nationalen Referenzzentrum für Influenzaviren während der COVID-19-Pandemie

Das Nationale Referenzzentrum für Influenzaviren gewinnt durch die fortlaufende Untersuchung von Proben aus den Sentinelpraxen der Arbeitsgemeinschaft Influenza einen umfassenden Überblick über die zirkulierenden respiratorischen Erreger in Deutschland. Dazu gehören neben SARS-CoV-2 und den Influenzaviren auch das Respiratorische Synzytialvirus, Parainfluenzaviren, humane Metapneumoviren, humane saisonale Coronaviren und humane Rhinoviren. Die Analyseergebnisse von 15.660 Sentinelproben sowie weiteren Isolaten im Zeitraum von Kalenderwoche 5/2020 bis 21/2022 werden im Epidemiologischen Bulletin 22/2022 vorgestellt. Beschrieben werden außerdem die Zirkulation respiratorischer Erreger im Vergleich zu vorpandemischen Saisons, die molekulare Charakterisierung und phylogenetische Analysen, die Überprüfung der Passgenauigkeit der eingesetzten Influenzaimpfstoffe und die Resistenzprüfung von Influenzaviren

Influenza viruses – antiviral therapy and resistance

Influenza is a serious and frequently underestimated, but vaccine preventable disease. The adamantane derivates rimantadine and amantadine and the neuraminidase inhibitors zanamivir and oseltamivir are the only antiviral drugs currently approved in Europe for therapy and prophylaxis of influenza infections. Resistance to these drugs occurs due to mutations within the therapeutic target proteins M2 ion channel protein and viral neuraminidase. An unexpected occurrence of oseltamivir-resistant seasonal A(H1N1) viruses was detected in winter 2007/2008. The prevalence of these viruses increased rapidly and nearby all viruses circulating during the following seasons were resistant to oseltamivir. The A(H1N1)pdm09 viruses replaced the former seasonal A(H1N1) subtype during the 2009–2010 influenza season. Fortunately, resistance to neuraminidase inhibitors was detected in A(H1N1)pdm09, A(H3N2) and influenza B viruses only sporadically and was treatment related mostly. Comprehensive analyses of circulating viruses showed a high prevalence of A(H3N2) influenza viruses that are resistant to adamantane derivates since 2004/2005 and a progressive trend in the prevalence of resistant viruses up to 100% in following seasons. The M2 ion channel protein of A(H1N1)pdm09 viruses is associated with the Eurasian avian-like swine lineage and thus show “natural” resistance to adamantane derivates. Therefore, only neuraminidase inhibitors are recommended for influenza treatment today. This manuscript summarizes the occurrence and spread of antiviral resistant influenza viruses and highlights the importance for developing and/or approving new antiviral compounds

Antiretroviral Therapy and Resistance of HIV-1

0\. Titelblatt, Inhaltsverzeichnis 1\. Einleitung 1 2\. Material 31 3\. Methoden 35 4\. Ergebnisse 62 5\. Diskussion 106 6\. Zusammenfassung 121 7\. Summary 123 8\. Literatur 125 9\. Abkürzungsverzeichnis 149 10\. Anhang 151 11\. Danksagung 159Ziel der vorliegenden Arbeit war, die potentielle Ausbreitung replikationskompetenter resistenter HIV in Deutschland zu untersuchen. Die genotypische Resistenzanalyse von 136 HIV-Infektionen aus Blutproben therapie- naiver PatientInnen mit dokumentierter Serokonversion zwischen Januar 1996 und Dezember 2001 zeigte, dass 47% der Viren mindestens eine resistenz-assoziierte Mutation in den thera-peutischen Zielenzymen Protease bzw. Reverse Transkriptase enthielten. In 15 Infektionen (11%) wurde anhand der phänotypischen Resistenzuntersuchung eine in vitro Resistenz der Viren nachgewiesen (Kooperation mit dem NRZ Retroviren, Erlangen), wobei der anhand des genotypischen Resistenzmusters erwartete Phänotyp mit dem tatsächlich gemessenen relativ gut überein stimmte (4 NRTI, 2 NNRTI, 3 PI). In sechs Fällen konnte Resistenz gegen zwei Wirkstoffklassen (NRTI/PI bzw. NRTI/NNRTI) nachgewiesen werden. Ein gehäuftes Auftreten der Übertragung dieser multiresistenten Viren in den Jahren 2000/2001 wurde beobachtet, erreichte jedoch keine statistische Signifikanz. Die PatientInnen aus dem Studienkollektiv, die im Rahmen der Serokonverterstudie des Robert Koch- Institutes rekrutiert wurden (Kooperation mit Infektionsepidemiologie), hatten sich fast ausschließlich in Deutschland - eine Infektion stammte aus den Niederlanden, eine aus Thailand - infiziert. Die meisten Infektionen wurden durch homosexuelle Kontakte über-tragen. Von 50% der Infizierten konnte eine Blutprobe während bzw. innerhalb von drei Monaten nach der Serokonversion erhalten werden. Fast alle der hier untersuchten Viren gehören dem HIV-1 Subtyp B an. HIV-1 non-B Infektionen wurden in zwei Fällen nachgewiesen (eine HIV-1 Subtyp C und eine Subtyp D Infektion). Anhand von Blutproben therapie- naiver nigerianischer PatientInnen (Kooperation mit University of Jos, Nigeria) wurde die in vitro Resistenz von Viren des HIV-1 Subtyps G bzw. CRF02-AG untersucht. Die hier nachgewiesenen subtypspezifischen resistenz- assoziierten Mutation im pol-Gen, führten jedoch nicht zu einer messbaren phänotypischen Resistenz der untersuchten Viren. Im zweiten Teil der vorliegenden Arbeit wurde die virale Fitness resistenter übertragener Viren in Wachstumskinetiken im Vergleich zu sensitiven Wildtypviren untersucht. Dazu wur-den NNRTI/NRTI und NRTI/PI resistente und sensitive, infektiöse, rekombinante HIV-1 Klone hergestellt, deren Vermehrungseffizienz in einem Reportergen Assay (SEAP), durch p24-Antigenbestimmung, Ermittlung des infektiösen Titers (TCID50) und Quantifizierung der viralen RNA bestimmt. Es wurde eine im Vergleich zum sensitiven Wildtypklon geringere Vermehrungseffizienz der resistenten Virusklone beobachtet. Zusätzlich konnte in einem real time RT-Assay (TaqMan) gezeigt werden, dass die in vitro Infektion von lymphoiden Zellen mit resistenten HIV Klonen im Vergleich zu Infektionen mit Wildtypklonen zu geringeren RT-Aktivitäten im Zellkulturüberstand führte. Ein Einfluss einzelner Schlüsselmutationen auf die virale Fitness, insbesondere der in der Literatur als fitnessmindernd beschriebenen M184V Substitution der RT, konnte mit den verwendeten Methoden nicht nachgewiesen werden. Die Ergebnisse dieser Arbeit zeigen, dass in Deutschland resistente, replikationskompetente HIV-1 übertragen werden. Die Überwachung der Ausbreitungsdynamik und die Unter-suchung der Vermehrungsfähigkeit antiretroviral resistenter HIV sowie die Langzeit- beobachtung der untersuchten Serokonverter wird im Rahmen des deutschen HIV- Kompetenznetzwerkes und des europäischen Programms SPREAD erfolgen.Subject of this work was to investigate the potential spreading of replication competent and resistant HIV in Germany. Genotypic resistance analysis of 136 HIV infections of therapy-naive patients with documented seroconversion between January 1996 and December 2001 showed that 47% of the viruses contained at least one resistance-associated mutation within the therapeutic target enzymes, protease and reverse transcriptase. Phenotypic analysis demonstrated 15 infections with in vitro resistant viruses (in cooperation with the National Reference Centre for Retroviruses, Erlangen). The expected phenotype, which was deduced from the genotype correlated mostly with the measured phenotype (4 NRTI, 2 NNRTI, 3 PI). In six cases resistance against two drug-classes could be detected (NRTI/PI and NRTI/NNRTI, respectively). Frequent transmission of these multi-drug resistant viruses was seen in 2000/2001, but it did not reach statistical significance. Patients were recruited within the seroconverter study (Robert Koch-Institut, in cooperation with the Department for Infection Epidemiology). They had been infected mostly in Germany, one infection was from Thailand, one from The Netherlands. Homosexual contacts represented the main risk of infection. In 50% of the therapy-naive seroconverters blood samples could be collected during seroconversion or within three months after seroconversion. In almost every case the examined viruses belonged to HIV-1 subtype B. HIV-1 non-B infections were detected in only two cases (one HIV-1 subtype C and one subtype D). The in vitro resistance of viruses belonging to HIV-1 subtype G and CRF02-AG was studied in blood samples of therapy-naive patients from Nigeria (in cooperation with University of Jos, Nigeria). The detected subtype specific resistance-associated mutations in the investigated viruses did not result in measurable resistance in the phenotype. In the second part of this work the viral fitness of resistant transmitted viruses was analyzed in comparison with sensitive wildtype virus. NNRTI/NRTI and NRTI/PI resistant recombinante HIV-1 clones were produced. The replication efficiency of these virus clones was examined employing a reportergene assay (SEAP), p24-antigene determination, quantification of the infectious titer (TCID50) and of the viral RNA. Compared with the sensitive wildtype HIV clone, the resistant virus clones showed a decreased replication efficiency. Additionally a decreased RT-activity in cell culture after infection of lymphoid cells with resistant HIV clones compared with wildtype could be detected. An influence of single key mutations, the M184V substitution of the RT for example, which is known to decrease viral fitness, could not be demonstrated. The results of this work show that resistant and replication competent HIV-1 were transmitted in Germany. Surveillance of the dynamic of spreading and the investigation of the replication efficiency of antiretroviral resistant HIV as well as the long- term follow up of the investigated seroconverters will take place within the HIV competence network and the European program SPREAD

Insights into the direct anti-influenza virus mode of action of Rhodiola rosea

Background: The anti-influenza A virus activities and contents of previously isolated most active flavonoids (rhodiosin and tricin) from a standardized hydro-ethanolic R. rosea root and rhizome extract (SHR-5®), did not fully explain the efficacy of SHR-5®. Moreover, the mode of antiviral action of SHR-5® is unknown. Purpose: To determine the anti-influenza viral principle of SHR-5® we evaluated i) the combined anti-influenza virus effect of rhodiosin and tricin, ii) the impact of its tannin-enriched fraction (TE), iii) its antiviral spectrum and mode of action, and iv) its propensity for resistance development in vitro. Methods: The combined anti-influenza virus effect of rhodiosin and tricin and the impact of TE were investigated with cytopathic effect (CPE)-inhibition assays in MDCK cells. A tannin-depleted fraction (TD) and TE were prepared by polyamide column chromatography and dereplicated by LC-MS. Plaque-reduction assays provided insights into the anti-influenza virus profile, the mode of action, and the propensity for resistance development of SHR-5®. Results: Our results i) did not reveal synergistic anti-influenza A virus effects of rhodiosin and tricin, but ii) proved a strong impact of TE mainly composed of prodelphinidin gallate oligomers. iii) TE inhibited the plaque-production of influenza virus A(H1N1)pdm09, A(H3N2), and B (Victoria and Yamagata) isolates (including isolates resistant to neuraminidase and/or M2 ion channel inhibitors) with 50% inhibitory concentration values between 0.12 - 0.53 µg/ml similar to SHR-5®. Mechanistic studies proved a virucidal activity, inhibition of viral adsorption, viral neuraminidase activity, and virus spread by SHR-5® and TE. iv) No resistance development was observed in vitro. Conclusion: For the first time a comprehensive analysis of the anti-influenza virus profile of a hydro-ethanolic R. rosea extract (SHR-5®) was assessed in vitro. The results demonstrating broad-spectrum multiple direct anti-influenza virus activities, and a lack of resistance development to SHR-5® together with its known augmentation of host defense, support its potential role as an adaptogen against influenza virus infection

Household transmissibility and other characteristics of seasonal oseltamivir-resistant influenza A(H1N1) viruses, Germany, 2007-8

During the influenza season 2007-8, the proportion of seasonal influenza A(H1N1) viruses resistant to the neuraminidase inhibitor oseltamivir increased worldwide. We conducted an investigation to compare patients infected with oseltamivir-resistant (ose-R) and oseltamivir- susceptible (ose-S) influenza A(H1N1) viruses regarding risk factors for resistance and the capability to transmit in the household setting. Within a cohort of 396 laboratory confirmed influenza patients from sentinel physicians we conducted a nested casecontrol study among patients infected with A(H1N1). Thirty patients in the cohort were infected with influenza B, none with influenza A(H3N2) and 366 with A(H1N1). Of the 366 A(H1N1) viruses 52 (14%) were ose-R. Demographic characteristics, oseltamivir exposure, travel history and outcome were not significantly different between ose-S and ose-R patients. Among 133 households in the nested case-control study, secondary household attack rates in households with ose-R cases and households with ose-S cases were similar (23 versus 26%; p-value=0.54). Ose-R household status and occurrence of secondary cases were associated with an odds ratio of 0.85 (95% confidence interval 0.38-1.88). We conclude that seasonal ose-R influenza A(H1N1) viruses have transmitted well in the household setting

Increase of Synergistic Secondary Antiviral Mutations in the Evolution of A(H1N1)pdm09 Influenza Virus Neuraminidases

The unexpected emergence of oseltamivir-resistant A(H1N1) viruses in 2008 was facilitated in part by the establishment of permissive secondary neuraminidase (NA) substitutions that compensated for the fitness loss due to the NA-H275Y resistance substitution. These viruses were replaced in 2009 by oseltamivir-susceptible A(H1N1)pdm09 influenza viruses. Genetic analysis and screening of A(H1N1)pdm09 viruses circulating in Germany between 2009 and 2024 were conducted to identify any potentially synergistic or resistance-associated NA substitutions. Selected viruses were then subjected to further characterization in vitro. In the NA gene of circulating A(H1N1)pdm09 viruses, two secondary substitutions, NA-V241I and NA-N369K, were identified. These substitutions demonstrated a stable lineage in phylogenetic analysis since the 2010–2011 influenza season. The data indicate a slight increase in viral NA bearing two additional potentially synergistic substitutions, NA-I223V and NA-S247N, in the 2023–2024 season, which both result in a slight reduction in susceptibility to NA inhibitors. The accumulation of secondary synergistic substitutions in the NA of A(H1N1)pdm09 viruses increases the probability of the emergence of antiviral-resistant viruses. Therefore, it is crucial to closely monitor the evolution of circulating influenza viruses and to develop additional antiviral drugs against different target proteins

Trends in respiratory virus circulation following COVID-19-targeted nonpharmaceutical interventions in Germany, January - September 2020: Analysis of national surveillance data

Background:During the initial COVID-19 response, Germany’s Federal Government implemented several nonpharmaceutical interventions (NPIs) that were instrumental in suppressing early exponential spread ofSARS-CoV-2. NPI effect on the transmission of other respiratory viruses has not been examined at the national level thus far.Methods:Upper respiratory tract specimens from 3580 patients with acute respiratory infection (ARI), collected within the nationwide German ARI Sentinel, underwent RT-PCR diagnostics for multiple respiratory viruses. The observation period (weeks 1-38 of 2020) included the time before, during and after a farreaching contact ban. Detection rates for different viruses were compared to 2017-2019 sentinel data (15350 samples; week 1-38, 11823 samples). Findings :The March 2020 contact ban, which was followed by a mask mandate, was associated with an unprecedented and sustained decline of multiple respiratory viruses. Among these, rhinovirus was the single agent that resurged to levels equalling those of previous years. Rhinovirus rebound was first observed in children, after schools and daycares had reopened. By contrast, other non enveloped viruses (i.e. gastroenteritisviruses reported at the national level) suppressed after the shutdown did not rebound.Interpretation:Contact restrictions with a subsequent mask mandate in spring may substantially reduce respiratory virus circulation. This reduction appears sustained for most viruses, indicating that the activity of influenza and other respiratory viruses during the subsequent winter season might be low,whereas rhinovirus resurgence, potentially driven by transmission in educational institutions in a setting of waning population immunity, might signal predominance of rhinovirus-related ARIs.Peer Reviewe

Virologische Analysen in der Influenzasaison 2019/20

Die hier vorgestellten Ergebnisse des Nationalen Referenzzentrums für Influenzaviren umfassen Daten zu innerhalb des Sentinels der AGI isolierten Viren und von Viren, die im Rahmen von Ausbrüchen, der Untersuchung schwerer Erkrankungsfälle und von Typisierungsanfragen oder Projekten untersucht wurden sowie von Isolaten aus Einsendungen von Instituten und Gesundheitsämtern. Untersuchungen zur weiterführenden Charakterisierung von RSV und weiteren respiratorischen Viren wurden darüber hinaus bei einer Subpopulation der Sentinelproben vom Konsiliarlabor für RSV, PIV und HMPV durchgeführt. Ab der 9. KW 2020 wurden Untersuchungen auf SARS-CoV-2 integriert