Emergence of highly pathogenic H5N2 and H7N1 influenza A viruses from low pathogenic precursors by serial passage in ovo.

Highly pathogenic (HPAI) strains emerge from their low pathogenic (LPAI) precursors and cause severe disease in poultry with enormous economic losses, and zoonotic potential. Understanding the mechanisms involved in HPAI emergence is thus an important goal for risk assessments. In this study ostrich-origin H5N2 and H7N1 LPAI progenitor viruses were serially passaged seventeen times in 14-day old embryonated chicken eggs and Ion Torrent ultra-deep sequencing was used to monitor the incremental changes in the consensus genome sequences. Both virus strains increased in virulence with successive passages, but the H7N1 virus attained a virulent phenotype sooner. Mutations V63M, E228V and D272G in the HA protein, Q357K in the nucleoprotein (NP) and H155P in the neuraminidase protein correlated with the increased pathogenicity of the H5N2 virus; whereas R584H and L589I substitutions in the polymerase B2 protein, A146T and Q220E in HA plus D231N in the matrix 1 protein correlated with increased pathogenicity of the H7N1 virus in embryos. Enzymatic cleavage of HA protein is the critical virulence determinant, and HA cleavage site motifs containing multibasic amino acids were detected at the sub-consensus level. The motifs PQERRR/GLF and PQRERR/GLF were first detected in passages 11 and 15 respectively of the H5N2 virus, and in the H7N1 virus the motifs PELPKGKK/GLF and PELPKRR/GLF were detected as early as passage 7. Most significantly, a 13 nucleotide insert of unknown origin was identified at passage 6 of the H5N2 virus, and at passage 17 a 42 nucleotide insert derived from the influenza NP gene was identified. This is the first report of non-homologous recombination at the HA cleavage site in an H5 subtype virus. This study provides insights into how HPAI viruses emerge from low pathogenic precursors and demonstrated the pathogenic potential of H5N2 and H7N1 strains that have not yet been implicated in HPAI outbreaks.

Characterization of highly pathogenic avian influenza H5Nx viruses in the ferret model.

Highly pathogenic avian influenza (HPAI) H5 viruses, of the A/goose/Guangdong/1/1996 lineage, have exhibited substantial geographic spread worldwide since the first detection of H5N1 virus in 1996. Accumulation of mutations in the HA gene has resulted in several phylogenetic clades, while reassortment with other avian influenza viruses has led to the emergence of new virus subtypes (H5Nx), notably H5N2, H5N6, and H5N8. H5Nx viruses represent a threat to both the poultry industry and human health and can cause lethal human disease following virus exposure. Here, HPAI H5N6 and H5N2 viruses (isolated between 2014 and 2017) of the 2.3.4.4 clade were assessed for their capacity to replicate in human respiratory tract cells, and to cause disease and transmit in the ferret model. All H5N6 viruses possessed increased virulence in ferrets compared to the H5N2 virus; however, pathogenicity profiles varied among the H5N6 viruses tested, from mild infection with sporadic virus dissemination beyond the respiratory tract, to severe disease with fatal outcome. Limited transmission between co-housed ferrets was observed with the H5N6 viruses but not with the H5N2 virus. In vitro evaluation of H5Nx virus replication in Calu-3 cells and the identification of mammalian adaptation markers in key genes associated with pathogenesis supports these findings.

Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses.

The panzootic caused by A/goose/Guangdong/1/96-lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human-to-human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human-to-human transmissibility and impact on human health should such human-to-human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.

Clade 2.3.4.4 H5 North American Highly Pathogenic Avian Influenza Viruses Infect, but Do Not Cause Clinical Signs in, American Black Ducks (Anas rubripes).

Highly pathogenic avian influenza virus (HPAIV) from the goose/Guangdong/1996 clade 2.3.4.4 H5 lineage spread from Asia into North America in 2014, most likely by wild bird migrations. Although several variants of the virus were detected, H5N8 and H5N2 were the most widespread in North American wild birds and domestic poultry. In early 2015, the H5N2 virus spread through commercial poultry in the Midwest, and >50 million chickens and turkeys died or had to be culled. Related H5 HPAIVs are still endemic in much of the Eastern Hemisphere. The wild bird species that were involved with dissemination of the virus in North America are not known. Dabbling ducks, especially mallards (Anas platyrhynchos), typically have the highest detection rates for avian influenza viruses. To better characterize the wild avian species that could spread the virus, American black ducks (Anas rubripes), which are closely related to mallards, were challenged with the North American H5N2 and H5N8 index HPAIV isolates: A/Northern Pintail/WA/40964/2014 H5N2 and A/Gyrfalcon/WA/41088/2014 H5N8. Although the American black ducks could be infected with low doses of both isolates (≤102 50% egg infective doses), ducks shed the H5N2 longer than the H5N8 (10 vs. 7 days) and the titers of virus shed were higher. Although there were too few ducks available on which to draw definitive conclusions, this suggests that American black ducks could serve as a more efficient reservoir for the H5N2 virus than the H5N8 virus.

Nota de investigación- Los virus de influenza aviar altamente patógenos de América del Norte H5 clado 2.3.4.4 son capaces de infectar pero no causan signos clínicos en ánades sombríos americanos (Anas rubripes). Los virus de la influenza aviar altamente patógena (HPAIV) subtipo H5pertenecientes al linaje ganso/Guangdong/1996 clado 2.3.4.4, se han propagado desde Asia a América del Norte en el año 2014, muy probablemente por migración de aves silvestres. Aunque se detectaron varias variantes del virus, los subtipos H5N8 y H5N2 fueron los más extendidas en aves silvestres y aves domésticas de América del Norte. A principios de 2015, el virus H5N2 se propagó a través de aves comerciales en el medio oeste de los Estados Unidos, y más de 50 millones de pollos y pavos murieron o tuvieron que ser eliminados. Virus de la influenza aviar de alta patogenicidad H5 relacionados aún son endémicos en gran parte del hemisferio oriental. Las especies de aves silvestres que participaron en la diseminación del virus en América del Norte no se conocen. Los patos chapoteadores, especialmente los patos silvestres de collar, suelen tener las tasas de detección más altas para los virus de la influenza aviar. Para caracterizar mejor las especies de aves silvestres que podrían propagar el virus, ánades sombríos americanos (Anas rubripes), que están estrechamente relacionados con los patos silvestres, se desafiaron con aislamientos índices del virus de la influenza aviar de alta patogenicidad de América del Norte H5N2 y H5N8: A/ánade rabudo/WA/40964/2014 H5N2 o con el virus A/halcón gerifalte/WA/41088/2014 H5N8. Aunque los ánades sombríos americanos podieron infectarse con dosis bajas de ambos aislamientos (≤102 50% de dosis infectivas para embrión de pollo), los patos eliminaron al virus H5N2 por más tiempo en comparación con el virus H5N8 (10 días y 7 días, respectivamente) y los títulos de virus fueron más altos. Aunque había muy pocos patos disponibles para sacar conclusiones definitivas, esto sugiere que los ánades sombríos americanos podrían servir como un reservorio más eficiente para el virus H5N2.

Estimating within-flock transmission rate parameter for H5N2 highly pathogenic avian influenza virus in Minnesota turkey flocks during the 2015 epizootic.

Better control of highly pathogenic avian influenza (HPAI) outbreaks requires deeper understanding of within-flock virus transmission dynamics. For such fatal diseases, daily mortality provides a proxy for disease incidence. We used the daily mortality data collected during the 2015 H5N2 HPAI outbreak in Minnesota turkey flocks to estimate the within-flock transmission rate parameter (ß). The number of birds in Susceptible, Exposed, Infectious and Recovered compartments was inferred from the data and used in a generalised linear mixed model (GLMM) to estimate the parameters. Novel here was the correction of these data for normal mortality before use in the fitting process. We also used mortality threshold to determine HPAI-like mortality to improve the accuracy of estimates from the back-calculation approach. The estimated ß was 3.2 (95% confidence interval (CI) 2.3-4.3) per day with a basic reproduction number of 12.8 (95% CI 9.2-17.2). Although flock-level estimates varied, the overall estimate was comparable to those from other studies. Sensitivity analyses demonstrated that the estimated ß was highly sensitive to the bird-level latent period, emphasizing the need for its precise estimation. In all, for fatal poultry diseases, the back-calculation approach provides a computationally efficient means to obtain reasonable transmission parameter estimates from mortality data.

Genetic basis of resistance to avian influenza in different commercial varieties of layer chickens.

An outbreak of H5N2 highly pathogenic avian influenza (HPAI) in 2015, resulting in mandatory euthanization of millions of chickens, was one of the most fatal in the US history. The aim of this study was to detect genes associated with survival following natural infection with HPAI during this outbreak. Blood samples were collected from 274 individuals from 3 commercial varieties of White Leghorn. Survivors and age and genetics matched non-affected controls from each variety were included in the comparison. All individuals were genotyped on the 600k SNP array. A genome-wide association study (GWAS) with the standard frequency test in PLINK was performed within each variety, whereas logistic regression with the first 3 multidimensional scaling components as covariates was used for joined analysis of all varieties. Several SNPs located within 3 regions reached the 5% Bonferroni genome-wide threshold of significance (P < 3.87E-06). The associations were identified for 2 varieties and only within genetic variety on chromosomes 11 (variety 1), 5, and 18 (variety 3). A genome-wide scan with FST was also performed for 40, 100, and 500 kb windows to support the genome-wide association analyses. The regions with highest FST values between cases and controls were located on chromosomes 1 and Z, and overlapped a number of genes with immunological function and QTL connected to health. Only a few regions were consistent between the analyses, and were significant in the FST genome-wide scan and approaching significance in GWAS. This study confirms that resistance to HPAI is a complex, polygenic trait and that mechanisms of resistance may be population specific. Further study utilizing much larger sample sizes and/or sequence data is needed to detect genes responsible for HPAI survival.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses.

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17.

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×106cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9.05 log TCID50/ml for A/Panama/2007/99 influenza H3N2. Porcine strains were also greatly rescued with titres from 4 to 7 log TCID50/ml depending of the subtypes. Interestingly, viral kinetics showed maximal titers reached at 24h post-infection for most of the strains, allowing early harvest time (Time Of Harvest: TOH). The B strains present specific production kinetics with a delay of 24h before reaching the maximal viral particle release. Process optimization on H1N1 2009 human pandemic strain allowed identifying best operating conditions for production (MOI, trypsin concentration, cell density at infection) allowing improving the production level by 2 log. Our results suggest that the DuckCelt®-T17 cell line is a very promising platform for industrial production of influenza viruses and particularly for avian viral strains.

Inter-Species Host Gene Expression Differences in Response to Human and Avian Influenza A Virus Strains.

Low pathogenic avian influenza (LPAI) viruses are a source of sporadic human infections and could also contribute to future pandemic outbreaks but little is known about inter-species differences in the host responses to these viruses. Here, we studied host gene expression signatures of cell lines from three species (human, chicken, and canine) in response to six different viruses (H1N1/WSN, H5N2/F59, H5N2/F118, H5N2/F189, H5N3 and H9N2). Comprehensive microarray probe set re-annotation and ortholog mapping of the host genes was necessary to allow comparison over extended functionally annotated gene sets and orthologous pathways. The annotations are made available to the community for commonly used microarray chips. We observe a strong tendency of the response being cell type- rather than virus-specific. In chicken cells, we found up-regulation of host factors inducing virus infectivity (e.g., oxysterol binding protein like 1A (OSBPL1A) and Rho GTPase activating protein 21 (ARHGAP21)) while reducing apoptosis (e.g., mitochondrial ribosomal protein S27 (MRPS27)) and increasing cell proliferation (e.g., COP9 signalosome subunit 2 (COPS2)). On the other hand, increased antiviral, pro-apoptotic and inflammatory signatures have been identified in human cells while cell cycle and metabolic pathways were down-regulated. This signature describes how low pathogenic avian influenza (LPAI) viruses are being tolerated and shed from chicken but potentially causing cellular disruption in mammalian cells.

Absence of clinical disease and contact transmission of HPAI H5NX clade 2.3.4.4 from North America in experimentally infected pigs.

BACKGROUND: In the fall of 2014, highly pathogenic avian influenza (HPAI) subtype H5N8 clade 2.3.4.4 was introduced into North America by migrating waterfowl from Asia where, through reassortment, novel HPAI H5N2 and H5N1 viruses emerged. OBJECTIVES: Assess the susceptibility of pigs to HPAI H5N1, H5N2, and H5N8 clade 2.3.3.3 from North America. METHODS: Pigs and trachea explants were inoculated with a representative panel of H5NX clade 2.3.4.4 HPAI viruses from North America. Nasal swabs, BALF, and sera were collected to assess replication and transmission in challenged and direct contact pigs by RRT-PCR, virus isolation, hemagglutination inhibition, and ELISA. RESULTS: Limited virus replication was restricted to the lower respiratory tract of challenged pigs, though absent in the nasal passages and trachea cultures, as determined by RRT-PCR in all samples. Seroconversion of inoculated pigs was detected by NP ELISA but was not reliably detected by antigen-specific hemagglutination inhibition. Boost with adjuvanted virus was required for the production of neutralizing antibodies to assess cross-reactivity between wild-type avian strains. All RRT-PCR and serology tests were negative for contact animals indicating a failure of transmission from primary inoculated pigs. CONCLUSIONS: H5NX clade 2.3.4.4 strains can replicate in the lower respiratory tract of swine upon high titer inoculation, though appear to be incapable of replication in swine nasal epithelium in vivo or ex vivo in trachea explants in culture. Infected pigs did not produce high levels of serum antibodies following infection. Collectively, our data show HPAI H5NX clade 2.3.4.4 viruses to be poorly adapted for replication and transmission in swine.

Highlighting the complexities of a groundwater pilot study during an avian influenza outbreak: Methods, lessons learned, and select contaminant results.

The highly pathogenic avian influenza (H5N2) outbreak in the Midwestern United States (US) in 2015 was historic due to the number of birds and poultry operations impacted and the corresponding economic loss to the poultry industry and was the largest animal health emergency in US history. The U.S. Geological Survey (USGS), with the assistance of several state and federal agencies, aided the response to the outbreak by developing a study to determine the extent of virus transport in the environment. The study goals were to: develop the appropriate sampling methods and protocols for measuring avian influenza virus (AIV) in groundwater, provide the first baseline data on AIV and outbreak- and poultry-related contaminant occurrence and movement into groundwater, and document climatological factors that may have affected both survival and transport of AIV to groundwater during the months of the 2015 outbreak. While site selection was expedient, there were often delays in sample response times due to both relationship building between agencies, groups, and producers and logistical time constraints. This study's design and sampling process highlights the unpredictable nature of disease outbreaks and the corresponding difficulty in environmental sampling of such events. The lessons learned, including field protocols and approaches, can be used to improve future research on AIV in the environment.

Systems-based approach to examine the cytokine responses in primary mouse lung macrophages infected with low pathogenic avian Influenza virus circulating in South East Asia.

BACKGROUND: Influenza A virus (IAV) is a major public health concern, being responsible for the death of approximately half a million people each year. Zoonotic transmissions of the virus from swine and avian origin have occurred in the past, and can potentially lead to the emgergence of new IAV stains in future pandemics. Pulmonary macrophages have been implicated in disease severity in the lower airway, and understanding the host response of macrophages infected with avian influenza viruses should provide new therapeutic strategies. RESULTS: We used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) infected with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ with similar efficiency to the H1N1/WSN virus. While all viruses induced antiviral responses, the H5N3 virus infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses. CONCLUSIONS: The LPAI H5N2 and H5N3 viruses are able to infect murine lung macrophages. However, the H5N3 virus was associated with increased expression of pro-inflammatory mediators. Although the H5N3 virus it is capable of inducing high levels of cytokines that are associated with inflammation, this property is distinct from its inability to efficiently replicate in a mammalian host.

Avian and human influenza virus compatible sialic acid receptors in little brown bats.

Influenza A viruses (IAVs) continue to threaten animal and human health globally. Bats are asymptomatic reservoirs for many zoonotic viruses. Recent reports of two novel IAVs in fruit bats and serological evidence of avian influenza virus (AIV) H9 infection in frugivorous bats raise questions about the role of bats in IAV epidemiology. IAVs bind to sialic acid (SA) receptors on host cells, and it is widely believed that hosts expressing both SA α2,3-Gal and SA α2,6-Gal receptors could facilitate genetic reassortment of avian and human IAVs. We found abundant co-expression of both avian (SA α2,3-Gal) and human (SA α2,6-Gal) type SA receptors in little brown bats (LBBs) that were compatible with avian and human IAV binding. This first ever study of IAV receptors in a bat species suggest that LBBs, a widely-distributed bat species in North America, could potentially be co-infected with avian and human IAVs, facilitating the emergence of zoonotic strains.

Differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts.

BACKGROUND: Innate immunity provides first line of defense against viral infections. The interactions between hosts and influenza A virus and the response of host innate immunity to viral infection are critical determinants for the pathogenicity or virulence of influenza A viruses. This study was designed to investigate global changes of gene expression and detailed responses of innate immune systems in human and avian hosts during the course of infection with various subtypes of influenza A viruses, using collected and self-generated transcriptome sequencing data from human bronchial epithelial (HBE), human tracheobronchial epithelial (HTBE), and A549 cells infected with influenza A virus subtypes, namely H1N1, H3N2, H5N1 HALo mutant, and H7N9, and from ileum and lung of chicken and quail infected with H5N1, or H5N2. RESULTS: We examined the induction of various cytokines and chemokines in human hosts infected with different subtypes of influenza A viruses. Type I and III interferons were found to be differentially induced with each subtype. H3N2 caused abrupt and the strongest response of IFN-ß and IFN-λ, followed by H1N1 (though much weaker), whereas H5N1 HALo mutant and H7N9 induced very minor change in expression of type I and III interferons. Similarly, differential responses of other innate immunity-related genes were observed, including TMEM173, MX1, OASL, IFI6, IFITs, IFITMs, and various chemokine genes like CCL5, CX3CL1, and chemokine (C-X-C motif) ligands, SOCS (suppressors of cytokine signaling) genes. Third, the replication kinetics of H1N1, H3N2, H5N1 HALo mutant and H7N9 subtypes were analyzed, H5N1 HALo mutant was found to have the highest viral replication rate, followed by H3N2, and H1N1, while H7N9 had a rate similar to that of H1N1 or H3N2 though in different host cell type. CONCLUSION: Our study illustrated the differential responses of innate immunity to infections of different subtypes of influenza A viruses. We found the influenza viruses which induced stronger innate immune responses replicate slower than those induces weaker innate immune responses. Our study provides important insight into links between the differential innate immune responses from hosts and the pathogenicity/ virulence of different subtypes of influenza A viruses.

H5N2 Highly Pathogenic Avian Influenza Viruses from the US 2014-2015 outbreak have an unusually long pre-clinical period in turkeys.

BACKGROUND: From December 2014 through June 2015, the US experienced the most costly highly pathogenic avian influenza (HPAI) outbreak to date. Most cases in commercial poultry were caused by an H5N2 strain which was a reassortant with 5 Eurasian lineage genes, including a clade 2.3.4.4 goose/Guangdong/1996 lineage hemagglutinin, and 3 genes from North American wild waterfowl low pathogenicity avian influenza viruses. The outbreak primarily affected turkeys and table-egg layer type chickens. Three isolates were selected for characterization in turkeys: the US index isolate from December 2014 (A/northern pintail/WA/40964/2014), and two poultry isolates from April 2015 (A/chicken/IA/13388/2015 and A/turkey/MN/12528/2015). RESULTS: Four week old broad-breasted white turkeys were inoculated with one of three doses (102, 104 or 106 50% egg infectious doses [EID50] per bird) of each of the isolates to evaluate infectious dose and pathogenesis. The mean bird infectious dose of A/northern pintail/WA/40964/2014 and A/turkey/MN/12528/2015 was 105 EID50 per bird, but was 103 EID50 per bird for A/chicken/IA/13388/2015, suggesting the latter had greater adaptation to gallinaceous birds. All three isolates had unusually long mean death time of 5.3-5.9 days post challenge, and the primary clinical signs were severe lethargy and neurological signs which started no more than 24 h before death (the average pre-clinical period was 4 days). Infected turkeys also shed high levels of virus by both the oropharyngeal and cloacal routes. CONCLUSIONS: The unusually long mean death times, high levels of virus in feces, and increased adaptation of the later viruses may have contributed to the rapid spread of the virus during the peak of the outbreak.

Investigation into the Airborne Dissemination of H5N2 Highly Pathogenic Avian Influenza Virus During the 2015 Spring Outbreaks in the Midwestern United States.

We investigated the plausibility of aerosol transmission of H5N2 highly pathogenic avian influenza (HPAI) virus during the 2015 spring outbreaks that occurred in the U.S. midwest. Air samples were collected inside and outside of infected turkey and layer facilities. Samples were tested to assess HPAI virus concentration (RNA copies/m(3) of air), virus viability, and virus distribution by particle size. HPAI virus RNA was detected inside and up to 1000 m from infected facilities. HPAI virus was isolated from air samples collected inside, immediately outside, up to 70 m from infected facilities, and in aerosol particles larger than 2.1 µm. Direct exposure to exhausted aerosols proved to be a significant source of environmental contamination. These findings demonstrate HPAI virus aerosolization from infected flocks, and that both the transport of infectious aerosolized particles and the deposition of particles on surfaces around infected premises represent a potential risk for the spread of HPAI.

Changes in adaptation of H5N2 highly pathogenic avian influenza H5 clade 2.3.4.4 viruses in chickens and mallards.

H5N2 highly pathogenic avian influenza (HPAI) viruses caused a severe poultry outbreak in the United States (U.S.) during 2015. In order to examine changes in adaptation of this viral lineage, the infectivity, pathogenicity and transmission of poultry H5N2 viruses were investigated in chickens and mallards in comparison to the wild duck 2014 U.S. index H5N2 virus. The four poultry isolates examined had a lower mean bird infectious dose than the index virus but still transmitted poorly to direct contacts. In mallards, two of the H5N2 poultry isolates had similar high infectivity and transmissibility as the index H5N2 virus, the H5N8 U.S. index virus, and a 2005 H5N1 clade 2.2 virus. Mortality occurred with the H5N1 virus and, interestingly, with one of two poultry H5N2 isolates. Increased virus adaptation to chickens was observed with the poultry H5N2 viruses; however these viruses retained high adaptation to mallards but pathogenicity was differently affected.

High Rates of Detection of Clade 2.3.4.4 Highly Pathogenic Avian Influenza H5 Viruses in Wild Birds in the Pacific Northwest During the Winter of 2014-15.

In 2014, clade 2.3.4.4 H5N8 highly pathogenic avian influenza (HPAI) viruses spread across the Republic of Korea and ultimately were reported in China, Japan, Russia, and Europe. Mortality associated with a reassortant HPAI H5N2 virus was detected in poultry farms in western Canada at the end of November. The same strain (with identical genetic structure) was then detected in free-living wild birds that had died prior to December 8, 2014, of unrelated causes in Whatcom County, Washington, U. S. A., in an area contiguous with the index Canadian location. A gyrfalcon (Falco rusticolus) that had hunted and fed on an American wigeon (Anas americana) on December 6, 2014, in the same area, and died 2 days later, tested positive for the Eurasian-origin HPAI H5N8. Subsequently, an active surveillance program using hunter-harvested waterfowl in Washington and Oregon detected 10 HPAI H5 viruses, of three different subtypes (four H5N2, three H5N8, and three H5N1) with four segments in common (HA, PB2, NP, and MA). In addition, a mortality-based passive surveillance program detected 18 HPAI (14 H5N2 and four H5N8) cases from Idaho, Kansas, Oregon, Minnesota, Montana, Washington, and Wisconsin. Comparatively, mortality-based passive surveillance appears to have detected these HPAI infections at a higher rate than active surveillance during the period following initial introduction into the United States.

Factors Associated with Highly Pathogenic Avian Influenza H5N2 Infection on Table-Egg Layer Farms in the Midwestern United States, 2015.

A case-control study was conducted among commercial table-egg layer and pullet operations in Iowa and Nebraska, United States, to investigate potential risk factors for infection with highly pathogenic avian influenza (HPAI) H5N2. A questionnaire was developed and administered to 28 case farms and 31 control farms. Data were collected at the farm and barn levels, enabling two separate analyses to be performed-the first a farm-level comparison of case farms vs. control farms, and the second a barn-level comparison between case barns on case farms and control barns on control farms. Multivariable logistic regression models were fit using a forward-selection procedure. Key risk factors identified were farm location in an existing control zone, rendering and garbage trucks coming near barns, dead-bird disposal located near barns, and visits by a company service person. Variables associated with a decreased risk of infection included visitors changing clothing, cleaning and disinfecting a hard-surface barn entryway, and ceiling/eaves ventilation in barns.

Case Series of Turkey Farms from the H5N2 Highly Pathogenic Avian Influenza Outbreak in the United States During 2015.

Between December 2014 and June 2015, an outbreak of H5N2 HPAI caused the largest and most expensive agriculture emergency in U.S. Department of Agriculture-Animal and Plant Health Inspection Service history. The outbreak affected 21 states; 232 poultry farms (211 commercial and 21 backyard) were affected, and approximately 49.6 million birds were depopulated on poultry farms. The majority of affected farms were commercial turkey operations (n = 160). This report is a case series describing 104 H5N2 HPAI-affected turkey farms in Iowa, Minnesota, Missouri, North Dakota, South Dakota, and Wisconsin that had H5N2 HPAI virus detected between March 5 and June 1, 2015. The farm manager or farm personnel voluntarily completed an epidemiologic questionnaire administered by state and federal animal health officials. Equipment and vehicle sharing with other farms was common, particularly for feed trucks (77% of farms shared feed trucks with other farms), live haul loaders (90.4%), poult trailers (72.0%), and preloaders (80.7%). Many farms had water bodies in proximity to the farm, such as a pond (42.6%) or stream (21.8%). About one-third of farms (33.7%) reported seeing wild birds inside the turkey barns. Only 44.2% of farms reported that third-party biosecurity audits or assessments had been conducted. Because the newly introduced Asian H5N8 HPAI and two new HPAI viruses, H5N2 and H5N1, are now circulating in U.S. wild birds, primarily migratory waterfowl, a greater potential for reoccurrence exists with the spring and fall migratory seasons, representing higher risk periods for outbreaks of HPAI in commercial poultry farms in the future. Eliminating exposure to wild birds, especially waterfowl or environments contaminated by wild waterfowl, will reduce risk of reintroduction of H5N2 HPAI virus, and ensuring good on-farm biosecurity will help the poultry industry avoid introduction of influenza and lateral spread between farms.