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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., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Respiratory disease is a significant economic issue in pig farming, with a complex aetiology that includes swine influenza A viruses (swIAV), which are common in European domestic pig populations. The most recent human influenza pandemic in 2009 showed swIAV's zoonotic potential. Monitoring pathogens and disease control are critical from a preventive standpoint, and are based on quick, sensitive, and specific diagnostic assays capable of detecting and distinguishing currently circulating swIAV in clinical samples. For passive surveillance, a set of multiplex quantitative reverse transcription real-time PCRs (mRT-qPCR) and MinION-directed sequencing was updated and deployed. Several lineages and genotypes of swIAV were shown to be dynamically developing, including novel reassortants between human pandemic H1N1 and the avian-derived H1 lineage of swIAV. Despite this, nearly 70% (842/1216) of individual samples from pigs with respiratory symptoms were swIAV-negative, hinting to different aetiologies. The complex and synergistic interactions of swIAV infections with other viral and bacterial infectious agents contribute to the aggravation of pig respiratory diseases. Using a newly developed mRT-qPCR for the combined detection of swIAV and the recently described porcine respirovirus 1 (PRV1) and swine orthopneumovirus (SOV) widespread co-circulation of PRV1 (19.6%, 238/1216 samples) and SOV (14.2%, 173/1216 samples) was evident. Because of the high incidence of PRV1 and SOV infections in pigs with respiratory disease, these viruses may emerge as new allies in the porcine respiratory disease syndrome.

Swine influenza A viruses (S-IAV) circulate in wild boar populations worldwide. Subtypes primarily reflect those actually present within the respective pig industry. Accordingly, infections with swine H1N1, H1N2 and H3N2 have been reported for several regions of Germany. As pigs are susceptible not only to S-IAV but also to avian and human influenza A viruses, it is necessary to consider the possibility that new reassortant viruses with pandemic potential may arise in these new hosts. Therefore, in this study the impact of recent IAV epidemics on antibody prevalences in Bavarian wild boar was assessed. Important events considered were the H1N1pdm09 pandemic, which affected humans and swine, and the highly pathogenic avian influenza (HPAI) H5N8 panzootic in 2016 and 2017, affecting wild and domestic birds. IAV seroprevalences were determined analysing 1,396 samples from before and after the H5N8 panzootic, from various regions in Bavaria, a large administrative region in the South of Germany. Taken together, seroprevalences varied markedly from 1.44% to 12.59%, relative to region and time. However, no discrete correlation was found to population density either in wild boar or in pigs. Antibodies against H1N1 were the most prevalent. In addition, antibodies were detected reacting against H1N2 and against H1pdmNx reassortant viruses, already known to circulate in domestic pigs in Bavaria and notably also against the avian influenza A virus H5N8; the latter in samples taken in 2017. These results confirm the exposure of wild boar to IAV of diverse origin and the increasing variability of S-IAV present in the field. The necessity for continuous IAV surveillance not only of domestic swine but also of wildlife is emphasized., (© 2021 Wiley-VCH GmbH.)

Analysis of 228 H3N2 swine influenza A virus isolates collected between 2003 and 2015 in Germany revealed important changes in molecular epidemiology. The data indicate that a novel reassortant, Rietberg/2014-like swine H3N2, emerged in February 2014 in Northern Germany. It is comprised of a hemagglutinin gene of seasonal H3N2 (A/Denmark/129/2005-like), a neuraminidase gene of Emmelsbuell/2009-like swine H1N2 and the internal gene cassette of pandemic H1N1 viruses. Together with Danish swine H3N2 strains of 2013-2015 with identical genome layout, the Rietberg/2014-like viruses represent a second swine H3N2 lineage which cocirculates with a variant of the Gent/1984-like swine H3N2 lineage. This variant, named Gent1984/Diepholz-like swine H3N2, has a Gent/1984-like HA and a Diepholz/2008-like NA; the origin of the internal gene cassette likely derived from avian-like swine H1N1. The first isolate of the Gent1984/Diepholz reassortant emerged in Northern Germany in September 2011 whereas the last German Gent/1984-like isolate was collected in October 2011.

The genome analysis of 328 H1N1 swine influenza virus isolates collected in a 13-year long-term swine influenza surveillance in Germany is reported. Viral genomes were sequenced with the Illumina next-generation sequencing technique and conventional Sanger methods. Phylogenetic analyses were conducted with Bayesian tree inference. The results indicate continued prevalence of Eurasian avian swine H1N1 but also emergence of a novel H1N1 reassortant, named Schneiderkrug/2013-like swine H1N1, with human-like hemagglutinin and avian-like neuraminidase and internal genes. Additionally, the evolution of an antigenic drift variant of A (H1N1) pdm09 was observed, named Wachtum/2014-like swine H1N1. Both variants were first isolated in northwest Germany, spread to neighboring German states and reached greater proportions of the H1N1 isolates of 2014 and 2015. The upsurge of Wachtum/2014-like swine H1N1 is of interest as this is the first documented persistent swine-to-swine spread of A (H1N1) pdm09 in Germany associated with antigenic variation. Present enzootic swine influenza viruses in Germany now include two or more co-circulating, antigenically variant viruses of each of the subtypes, H1N1 and H1N2., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

A swine influenza survey was conducted between 2003 and 2015 in Germany. During this period, 8122 snout swabs or other respiratory specimens from pigs of 5178 herds, mainly from Germany, were investigated for the presence of swine influenza A virus (S-IAV). In total, 1310 S-IAV isolates were collected. Of this collection, the complete genome of 267 H1N2 S-IAV isolates was sequenced and phylogenetically analyzed. The data demonstrate the incursion of human-like swine H1N2 viruses (Gent/1999-like) in 2000 and prevalent circulation until 2010. From 2008 onward, a sustained and broad change of the genetic constellation of the swine H1N2 subtype commenced. The Gent/1999-like swine H1N2 viruses ceased and several new swine H1N2 reassortants emerged and became prevalent in Germany. Of these, the upsurge of the Diepholz/2008-like, Emmelsbuell/2009-like and Papenburg/2010-like viruses is notable. The data reveal the importance of reassortment events in S-IAV evolution. The strong circulation of S-IAV of different lineages in the swine population throughout the year underlines that pigs are important reservoir hosts.

Influenza A viruses are one of the most important and most studied pathogens in humans and domestic animals but little is known about viral prevalence in non-avian wildlife. Serum samples from three free-ranging cervid species (red [ Cervus elaphus], fallow [ Dama dama] , and roe deer [ Capreolus capreolus]) were collected from six German national parks between 2000 and 2002. The serum was tested for the presence of influenza A antibodies using a commercial competitive enzyme-linked immunosorbent assay. Only one of 137 samples tested positive.

The aim of this study was to investigate the prevalence of CIV H3N8 in dogs in Germany. Blood samples from 272 clinically healthy dogs and 35 dogs with acute respiratory signs were screened for antibodies against influenza virus by ELISA and haemagglutination inhibition (HI) assay; positive samples were further tested by fluorescent antibody test (FAT) and subtype-specific HI assay. Nasal and pharyngeal swabs from all sick dogs were submitted for real-time (RT)-PCR for influenza virus RNA; PCR results were negative in all cases. One healthy and one sick dog tested positive for antibodies against influenza virus nucleoprotein by ELISA, but both were negative by subtype-specific HI assay and FAT, and were therefore considered false positive results. Thus, antibody prevalence against CIV H3N8 was 0% (0-1.24%, 95% CI). Currently, the risk for CIV H3N8 infection in the German dog population seems very low., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

A serological survey for the detection of antibodies to influenza A(H1N1)pdm09 was carried out in a population of dogs and cats in Germany. A total of 1150 sera collected in 2010 and 2011 were screened using an ELISA targeting anti-nucleoprotein NP antibodies. Those initially screened positive samples were subsequently tested for antibodies to N1 neuraminidase followed by a virus neutralization test using A/Bayern/74/2009 strain. A prevalence of A(H1N1)pdm09-specific antibodies of 0.13% and 1.93% was estimated among dogs and cats, respectively. Evidence of exposure to other influenza A virus subtypes was also observed., (2012 Blackwell Verlag GmbH)

The incursion of the human pandemic influenza A virus H1N1 (2009) (H1N1 pdm) into pig populations and its ongoing co-circulation with endemic swine influenza viruses (SIVs) has yielded distinct human-porcine reassortant virus lineages. The haemagglutinin (HA) gene of H1N1 pdm was detected in 41 influenza virus-positive samples from seven swine herds in north-west Germany in 2011. Eight of these samples yielded virus that carried SIV-derived neuraminidase N2 of three different porcine lineages in an H1N1 pdm backbone. The HA sequences of these viruses clustered in two distinct groups and were distinguishable from human and other porcine H1 pdm by a unique set of eight non-synonymous mutations. In contrast to the human population, where H1N1 pdm replaced seasonal H1N1, this virus seems to co-circulate and interact more intensely with endemic SIV lineages, giving rise to reassortants with as-yet-unknown biological properties and undetermined risks for public health.

A total of 1026 serum samples from 388 pigs from three Bavarian rearing farms in the region of Swabia were investigated in the course of investigations into the development of antibodies against Influenza A virus subtypes H1N1, H1N2 and H3N2 by haemagglutination inhibition test during the period from November 2002 to February 2004. There were no signs of respiratory disease during this period. The antibody titres decreased steadily in this period which corresponds to the kinetics of maternally-derived antibodies. Therefore the antibodies reflect the situation of influenza in the farms of origin from which the piglets were purchased. These farms were located in Bavaria and Baden-Wuerttemberg. There was a high activity of H1N1 influenza A viruses in this region whereas the antibody profiles against H1N2 and H3N2 varied between the different farms, which can be attributed to past vaccinations and infections. Thus there was a uniform immunological situation within the regions against H1N1 whereas that against H1N2 and H3N2 differed. The analysis of the antibody profiles allows conclusions to be drawn about the epidemiological situation and means of immunoprophylaxis.

The aim of this study was to evaluate the epidemiological situation of swine influenza virus (SIV) infections in different wild boar populations in Germany based on a serological surveillance in some Bundeslaender (federal states) in connection with virological investigations in wild boar shot in Northern Germany (Mecklenburg-Western Pomerania, district of Nordvorpommern). Altogether, 1245 sera from wild boar were tested using the hemagglutination inhibition test. The established seroprevalence rate was low (on average 5.2%). Antibodies were only detected against the subtypes H1N1 and H3N2 showing differences between wild boar populations and age classes. The virological investigation of samples derived from lungs of wild boar shot in Mecklenburg-Western Pomerania, district of Nordvorpommern (n=242), revealed that the virus prevalence (two virologically positive animals, 0.8%) was very low. Based on serological typing, the isolated SIV was identified as subtype H3N2. Molecular biological investigations of the hemagglutinin (HA) and neuraminidase (NA) genes confirmed this result. This study suggests that SIV infections in wild boar seem to be no serious threat for domestic pigs.

Natural and experimental infections have shown that cats are susceptible to highly pathogenic avian influenza A virus subtype H5N1 (HPAIV H5N1). Cats can be severely affected and die from the disease, but subclinical infections have also been reported. To learn more about the role of cats in the spread of the virus and about the risk posed to cats, the prevalence of H5N1 virus was examined in 171 cats from areas in Germany and Austria in which birds infected with HPAIV H5N1 had been found. Pharyngeal swabs were examined for H5N1 virus using real-time polymerase chain reaction, and serum samples were tested for antibodies to influenza virus. None of the cats showed evidence of infection with H5N1 virus. Prevalence of H5N1 virus was determined to be <1.8% (95% confidence interval (CI): 0.000000-0.017366); prevalence of antibodies was <2.6% (95% CI: 0.000000-0.025068).

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Germany in 343 dead wild birds, as well as in a black swan (Cygnus atratus) kept in a zoo, three stray cats, one stone marten (Martes foina), and in a single turkey farm. In 2007 (June-July) the virus reoccurred in 96 wild birds at six geographically separate locations in the Southeast of Germany. In addition, a backyard mixed duck and goose holding was affected. Real-time RT-PCR [Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600-603] and nucleotide sequencing confirmed that these H5-viruses belonged to the Qinghai lineage of HPAIV H5N1 (clade 2.2). For a more detailed analysis, the hemagglutinin and neuraminidase genes of 27 selected German H5N1 viruses isolated 2006 or 2007 and originating from different regions and animal species were sequenced and analysed phylogenetically. As a result, three closely related but distinguishable H5N1 subclades could be defined: In 2006 a 'Northern type' (subclade 2.2.2), representing virus isolates from the German federal states Mecklenburg-Western Pomerania, Schleswig-Holstein, Brandenburg, and Lower Saxony, and a 'Southern type' (subclade 2.2.1) from Baden-Württemberg and Bavaria were detected. Interestingly, representatives of both types were present in Central Germany and caused the outbreak in turkeys (subclade 2.2.2) and in a case in a tufted duck (Aythya fuligula) (subclade 2.2.1) in Saxony. Furthermore, one isolate from the South of Germany was identified as 2.2.2 and vice versa a 2.2.1-like isolate was found in Northern Germany. H5N1 viruses isolated in 2007 belonged to a third type (subclade 2.2.3) which was not detected in 2006. Our data suggest the introduction of three distinct H5N1 variants into the wild bird population of Germany. The source of these viruses and the exact time of introduction remain obscure. Based on the identification of closely related H5N1 viruses from Southern and Central Russia, a recent introduction via wild birds on winter escape from these regions, early in 2006 constitutes the most likely scenario for the 2006 outbreaks. The viruses detected in 2007 most likely represent another new incursion from an as yet unknown source.

European porcine H1N2 influenza viruses arose after multiple reassortment steps involving a porcine influenza virus with avian-influenza-like internal segments and human H1N1 and H3N2 viruses in 1994. In Germany, H1N2 swine influenza viruses first appeared in 2000. Two German H1N2 swine influenza virus strains isolated from pigs with clinical symptoms of influenza are described. They were characterized by the neutralization test, haemagglutination inhibition (HI) test and complete sequencing of the viral genomes. The data demonstrate that these viruses represent a novel H1N2 reassortant. The viruses showed limited neutralization by sera raised against heterologous A/sw/Bakum/1,832/00-like H1N2 viruses. Sera pools from recovered pigs showed a considerably lower HI reaction, indicative of diagnostic difficulties in using the HI test to detect these viruses with A/sw/Bakum/1,832/00-like H1N2 antigens. Genome sequencing revealed the novel combination of the human-like HAH1 gene of European porcine H1N2 influenza viruses and the NAN2 gene of European porcine H3N2 viruses.

Analysis of the full-length sequences of all eight segments of the German wild-bird H5N1 highly pathogenic avian influenza virus index isolate, A/Cygnus cygnus/Germany/R65/2006, and an H5N1 isolate from a cat (A/cat/Germany/R606/2006) obtained during an outbreak in February 2006 revealed a very high similarity between these two sequences. One amino acid substitution in the PA gene, encoding a protein involved in virus RNA replication, and one amino acid substitution in the haemagglutinin (HA) protein were observed. Phylogenetic analyses of the HA and neuraminidase nucleotide sequences showed that avian influenza H5N1 isolates from the Astrakhan region located in southern Russia were the closest relatives. Reassortment events could be excluded in comparison with other 'Qinghai-like' H5N1 viruses. In addition, an H5N1 isolate originating from a single outbreak in poultry in Germany was found to be related closely to the H5N1 viruses circulating at that time in the wild-bird population.

Data on the economic burden of lower respiratory tract infections (LRTI) in young children are lacking in Germany. The objective of the cost-of-illness study was to estimate the economic impact of community-acquired LRTI and nosocomial LRTI as well as of infections due to respiratory syncytial virus (RSV), parainfluenza viruses (PIV) and influenza viruses (IV). The economic analysis is part of the PRIDE study, a prospective, multi-centre, population-based epidemiological study on the impact of LRTI in children aged 0 to 36 months in Germany. The analysis includes children with community-acquired infections (1329 cases treated as outpatients, 2039 cases treated as inpatients) and nosocomial infections (90 cases). Medical services consumed were generated by chart abstraction and parental expenses data by telephone interviews within four weeks after physician visit or hospitalisation. Costs were evaluated from following perspectives: third party payer, parent and society. Total costs for outpatient treatment are Euro 123 per LRTI case. Stratified by virus type, total costs per case are Euro 163 (RSV), Euro 100 (PIV) and Euro 223 (IV). Total costs per hospitalised LRTI case amount to Euro 2579. Stratified by virus type, total costs per case are Euro 2772 (RSV), Euro 2374 (PIV) and Euro 2597 (IV). Total costs per nosocomial case are Euro 2814. Economic burden due to LRTI is Euro 213 million annually. It is concluded that treatment of LRTI in children up to age three causes a considerable economic burden in Germany. Presented results are the first data describing the economic burden of LRTI in young children assessed by means of the incidence data for Germany. This cost-of-illness study provides basic data for further decision-making, focusing on the economic assessment of preventive strategies for RSV, PIV and IV infections.

Reported here are the results of antigenic and genetic characterisation of equine influenza strains causing local outbreaks reported to the Equine Diagnostic Centre in Berlin, Germany. In 2000, equine influenza virus was detected in a nasal swab from a non-vaccinated horse using a rapid diagnostic kit, but was not successfully isolated. Partial direct sequencing of the haemagglutinin (HA1) gene, indicated that the virus was a European lineage H3N8 subtype strain representative of strains isolated in several European countries during 2000. In 2002, two equine influenza viruses were isolated from nasal swabs both taken from unvaccinated horses with acute respiratory symptoms housed at the same stables. Antigenic characterisation using a panel of ferret antisera suggested that these isolates also belonged to the European lineage of H3N8 viruses. Analysis of deduced HA1 amino acid sequences confirmed that the HA1 of both isolates were identical and belonged to the European lineage. However, from phylogenetic analysis, both strains appeared to be more closely related to viruses isolated between 1989 and 1995 than to viruses isolated more recently in Europe. These results suggested that viruses with fewer changes than those on the main evolutionary lineage may continue to circulate. The importance of expanding current equine influenza surveillance efforts is emphasised.

We examined influenza virus strains of the subtype H3N2 from outbreaks of respiratory diseases in swine herds in Germany. Four different clusters can be distinguished when comparing parts of the HA1 gene from porcine H3N2 isolates analyzed between 1982 and 2001. Comparison between these clusters reveals a bp homology of less than 90%. In contrast, the homology within the clusters is between 93.7 and 100%. Each of these clusters was confined to a specific time period. For the NA gene an additional cluster is formed by the porcine H1N2 isolate. The findings that different subtypes and drift variants are circulating in the German pig population explain the emergence of new influenza virus variants and the need for continued surveillance of swine., (Copyright 2004 S. Karger AG, Basel)

150 dog and 240 pig sera were tested for antibodies against influenza C virus in the hemagglutination inhibition test and confirmed by immunofluorescence and Western blotting tests. Virus strain C/JHB/1/66 was utilized as a standard antigen. It was found that 50.6% of the total number of dogs in the age between one month and 14 years possessed antibody titers of 1:20 or more, which was set as the general cut-off. In comparison, 24.0% of pigs in the age between one day and one month (n = 90) and 25.9% of pigs in the age between one month and one year (n = 150) were found to be positive. The highest hemagglutination inhibition titer was determined as 1:320 for of the dog sera and 1:160 for of the pig sera, respectively.

In August of 1989 sera from 2115 finishing pigs out of 214 herds in the north of Schleswig- Holstein (Germany) were tested for antibodies against three strains of Influenza A viruses by using the haemagglutination inhibition test. Seroprevalences of A/Swine/Nederland/25/80 (H1N1), A/Philippines/2/82 (H3N2), and A/Port Chalmers/1/73 (H3N2) on herd level were 23.4%, 20.6% and 5.1% respectively. The mean within herd prevalences were 9.6%, 14.7% and 0.6%, respectively. Antibodies against H1N1 and H3N3 strains were simultaneously found in 7.5% of the herds and in 1.6% of the pigs. In 485 (22.9%) of the pigs and in 83 (38.3%) of the herds at least one of the strains was prevalent. In 61 (73.5%) of those herds there was no evidence of a clinical outbreak before testing. A higher risk of infection with influenza virus was associated with high pig density (> or = 200 pigs per km2) and with management factors as greater herd size (> 300 finishing pigs), slatted floors (compared to straw bedding) and all in-all out system (compared to continuous purchase).