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Human metapneumovirus (HMPV) is a cause of respiratory tract illness at all ages. In this study the epidemiological and molecular diversity among patients of different ages was investigated. Between 2000–2001 and 2009–2010, HMPV was detected in 3% (138/4,549) of samples from outpatients with influenza-like illness with a new, sensitive real-time RT-PCR assay. Several hundred (797) clinical specimens from hospitalized children below the age of 4 years with acute respiratory illness were investigated and HMPV was detected in 11.9% of them. Investigation of outpatients revealed that HMPV infections occurred in individuals of all ages but were most prevalent in children (0–4 years) and the elderly (>60 years). The most present clinical features of HMPV infections were cough, bronchitis, fever/shivers and pneumonia. About two thirds of HMPV-positive samples were detected in February and March throughout the study period. Molecular characterization of HMPV revealed a complex cyclic pattern of group dominance where HMPV subgroup A and B viruses predominated in general for three consecutive seasons. German HMPV represented all genetic lineages including A1, A2, B1, B2, sub-clusters A2a and A2b. For Germany, not only time-dependent circulation of lineages and sub-clusters was observed but also co-circulation of two or three predominant lineages. Two newly emerging amino acid substitutions (positions 223 and 280) of lineage B2 were detected in seven German HMPV sequences. Our study gives new insights into the molecular epidemiology of HMPV in in- and outpatients over a time period of 10 years for the first time. It is one of only few long-term surveillance studies in Europe, and allows comparative molecular analyses of HMPV circulating worldwide. [ABSTRACT FROM AUTHOR]

Influenza A(H1N1)pdm09 viruses cause sporadically very severe disease including fatal clinical outcomes associated with pneumonia, viremia and myocarditis. A mutation characterized by the substitution of aspartic acid (wild-type) to glycine at position 222 within the haemagglutinin gene (HA-D222G) was recorded during the 2009 H1N1 pandemic in Germany and other countries with significant frequency in fatal and severe cases. Additionally, A(H1N1)pdm09 viruses exhibiting the polymorphism HA-222D/G/N were detected both in the respiratory tract and in blood. Specimens from mild, fatal and severe cases were collected to study the heterogeneity of HA-222 in A(H1N1)pdm09 viruses circulating in Germany between 2009 and 2011. In order to enable rapid and large scale analysis we designed a pyrosequencing (PSQ) assay. In 2009/2010, the 222D wild-type of A(H1N1)pdm09 viruses predominated in fatal and severe outcomes. Moreover, co-circulating virus mutants exhibiting a D222G or D222E substitution (8/6%) as well as HA-222 quasispecies were identified (10%). Both the 222D/G and the 222D/G/N/V/Y polymorphisms were confirmed by TA cloning. PSQ analyses of viruses associated with mild outcomes revealed mainly the wild-type 222D and no D222G change in both seasons. However, an increase of variants with 222D/G polymorphism (60%) was characteristic for A(H1N1)pdm09 viruses causing fatal and severe cases in the season 2010/2011. Pure 222G viruses were not observed. Our results support the hypothesis that the D222G change may result from adaptation of viral receptor specificity to the lower respiratory tract. This could explain why transmission of the 222G variant is less frequent among humans. Thus, amino acid changes at HA position 222 may be the result of viral intra-host evolution leading to the generation of variants with an altered viral tropism. [ABSTRACT FROM AUTHOR]