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.

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.

Evidence of exposure of domestic pigs to Highly Pathogenic Avian Influenza H5N1 in Nigeria.

Avian influenza viruses (AIV) potentially transmit to swine as shown by experiments, where further reassortment may contribute to the generation of pandemic strains. Associated risks of AIV inter-species transmission are greater in countries like Nigeria with recurrent epidemics of highly pathogenic AI (HPAI) in poultry and significant pig population. Analysis of 129 tracheal swab specimens collected from apparently healthy pigs at slaughterhouse during presence of HPAI virus H5N1 in poultry in Nigeria for influenza A by RT-qPCR yielded 43 positive samples. Twenty-two could be determined by clade specific RT-qPCR as belonging to the H5N1 clade 2.3.2.1c and confirmed by partial hemagglutinin (HA) sequence analysis. In addition, 500 swine sera were screened for antibodies against influenza A virus nucleoprotein and H5 HA using competition ELISAs and hemagglutination inhibition (HI) tests. Serologically, 222 (44.4%) and 42 (8.4%) sera were positive for influenza A virus NP and H5 antibodies, respectively. Sera reacted to H5N1 and A/H1N1pdm09 strains by HI suggesting exposure of the Nigerian domestic pig population to these viruses. We report for the first time in Nigeria, exposure of domestic pigs to H5N1 virus. This poses potential public health and pandemic risk due to interspecies transmission of avian and human influenza viruses.

Swarm incursions of reassortants of highly pathogenic avian influenza virus strains H5N8 and H5N5, clade 2.3.4.4b, Germany, 2016/17.

The outbreak of highly pathogenic avian influenza H5Nx viruses in winter 2016/2017 was the most severe HPAI epizootic ever reported in Germany. The H5N8 and H5N5 viruses detected in birds in Germany in 2016/2017 represent a reassortant swarm of at least five distinct genotypes, which carried closely related HA segments derived from clade 2.3.4.4b. The genotypes of these viruses and their spatio-temporal distribution indicated a unique situation with multiple independent entries of HPAIV into Germany.

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.

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.

Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4b in Germany in 2016/2017.

Here, we report on the occurrence of highly pathogenic avian influenza (HPAI) H5Nx clade 2.3.4.4b in Germany. Between November 8, 2016, and September 30, 2017, more than 1,150 cases of HPAI H5Nx clade 2.3.4.4b in wild birds and 107 outbreaks in birds kept in captivity (92 poultry holdings and 15 zoos/animal parks) were reported in Germany. This HPAI epidemic is the most severe recorded in Germany so far. The viruses were apparently introduced by migratory birds, sparking an epidemic among wild birds across Germany with occasional incursions into poultry holdings, zoos and animal parks, which were usually rapidly detected and controlled by stamping out. HPAI viruses (mainly subtype H5N8, in a few cases also H5N5) were found in dead wild birds of at least 53 species. The affected wild birds were water birds (including gulls, storks, herons, and cormorants) and scavenging birds (birds of prey, owls, and crows). In a number of cases, substantial gaps in farm biosecurity may have eased virus entry into the holdings. In a second wave of the epidemic starting from February 2017, there was epidemiological and molecular evidence for virus transmission of the infections between commercial turkey holdings in an area of high poultry density, which caused approximately 25% of the total number of outbreaks in poultry. Biosecurity measures in poultry holdings should be adapted. This includes, inter alia, wearing of stable-specific protective clothing and footwear, cleaning, and disinfection of equipment that has been in contact with birds and prevention of contacts between poultry and wild water birds.

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.

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs.

BACKGROUND: A diversifying pool of mammalian-adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. OBJECTIVES: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost-effective large-scale analysis. METHODS: New SIV haemagglutinin (HA) and neuraminidase (NA) subtype- and lineage-specific multiplex real-time RT-PCRs (RT-qPCR) have been developed and validated with reference virus isolates and clinical samples. RESULTS: A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M-gene-specific influenza A virus RT-qPCR. In a second step, positive samples are examined by tetraplex HA- and triplex NA-specific RT-qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages "av" (European avian-derived), "hu" (European human-derived) and "pdm" (human pandemic A/H1N1, 2009) are distinguished by RT-qPCRs, and within the NA subtype N1, lineage "pdm" is differentiated. An RT-PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT-qPCR subtyping. CONCLUSIONS: These new multiplex RT-qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe.

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.

Complete Genome Sequence of Avian Paramyxovirus Strain APMV-6/red-crested pochard/Balkhash/5842/2013 from Kazakhstan.

An avian paramyxovirus 6 strain was isolated during a wild bird monitoring study in Kazakhstan in 2013. The virus was isolated from a wild duck red-crested pochard (Netta rufina) in southeastern Kazakhstan. Here, we present the complete genome sequence of the virus.

Sample preparation for avian and porcine influenza virus cDNA amplification simplified: Boiling vs. conventional RNA extraction.

RNA extraction and purification is a fundamental step that allows for highly sensitive amplification of specific RNA targets in PCR applications. However, commercial extraction kits that are broadly used because of their robustness and high yield of purified RNA are expensive and labor-intensive. In this study, boiling in distilled water or a commercial lysis buffer of different sample matrices containing avian or porcine influenza viruses was tested as an alternative. Real-time PCR (RTqPCR) for nucleoprotein gene fragment was used as read out. Results were compared with freshly extracted RNA by use of a commercial extraction kit. Different batches of virus containing materials, including diluted virus positive allantoic fluid or cell culture supernatant, and avian faecal, cloacal or oropharyngeal swab samples were used in this study. Simple boiling of samples without any additional purification steps can be used as an alternative RNA preparation method to detect influenza A virus nucleoprotein RNA in oropharyngeal swab samples, allantoic fluid or cell-culture supernatant. The boiling method is not applicable for sample matrices containing faecal material.

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.

Failure of productive infection of Mallards (Anas platyrhynchos) with H16 subtype of avian influenza viruses.

BACKGROUND: Mallard ducks and other waterfowl represent the most important reservoirs of low pathogenic avian influenza viruses (LPAIV). In addition, mallards are the most abundant duck species in Eurasia that migrate over long distances. Despite extended wild bird monitoring studies over the past decade in many Eurasian countries and investigating hundreds of thousands of wild bird samples, no mallard duck was found to be positive for avian influenza virus of subtype H16 in faecal, cloacal or oropharyngeal samples. Just three cases of H16 infections in Anseriformes species were described worldwide. In contrast, H16 viruses have been repeatedly isolated from birds of the Laridae family. OBJECTIVE: Here, we tested the hypothesis that mallards are less permissive to infection with H16 viruses. METHODS: Groups of mallard ducks of different age were inoculated via the oculo-nasal-oral route with different infectious doses of an H16N3 AIV. RESULTS: The ducks did not show any clinical symptoms, and no virus shedding was evident from cloacal and respiratory routes after experimental infection as shown by negative RT-qPCR results. In addition, all serum samples taken on days 8, 21 and 24 post-inoculation were negative by competitive NP-ELISA. CONCLUSIONS: This study provided evidence that mallards are resistant to infection with H16N3 LPAIV.

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.

Saving resources: avian influenza surveillance using pooled swab samples and reduced reaction volumes in real-time RT-PCR.

The occurrence of highly pathogenic (HP) avian influenza (AI) H5N1 in Asia and its spread to Africa and Europe prompted costly monitoring programs of wild birds and domestic poultry. AI virus excretion is tested by examining avian swab samples by real-time reverse transcription PCR (RT-qPCR). In this study, pools of swab samples and a reagents volume reduction per RT-qPCR were evaluated as measures of economization. Viral transport medium and faecal matrices were spiked with different low pathogenic AI virus strains and tested for loss of target RNA during all processing steps as individual rayon swabs or in sample pools of 5, 10 and 15 swabs. Fresh faeces from Mallard ducks and other aquatic bird species as sample matrix resulted in loss of AIV RNA of about 90% compared to transport medium. Due to sample RNA dilution in pools the likelihood of detection of single positive samples is decreasing with increasing size of sample pools. However, pools of five samples containing only one positive sample consistently gave positive results. Similarly, no differences in detection rates were obtained when analyzing 1030 wild bird swab samples either individually or in pools of five. Reducing the reaction volume of influenza A virus generic as well as of subtype-specific RT-qPCRs to 12.5 µl (2.5 µl template) instead of 25 µl did not adversely affect the limit of detection of these RT-qPCRs. A significant economic benefit without impeding detection efficacy can be achieved when sample pools of five samples are analyzed by RT-qPCR using a reduction of the reaction mix to the half of the original volume.

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.

Genetic data from avian influenza and avian paramyxoviruses generated by the European network of excellence (EPIZONE) between 2006 and 2011--review and recommendations for surveillance.

Since 2006, the members of the molecular epidemiological working group of the European "EPIZONE" network of excellence have been generating sequence data on avian influenza and avian paramyxoviruses from both European and African sources in an attempt to more fully understand the circulation and impact of these viruses. This review presents a timely update on the epidemiological situation of these viruses based on sequence data generated during the lifetime of this project in addition to data produced by other groups during the same period. Based on this information and putting it all into a European context, recommendations for continued surveillance of these important viruses within Europe are presented.