Outbreaks among Wild Birds and Domestic Poultry Caused by Reassorted Influenza A(H5N8) Clade 2.3.4.4 Viruses, Germany, 2016.

In November 2016, an influenza A(H5N8) outbreak caused deaths of wild birds and domestic poultry in Germany. Clade 2.3.4.4 virus was closely related to viruses detected at the Russia-Mongolia border in 2016 but had new polymerase acidic and nucleoprotein segments. These new strains may be more efficiently transmitted to and shed by birds.

Peste des Petits Ruminants Virus in Vulnerable Wild Small Ruminants, Iran, 2014-2016.

In 2014-2016, >1,000 wild goats and sheep in 4 northern and central provinces of Iran died from peste des petits ruminants virus (PPRV) infection. Partial nucleoprotein sequencing of PPRV from 3 animals showed a close relationship to lineage 4 strains from China. Control measures are needed to preserve vulnerable ruminant populations.

Influenza A Virus Infection in Pigs Attracts Multifunctional and Cross-Reactive T Cells to the Lung.

UNLABELLED: Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67(+)) CD8(+) T cells with an early effector phenotype (perforin(+) CD27(+)) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4(+) and CD8(+) T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4(+) and CD8(+) T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4(+) and CD8(+) memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs. IMPORTANCE: Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

Circulation of avian paramyxoviruses in wild birds of Kazakhstan in 2002-2013.

BACKGROUND: Screening wild birds for avian paramyxoviruses is of increasing importance. 6913 samples of tracheal and cloacal swabs were collected during 2002-2013 and tested to study the prevalence of APMVs in wild avifauna of Kazakhstan. As a result, 45 isolates were obtained during this period and their ecological niches and genetic relationships were defined. METHODS: Tracheal and cloacal samples from wild birds were collected using sterile swabs placed in viral transport medium and kept in liquid nitrogen until delivery to the laboratory. Samples were inoculated into 10-day-old embryonated chicken eggs and reverse transcription PCR (RT-PCR) assays were performed via a one-step protocol. The PCR products were sequenced and phylogenetic trees were constructed using the 'Neighbour Joining' method. RESULTS: Six thousand nine hundred thirteen samples from 183 bird species were investigated and 45 isolates belonging to four different serotypes APMV-1, APMV-4, APMV-6 and APMV-8 were identified. All APMVs were isolated predominantly from birds belonging to Anatidae family (ducks and geese) and only one APMV-4 isolate was obtained from shorebird (Curlew) on the Caspian seashore. Genetic studies showed that the recovered APMV-1 strains had highest homology with European isolates. APMV-4 strains isolated in 2003, and APMV-6 and APMV-8 isolated in 2013 were 99 % identical to isolates from Far East. CONCLUSION: This is the first reported characterization of avian paramyxoviruses from wild birds isolated in Kazakhstan. These data confirm the wide distribution of APMV-1, APMV-4 and APMV-6 in the Asian subcontinent. The obtained data contribute to the accumulation of knowledge on the genetic diversity and prevalence of APMVs in wild bird populations.

Evidence for genetic variation of Eurasian avian influenza viruses of subtype H15: the first report of an H15N7 virus.

Since the first detection of H15 avian influenza viruses (AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against it reacted strongly with the homologous antigen, but with lower titers when using the reference Australian antigen. The amino acid motifs of the HA cleavage site and receptor-binding site were different from those in the Australian viruses. The new virus, together with an H15 virus from Siberia from 2008, constitutes a new clade of H15 AIV isolates.

Influenza A(H5N8) Virus Similar to Strain in Korea Causing Highly Pathogenic Avian Influenza in Germany.

Highly pathogenic avian influenza (H5N8) virus, like the recently described H5N8 strain from Korea, was detected in November 2014 in farmed turkeys and in a healthy common teal (Anas crecca) in northeastern Germany. Infected wild birds possibly introduced this virus.

Avian influenza H7N9/13 and H7N7/13: a comparative virulence study in chickens, pigeons, and ferrets.

UNLABELLED: Human influenza cases caused by a novel avian H7N9 virus in China emphasize the zoonotic potential of that subtype. We compared the infectivity and pathogenicity of the novel H7N9 virus with those of a recent European avian H7N7 strain in chickens, pigeons, and ferrets. Neither virus induced signs of disease despite substantial replication in inoculated chickens and rapid transmission to contact chickens. Evidence of the replication of both viruses in pigeons, albeit at lower levels of RNA excretion, was also detected. No clear-cut differences between the two H7 isolates emerged regarding replication and antibody development in avian hosts. In ferrets, in contrast, greater replication of the avian H7N9 virus than of the H7N7 strain was observed with significant differences in viral presence, e.g., in nasal wash, lung, and cerebellum samples. Importantly, both viruses showed the potential to spread to the mammal brain. We conclude that efficient asymptomatic viral replication and shedding, as shown in chickens, facilitate the spread of H7 viruses that may harbor zoonotic potential. Biosafety measures are required for the handling of poultry infected with avian influenza viruses of the H7 subtype, independently of their pathogenicity for gallinaceous poultry. IMPORTANCE: This study is important to the field since it provides data about the behavior of the novel H7N9 avian influenza virus in chickens, pigeons, and ferrets in comparison with that of a recent low-pathogenicity H7N7 strain isolated from poultry. We clearly show that chickens, but not pigeons, are highly permissive hosts of both H7 viruses, allowing high-titer replication and virus shedding without any relevant clinical signs. In the ferret model, the potential of both viruses to infect mammals could be demonstrated, including infection of the brain. However, the replication efficiency of the H7N9 virus in ferrets was higher than that of the H7N7 strain. In conclusion, valuable data for the risk analysis of low-pathogenicity avian influenza viruses of the H7 subtype are provided that could also be used for the risk assessment of zoonotic potentials and necessary biosafety measures.

Expanded cocirculation of stable subtypes, emerging lineages, and new sporadic reassortants of porcine influenza viruses in swine populations in Northwest Germany.

The emergence of the human 2009 pandemic H1N1 (H1N1pdm) virus from swine populations refocused public and scientific attention on swine as an important source of influenza A viruses bearing zoonotic potential. Widespread and year-round circulation of at least four stable lineages of porcine influenza viruses between 2009 and 2012 in a region of Germany with a high-density swine population is documented here. European avian influenza virus-derived H1N1 (H1N1av) viruses dominated the epidemiology, followed by human-derived subtypes H1N2 and H3N2. H1N1pdm viruses and, in particular, recently emerging reassortants between H1N1pdm and porcine HxN2 viruses (H1pdmN2) were detected in about 8% of cases. Further reassortants between these main lineages were diagnosed sporadically. Ongoing diversification both at the phylogenetic and at the antigenic level was evident for the H1N1av lineage and for some of its reassortants. The H1avN2 reassortant R1931/11 displayed conspicuously distinct genetic and antigenic features and was easily transmitted from pig to pig in an experimental infection. Continuing diverging evolution was also observed in the H1pdmN2 lineage. These viruses carry seven genome segments of the H1N1pdm virus, including a hemagglutinin gene that encodes a markedly antigenically altered protein. The zoonotic potential of this lineage remains to be determined. The results highlight the relevance of surveillance and control of porcine influenza virus infections. This is important for the health status of swine herds. In addition, a more exhaustive tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required.

Genetic characterization of a new candidate hemagglutinin subtype of influenza A viruses.

ABSTRACTAvian influenza viruses (AIV) have been classified on the basis of 16 subtypes of hemagglutinin (HA) and 9 subtypes of neuraminidase. Here we describe genomic evidence for a new candidate HA subtype, nominally H19, with a large genetic distance to all previously described AIV subtypes, derived from a cloacal swab sample of a Common Pochard (Aythya ferina) in Kazakhstan, in 2008. Avian influenza monitoring in wild birds especially in migratory hotspots such as central Asia is an important approach to gain information about the circulation of known and novel influenza viruses. Genetically, the novel HA coding sequence exhibits only 68.2% nucleotide and 68.5% amino acid identity with its nearest relation in the H9 (N2) subtype. The new HA sequence should be considered in current genomic diagnostic AI assays to facilitate its detection and eventual isolation enabling further study and antigenic classification.

Reassortants of pandemic influenza A virus H1N1/2009 and endemic porcine HxN2 viruses emerge in swine populations in Germany.

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.

Effect of swab matrix, storage time, and temperature on detection of avian influenza virus RNA in swab samples.

Virologic monitoring of avian influenza viruses (AIV) mainly relies on the collection of oropharyngeal, cloacal, or fecal swab samples. The quality of swab samples, therefore, contributes to limitations of the informative value of such monitoring, but the cost of sampling has a great impact on the feasibility of wild bird monitoring studies or poultry surveillance programs. Here, the effect of different swab material and storage conditions on quality and quantity of AIV RNA detection in swab samples by real-time reverse-transcription quantitative PCR has been studied. Two commercial swab products, a rayon-tipped and a flocked nylon type, were compared. Similar suitability of the two swab types, despite a huge price difference, was observed. Superior results by using both types of swab were gained provided that 1) swabs stayed immersed overnight in an appropriate viral transport medium (VTM), or that 2) swabs were vigorously shaken in VTM for at least 1 min and up to 1 hr to release as much trapped virus material as possible. Degradation of RNA over a period of 2 wk for virus-containing samples is negligible when using constant storage conditions at 4 C or 20 C; temperature shifts proved to be more harmful.

Reassorted pandemic (H1N1) 2009 influenza A virus discovered from pigs in Germany.

A natural reassortant influenza A virus consisting of seven genome segments from pandemic (H1N1) 2009 virus and a neuraminidase segment from a Eurasian porcine H1N1 influenza A virus was detected in a pig herd in Germany. The obvious reassortment compatibility between the pandemic (H1N1) 2009 and H1N1 viruses of porcine origin raises concern as to whether swine may become a reservoir for further reassortants of pandemic (H1N1) 2009 viruses with unknown implications for human health and swine production.

Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007.

BACKGROUND: Virological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran. RESULTS: Overall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals.Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%. CONCLUSION: Results of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected.

Highly pathogenic avian influenza virus subtype H5N1 in mute swans (Cygnus olor) in Central Bosnia.

In order to determine the actual prevalence of avian influenza viruses (AIVs) in wild birds in Bosnia and Herzegovina, extensive surveillance was carried out between October 2005 and April 2006. A total of 394 samples representing 41 bird species were examined for the presence of influenza A virus using virus isolation in embryonated chicken eggs, PCR, and nucleotide sequencing. AIV subtype H5N1 was detected in two mute swans (Cygnus olor). The isolates were determined to be highly pathogenic avian influenza (HPAI) virus and the hemagglutinin sequence was closely similar to A/Cygnus olor/Astrakhan/ Ast05-2-10/2005 (H5N1). This is the first report of HPAI subtype H5N1 in Bosnia and Herzegovina.

Dynamics of specific antibody responses induced in mallards after infection by or immunization with low pathogenicity avian influenza viruses.

Natural infections with different subtypes of low pathogenicity avian influenza viruses (LPAIVs) are very common in wild duck populations. Recent outbreaks of high pathogenicity avian influenza virus (HPAIV) H5N1 in Eurasian and African countries stimulated monitoring activities in aquatic wild bird populations. Surveillance mainly focused on virus detection. Only a few serologic investigations have been conducted so far, although such data may retrospectively elucidate epidemiologic patterns of different AIV subtypes in the populations under study. To better understand the immunologic and serologic reactions of mallards after infection with LPAIV, we investigated the AIV type- and subtype-specific antibody dynamics in mallards after different LPAIV infections by hemagglutination inhibition, competitive enzyme-linked immunosorbent assay, and western blot analysis, as appropriate. Four groups of mallard ducks were used: 1) naturally infected birds, 2) birds that were experimentally infected with LPAIV, 3) birds that were immunized with inactivated virus preparations, and 4) negative control birds. Ducks were monitored for up to 15 mo, and serum samples were investigated every 1-4 wk. It could be shown that infection with LPAIV in mallards can be traced serologically over prolonged periods of time.

Surveillance of European Domestic Pig Populations Identifies an Emerging Reservoir of Potentially Zoonotic Swine Influenza A Viruses.

Swine influenza A viruses (swIAVs) can play a crucial role in the generation of new human pandemic viruses. In this study, in-depth passive surveillance comprising nearly 2,500 European swine holdings and more than 18,000 individual samples identified a year-round presence of up to four major swIAV lineages on more than 50% of farms surveilled. Phylogenetic analyses show that intensive reassortment with human pandemic A(H1N1)/2009 (H1pdm) virus produced an expanding and novel repertoire of at least 31 distinct swIAV genotypes and 12 distinct hemagglutinin/neuraminidase combinations with largely unknown consequences for virulence and host tropism. Several viral isolates were resistant to the human antiviral MxA protein, a prerequisite for zoonotic transmission and stable introduction into human populations. A pronounced antigenic variation was noted in swIAV, and several H1pdm lineages antigenically distinct from current seasonal human H1pdm co-circulate in swine. Thus, European swine populations represent reservoirs for emerging IAV strains with zoonotic and, possibly, pre-pandemic potential.

Highly pathogenic avian influenza virus (H5N1) in frozen duck carcasses, Germany, 2007.

We conducted phylogenetic and epidemiologic analyses to determine sources of outbreaks of highly pathogenic avian influenza virus (HPAIV), subtype H5N1, in poultry holdings in 2007 in Germany, and a suspected incursion of HPAIV into the food chain through contaminated deep-frozen duck carcasses. In summer 2007, HPAIV (H5N1) outbreaks in 3 poultry holdings in Germany were temporally, spatially, and phylogenetically linked to outbreaks in wild aquatic birds. Detection of HPAIV (H5N1) in frozen duck carcass samples of retained slaughter batches of 1 farm indicated that silent infection had occurred for some time before the incidental detection. Phylogenetic analysis established a direct epidemiologic link between HPAIV isolated from duck meat and strains isolated from 3 further outbreaks in December 2007 in backyard chickens that had access to uncooked offal from commercial deep-frozen duck carcasses. Measures that will prevent such undetected introduction of HPAIV (H5N1) into the food chain are urgently required.

Sequence diversity of the haemagglutinin open reading frame of recent highly pathogenic avian influenza H5N1 isolates from Egypt.

The sequences encoding the haemagglutinin (HA) of twelve H5N1 isolates obtained in 2006 and 2007 from different avian species in backyard holdings and poultry farms in Egypt revealed amino acid variations across the polypeptide and also in the polybasic cleavage motif of three of the isolates from backyard poultry with one, so far, unique mutation in an isolate from a chicken. The HAs of two isolates (A/goose/Egypt/R4/2007, A/chicken/Egypt/R3/2007) collected on the same day in the same village from two neighbouring houses were found to differ from each other. Five out of the seven nucleotide exchanges in these two isolates were translationally silent, and two resulted in amino acid substitutions: one in the polybasic cleavage motif and the other in the signal peptide. Circulation of different H5N1 strains possessing considerable variations in backyard poultry, particularly domestic waterfowl, draws attention to the evolution of H5N1 subtypes in Egypt.

Rapid pathotyping of recent H5N1 highly pathogenic avian influenza viruses and of H5 viruses with low pathogenicity by RT-PCR and restriction enzyme cleavage pattern (RECP).

Rapid and accurate diagnosis of avian influenza virus infections of poultry and humans comprises detection, subtyping, and, in case of subtypes H5 and H7, pathotyping of such viruses. Reliable methods for pathotyping of H5 avian influenza viruses (AIV) are based on determination of the intravenous pathogenicity index (IVPI) in specific pathogen free (SPF) chickens and on characterization of the hemagglutinin (HA) gene cleavage site by sequencing. The number of basic amino acids (arginine and lysine) at the cleavage site is an important indicator of pathogenicity. In this paper, a new rapid method for pathotyping of H5 subtype avian influenza viruses is described which is based on RT-PCR and restriction enzyme cleavage pattern (RECP) assay using the MboII restriction enzyme. Validation of the method using 28 H5 subtype reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity, especially when targeting recent highly pathogenic AIV (HPAIV) of subtype H5N1 and Asian origin. In addition, RECP results were validated by testing 47 field samples from different sources and by sequencing of their RT-PCR products. This approach for H5 AIV pathotyping proved to be fast, reliable, and comparatively sensitive and is suitable especially for laboratories lacking sequencing or in vivo pathotyping facilities.

Epidemiological survey of swine influenza A virus in selected wild boar populations in Germany.

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.