Characteristics of two zoonotic swine influenza A(H1N1) viruses isolated in Germany from diseased patients.

Interspecies transmission of influenza A viruses (IAV) from pigs to humans is a concerning event as porcine IAV represent a reservoir of potentially pandemic IAV. We conducted a comprehensive analysis of two porcine A(H1N1)v viruses isolated from human cases by evaluating their genetic, antigenic and virological characteristics. The HA genes of those human isolates belonged to clades 1C.2.1 and 1C.2.2, respectively, of the A(H1N1) Eurasian avian-like swine influenza lineage. Antigenic profiling revealed substantial cross-reactivity between the two zoonotic H1N1 viruses and human A(H1N1)pdm09 virus and some swine viruses, but did not reveal cross-reactivity to H1N2 and earlier human seasonal A(H1N1) viruses. The solid-phase direct receptor binding assay analysis of both A(H1N1)v showed a predominant binding to α2-6-sialylated glycans similar to human-adapted IAV. Investigation of the replicative potential revealed that both A(H1N1)v viruses grow in human bronchial epithelial cells to similar high titers as the human A(H1N1)pdm09 virus. Cytokine induction was studied in human alveolar epithelial cells A549 and showed that both swine viruses isolated from human cases induced higher amounts of type I and type III IFN, as well as IL6 compared to a seasonal A(H1N1) or a A(H1N1)pdm09 virus. In summary, we demonstrate a remarkable adaptation of both zoonotic viruses to propagate in human cells. Our data emphasize the needs for continuous monitoring of people and regions at increased risk of such trans-species transmissions, as well as systematic studies to quantify the frequency of these events and to identify viral molecular determinants enhancing the zoonotic potential of porcine IAV.

Alterations in hemagglutinin receptor-binding specificity accompany the emergence of highly pathogenic avian influenza viruses.

UNLABELLED: Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galß1-4GlcNAcß (3'SLN) and Neu5Acα2-3Galß1-3GlcNAcß (SiaLe(c)) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acα2-3Galß1-4(Fucα1-3)GlcNAcß (SiaLe(x)), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE: Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.

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.

Molecular characterization and evolution dynamics of influenza B viruses circulating in Germany from season 1996/1997 to 2019/2020.

Influenza B viruses are responsible for significant disease burden caused by viruses of both the Yamagata- and Victoria-lineage. Since the circulating patterns of influenza B viruses in different countries vary we investigated molecular properties and evolution dynamics of influenza B viruses circulating in Germany between 1996 and 2020. A change of the dominant lineage occurred in Germany in seven seasons in over past 25 years. A total of 676 sequences of hemagglutinin coding domain 1 (HA1) and 516 sequences of neuraminidase (NA) genes of Yamagata- and Victoria-lineage viruses were analyzed using time-scaled phylogenetic tree. Phylogenetic analysis demonstrated that Yamagata-lineage viruses are more diverse than the Victoria-lineage viruses and could be divided into nine genetic groups whereas Victoria-lineage viruses presented six genetic groups. Comparative phylogenetic analyses of both the HA and NA segments together revealed a number of inter-lineage as well as inter- and intra-clade reassortants. We identified key amino acid substitutions in major HA epitopes such as in four antigenic sites and receptor-binding sites (RBS) and in the regions close to them, with most substitutions in the 120-loop of both lineage viruses. Altogether, seventeen substitutions were fixed over time within the Yamagata-lineage with twelve of them in the antigenic sites. Thirteen substitutions were identified within the Victoria-lineage, with eleven of them in the antigenic sites. Moreover, all Victoria-lineage viruses of the 2017/2018 season were characterized by a deletion of two amino acids at the position 162-163 in the antigenic site of HA1. The viruses with triple deletion Δ162-164 were found in Germany since season 2018/2019. We highlighted the interplay between substitutions in the glycosylation sites and RBS and antigenic epitope during HA evolution. The results obtained underscore the need for continuous monitoring of circulating influenza B viruses. Early detection of strains with genetic and antigenic variation is essential to predict the circulation patterns for the following season. Such information is important for the development of optimal vaccines and strategies for prevention and control of influenza.

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.

Pharmacokinetics of Oral and Intravenous Oseltamivir Treatment of Severe Influenza B Virus Infection Requiring Organ Replacement Therapy.

BACKGROUND AND OBJECTIVES: Patients with severe influenza virus infection, multi-organ failure and organ replacement therapy may absorb and metabolize neuraminidase inhibitors differently. Systematic pharmacokinetic/pharmacodynamic clinical trials are currently lacking in this high-risk group. Inadequate dosing increases the risk of treatment failure and drug resistance, especially in severely ill patients with elevated virus loads. This study aims to explore the impact of organ replacement therapy on oseltamivir drug concentrations. METHODS: Serial pharmacokinetic/pharmacodynamic measurements and Sieving coefficients were assessed in two patients with severe influenza B infection requiring organ replacement therapy. RESULTS: Patient #1, a 9-year-old female with severe influenza B virus infection, biventricular assist device, and continuous veno-venous hemodiafiltration, received 75 mg oral oseltamivir twice-daily for 2 days, then intravenous oseltamivir with one-time renoprotective dosing (40 mg), followed by regular intravenous administration of 100 mg twice-daily. Plasma oseltamivir carboxylate concentrations were stable initially, but only regular administration of 100 mg resulted in virus load decline and clinical improvement. Patient #2, a 28-year-old female with influenza B virus infection requiring extracorporeal membrane oxygenation, received 75 mg oral oseltamivir twice-daily, resulting in erratic oseltamivir blood concentrations. In both patients, drug concentrations remained well within safety margins. CONCLUSIONS: In severe cases with multi-organ failure, administration of 100 mg intravenous oseltamivir twice-daily provided reliable drug concentrations, as opposed to renoprotective and oral dosing, thereby minimizing the risk of treatment failure and drug resistance. Evidence-based pediatric dosing recommendations and effective intravenous antiviral treatment modalities are needed for intensive care patients with life-threatening influenza disease.

Laboratory investigations are indispensable to monitor the progress of measles elimination--results of the German Measles Sentinel 1999-2003.

BACKGROUND: The elimination of measles is a goal set by the World Health Organisation to be reached by 2010 in the European region. OBJECTIVES: To enhance the measles surveillance in Germany, a country-wide laboratory supported a sentinel was established. STUDY DESIGN: A network of >1200 representatively distributed practitioners reported detailed data on all clinically diagnosed cases and provided specimens for laboratory diagnosis. RESULTS: A total of 3225 suspected cases were reported between October 1999 and December 2003. The incidence in Western Germany decreased from >15 cases per 100,000 population to one case in 2003, while in Eastern Germany <1 case per 100,000 population was observed during these years. Laboratory investigations were undertaken in 40% of cases in 2000/2001. This rate increased to 79% in 2003. Simultaneously, the rate of confirmed cases dropped from 60% in the former years to 23% in 2003. Measles virus (MV) detection by serology and by PCR revealed concordant results in 92%. Most suspected cases (85%) were unvaccinated with 66% being laboratory confirmed. Only 10% of suspected cases occurred in vaccinated individuals and very few (22%) could be confirmed. Analyses of confirmed measles in vaccinated patients (n = 49) revealed 24.5% primary vaccine failures, 24.5% reinfections after successful vaccination and 31% MV infection before or shortly after vaccination. The genetic characterisation of 389 MV isolates identified eight genotypes: B3, C2, D4, D5, D6, D7, G2 and H1. Only the C2, D6 and D7 MV genotypes circulated endemically in Western Germany. The newly emerged MV D7 almost completely replaced the pre-existing C2 and D6 MVs in 2001. The few measles cases detected in Eastern Germany were mostly caused by imported MVs. CONCLUSION: The data demonstrate that laboratory investigations including molecular methods are an indispensable tool for surveillance in all countries advanced in measles elimination.

Comparison of shedding characteristics of seasonal influenza virus (sub)types and influenza A(H1N1)pdm09; Germany, 2007-2011.

BACKGROUND: Influenza viral shedding studies provide fundamental information for preventive strategies and modelling exercises. We conducted a prospective household study to investigate viral shedding in seasonal and pandemic influenza between 2007 and 2011 in Berlin and Munich, Germany. METHODS: Study physicians recruited index patients and their household members. Serial nasal specimens were obtained from all household members over at least eight days and tested quantitatively by qRT-PCR for the influenza virus (sub)type of the index patient. A subset of samples was also tested by viral culture. Symptoms were recorded daily. RESULTS: We recruited 122 index patients and 320 household contacts, of which 67 became secondary household cases. Among all 189 influenza cases, 12 were infected with seasonal/prepandemic influenza A(H1N1), 19 with A(H3N2), 60 with influenza B, and 98 with A(H1N1)pdm09. Nine (14%) of 65 non-vaccinated secondary cases were asymptomatic/subclinical (0 (0%) of 21 children, 9 (21%) of 44 adults; p = 0.03). Viral load among patients with influenza-like illness (ILI) peaked on illness days 1, 2 or 3 for all (sub)types and declined steadily until days 7-9. Clinical symptom scores roughly paralleled viral shedding dynamics. On the first day prior to symptom onset 30% (12/40) of specimens were positive. Viral load in 6 asymptomatic/subclinical patients was similar to that in ILI-patients. Duration of infectiousness as measured by viral culture lasted approximately until illness days 4-6. Viral load did not seem to be influenced by antiviral therapy, age or vaccination status. CONCLUSION: Asymptomatic/subclinical infections occur infrequently, but may be associated with substantial amounts of viral shedding. Presymptomatic shedding may arise in one third of cases, and shedding characteristics appear to be independent of (seasonal or pandemic) (sub)type, age, antiviral therapy or vaccination; however the power to find moderate differences was limited.

Probing neutralizing-antibody responses against emerging measles viruses (MVs): immune selection of MV by H protein-specific antibodies?

Measles virus (MV) infection and vaccination induce long-lasting immunity and neutralizing-antibody responses that are directed against the MV haemagglutinin (H) and the fusion (F) protein. A new MV genotype, D7, emerged recently in western Germany and rapidly replaced the long-term endemically circulating genotypes C2 and D6. Analysis of the H gene of C2, D6, D7 and vaccine viruses revealed uniform sequences for each genotype. Interestingly, a consistent exchange of seven distinct amino acids in the D7 H was observed when compared with residues shared between C2, D6 and vaccine viruses, and one exchange (D416-->N) in the D7 H was associated with an additional N-linked glycosylation. In contrast, the F gene is highly conserved between MVs of these genotypes. To test whether the D7 H protein escapes from antibody responses that were raised against earlier circulating or vaccine viruses, the neutralizing capacity of mAbs recognizing seven distinct domains on the H of an Edmonston-related MV was compared. The mAbs revealed a selective and complete loss of two neutralizing epitopes on the D7 H when compared with C2, D6 and vaccine viruses. To assess whether these alterations of the D7 H affect the neutralizing capacity of polyclonal B-cell responses, genotype-specific antisera were produced in cotton rats. However, no significant genotype-dependent difference was found. Likewise, human sera obtained from vaccinees (n=7) and convalescents (n=6) did not distinguish between the MV genotypes. Although the hypothesis of selection of D7 viruses by pre-existing neutralizing antibodies is compatible with the differing pattern of neutralizing epitopes on the H protein, it was not confirmed by the results of MV neutralization with polyclonal sera.

Rapid replacement of endemic measles virus genotypes.

Although vaccination campaigns have significantly reduced the number of measles cases worldwide, endemic transmission of measles virus (MV) continues to occur in several continents, including Europe. To obtain current information on measles incidence and molecular data on circulating MVs in Germany, a nationwide measles sentinel was established. Phylogenetic analysis based on the variable part of the N gene from 80 MVs isolated between November 1999 and October 2001 revealed the presence of at least six distinct MV genotypes: B3, C2, D4, D6, G2 and a new variant of D7. Both the incidence and the pattern of MV genotypes differed markedly between the former East and West Germany. In the eastern part, few measles cases, mainly caused by genotypes originating from other countries (B3, D4, G2), were detected. In the western and southern parts, genotypes C2, D6 and D7 were associated with endemic transmission. Surprisingly, the indigenous genotypes predominant during the 1990s - C2 and D6 - disappeared simultaneously over the period of observation coinciding with the emergence and the wide spread of D7 viruses. While the incidence of measles remained constant, all MVs isolated in 2001 were assigned to D7. We note that the haemagglutinin (H) sequence of D7 viruses shows distinct exchanges of certain amino acids in the stem and propeller domain compared to C2, D6 and the MV vaccine strains used. This raises the possibility of a selective advantage of D7 viruses transmitted in the presence of H-specific antibodies.