Emergence, prevalence, and evolution of H5N8 avian influenza viruses in central China, 2020.

Highly pathogenic influenza A(H5N8) viruses have caused several worldwide outbreaks in birds and are able cross the species barrier to infect humans, posing a substantial threat to public health. After the first detection of H5N8 viruses in deceased swans in Inner Mongolia, we performed early warning and active monitoring along swan migration routes in central China. We isolated and sequenced 42 avian influenza viruses, including 40 H5N8 viruses, 1 H5N2 virus, and 1 H9N2 virus, in central China. Our H5N8 viruses isolated in swan stopover sites and wintering grounds showed high nucleotide homologies in the whole genome, revealing a common evolutionary source. Phylogenetic analysis revealed that the H5 viruses of clade 2.3.4.4b prevalent in 2020 have further diverged into two sub-clades: b1 and b2. The phylogeographic analysis also showed that the viruses of sub-clade b2 most likely originated from poultry in Russia. Notably, whooper swans were found to be responsible for the introduction of sub-clade b2 viruses in central China; whooper and tundra swans play a role in viral spread in the Yellow River Basin and the Yangtze River Basin, respectively. Our findings highlight swans as an indicator species for transborder spreading and monitoring of the H5N8 virus.

Genetic Characterization and Pathogenesis of Three Novel Reassortant H5N2 Viruses in South Korea, 2018.

The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds (KNU18-28: A/Wild duck/South Korea/KNU18-28/2018, KNU18-86: A/Bean Goose/South Korea/KNU18-86/2018, and KNU18-93: A/Wild duck/South Korea/KNU18-93/2018). Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia-Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China's H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health.

Estimating epidemiological parameters using diagnostic testing data from low pathogenicity avian influenza infected turkey houses.

Limiting spread of low pathogenicity avian influenza (LPAI) during an outbreak is critical to reduce the negative impact on poultry producers and local economies. Mathematical models of disease transmission can support outbreak control efforts by estimating relevant epidemiological parameters. In this article, diagnostic testing data from each house on a premises infected during a LPAI H5N2 outbreak in the state of Minnesota in the United States in 2018 was used to estimate the time of virus introduction and adequate contact rate, which determines the rate of disease spread. A well-defined most likely time of virus introduction, and upper and lower 95% credibility intervals were estimated for each house. The length of the 95% credibility intervals ranged from 11 to 22 with a mean of 17 days. In some houses the contact rate estimates were also well-defined; however, the estimated upper 95% credibility interval bound for the contact rate was occasionally dependent on the upper bound of the prior distribution. The estimated modes ranged from 0.5 to 6.0 with a mean of 2.8 contacts per day. These estimates can be improved with early detection, increased testing of monitored premises, and combining the results of multiple barns that possess similar production systems.

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.

Ecological factors associated with persistent circulation of multiple highly pathogenic avian influenza viruses among poultry farms in Taiwan during 2015-17.

Emergence and intercontinental spread of highly pathogenic avian influenza A (HPAI) H5Nx virus clade 2.3.4.4 has resulted in substantial economic losses to the poultry industry in Asia, Europe, and North America. The long-distance migratory birds have been suggested to play a major role in the global spread of avian influenza viruses during this wave of panzootic outbreaks since 2013. Poultry farm epidemics caused by multiple introduction of different HPAI novel subtypes of clade 2.3.4.4 viruses also occurred in Taiwan between 2015 and 2017. The mandatory and active surveillance detected H5N3 and H5N6 circulation in 2015 and 2017, respectively, while H5N2 and H5N8 were persistently identified in poultry farms since their first arrival in 2015. This study intended to assess the importance of various ecological factors contributed to the persistence of HPAI during three consecutive years. We used satellite technology to identify the location of waterfowl flocks. Four risk factors consistently showed strong association with the spatial clustering of H5N2 and H5N8 circulations during 2015 and 2017, including high poultry farm density (aOR:17.46, 95%CI: 5.91-74.86 and 8.23, 95% CI: 2.12-54.86 in 2015 and 2017, respectively), poultry heterogeneity index (aOR of 12.28, 95%CI: 5.02-31.14 and 2.79, 95%CI: 1.00-7.69, in 2015 and 2017, respectively), non-registered waterfowl flock density (aOR: 6.8, 95%CI: 3.41-14.46 and 9.17, 95%CI: 3.73-26.20, in 2015 and 2017, respectively) and higher percentage of cropping land coverage (aOR of 1.36, 95%CI: 1.10-1.69 and 1.04, 95%CI: 1.02-1.07, in 2015 and 2017, respectively). Our study highlights the application of remote sensing and clustering analysis for the identification and characterization of environmental factors in facilitating and contributing to the persistent circulation of certain subtypes of H5Nx in poultry farms in Taiwan.

Evolution and extensive reassortment of H5 influenza viruses isolated from wild birds in China over the past decade.

Lethal infection of wild birds with different subtypes of H5 viruses continuously occur. To investigate the genetic evolution and pathogenicity of H5 viruses in wild birds, we performed a detailed genetic and biologic analysis of 27 viruses, including H5N1, H5N2, H5N6, and H5N8 subtypes, that were responsible for avian influenza outbreaks in wild birds in China over the past decade. We found that these 27 viruses, bearing different clades/subclades of HA, were complicated reassortants and formed 12 different genotypes. Ten of the viruses tested were highly pathogenic in chickens, but showed distinct pathotypes in ducks and mice. Five of these 10 viruses, which were all from clade2.3.4.4, could bind human-type receptors. Our findings reveal the diversity of the genetic and biologic properties of H5 viruses circulating in wild birds and highlight the need to carefully monitor and evaluate the risks these viruses pose to animal and public health.

Eco-Epidemiological Evidence of the Transmission of Avian and Human Influenza A Viruses in Wild Pigs in Campeche, Mexico.

Influenza, a zoonosis caused by various influenza A virus subtypes, affects a wide range of species, including humans. Pig cells express both sialyl-α-2,3-Gal and sialyl-α-2,6-Gal receptors, which make them susceptible to infection by avian and human viruses, respectively. To date, it is not known whether wild pigs in Mexico are affected by influenza virus subtypes, nor whether this would make them a potential risk of influenza transmission to humans. In this work, 61 hogs from two municipalities in Campeche, Mexico, were sampled. Hemagglutination inhibition assays were performed in 61 serum samples, and positive results were found for human H1N1 (11.47%), swine H1N1 (8.19%), and avian H5N2 (1.63%) virus variants. qRT-PCR assays were performed on the nasal swab, tracheal, and lung samples, and 19.67% of all hogs were positive to these assays. An avian H5N2 virus, first reported in 1994, was identified by sequencing. Our results demonstrate that wild pigs are participating in the exposure, transmission, maintenance, and possible diversification of influenza viruses in fragmented habitats, highlighting the synanthropic behavior of this species, which has been poorly studied in Mexico.

Development and Evaluation of Real-Time Reverse Transcription Polymerase Chain Reaction Test for Quantitative and Qualitative Recognition of H5 Subtype of Avian Influenza Viruses.

Avian influenza viruses (AIV) affect a wide range of birds and mammals, cause severe economic damage to the poultry industry, and pose a serious threat to humans. Highly pathogenic avian influenza viruses (HPAI) H5N1 were first identified in Southeast Asia in 1996 and spread to four continents over the following years. The viruses have caused high mortality in chickens and various bird species and deadly infections in humans. Multiple conventional methods have been so far introduced for the detection and identification of avian influenza viruses. Traditional virus isolation methods are gold standard protocol in AI detection; nonetheless, virus isolation in embryonating chicken eggs (ECE) is not a rapid method for the detection of influenza viruses since it is time-consuming and labor-intensive. Furthermore, the isolation of highly pathogenic viruses, such as H5, needs BSL3 laboratories. Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR) is a sensitive and specific method for the detection of influenza viruses. The application of these nucleic acid-based techniques has increased our ability to identify and perform influenza virus care programs, especially in surveillance programs. The current study aimed to detect H5 subtype of avian influenza (AI) virus using fast, specific, and sensitive TaqMan RRT-PCR. Notably, single step RRT-PCR was used to prevent possible laboratory contamination. The specificity of this test was evaluated using nucleic acid extracted from several poultry pathogenic microorganisms and negative clinical specimens from AI-uninfected birds. The sensitivity analysis of the RRT-PCR assay was performed using in vitro-transcribed RNA copy and 10-fold serial dilution of standard AI virus with specific titer. The results indicated the high sensitivity of this method and the lowest detectable dilution of this method based on RNA copies and 1:10 serial dilutions of the standard virus was 10 1.9 EID50 /100.

Agricultural and geographic factors shaped the North American 2015 highly pathogenic avian influenza H5N2 outbreak.

The 2014-2015 highly pathogenic avian influenza (HPAI) H5NX outbreak represents the largest and most expensive HPAI outbreak in the United States to date. Despite extensive traditional and molecular epidemiological studies, factors associated with the spread of HPAI among midwestern poultry premises remain unclear. To better understand the dynamics of this outbreak, 182 full genome HPAI H5N2 sequences isolated from commercial layer chicken and turkey production premises were analyzed using evolutionary models able to accommodate epidemiological and geographic information. Epidemiological compartmental models embedded in a phylogenetic framework provided evidence that poultry type acted as a barrier to the transmission of virus among midwestern poultry farms. Furthermore, after initial introduction, the propagation of HPAI cases was self-sustainable within the commercial poultry industries. Discrete trait diffusion models indicated that within state viral transitions occurred more frequently than inter-state transitions. Distance and sample size were very strongly supported as associated with viral transition between county groups (Bayes Factor > 30.0). Together these findings indicate that the different types of midwestern poultry industries were not a single homogenous population, but rather, the outbreak was shaped by poultry industries and geographic factors.

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Virus de influenza aviar del subtipo H5 altamente patógenos y de baja patogenicidad en aves silvestres en Ucrania. Ha habido tres oleadas de brotes de influenza aviar altamente patógena en aves comerciales, de traspatio y en aves silvestres en Ucrania. La primera (2005-2006) y la segunda (2008) fueron causadas por el virus de influenza aviar de alta patogenicidad H5N1, con 45 brotes en aves comerciales (pollos) y aves de traspatio (pollos, patos y gansos) en cuatro regiones de Ucrania (AR Crimea, Kherson, Odesa y Sumy Oblast). Los virus de alta patogenicidad H5N1se aislaron de aves silvestres muertas: cormoranes (Phalacrocorax carbo) y de somormujos lavanco (Podiceps cristatus) en 2006 y 2008. La tercera ola del virus de influenza aviar de alta patogenicidad consistió en nueve brotes del virus de alta patogenicidad subtipo H5N8 en aves silvestres y domésticas, a partir de noviembre de 2016 en las regiones central y sur (Kherson, Odesa, Chernivtsi, Ternopil y Mykolaiv Oblast). Se detectó el virus al patogenicidad H5N8 en cisnes blancos muertos (Cygnus olor), pavos reales (Pavo cristatus) (en zoológicos), tarros canelos (Tadorna ferruginea), gansos caretos (Anser albifrons) y en muestras ambientales en 2016 y 2017. Una vigilancia más amplia de aves silvestres para detectar el virus de la influenza aviar se realizó entre 2006 y 2016 en las regiones de Ucrania sospechosas de ser rutas migratorias intercontinentales (norte-sur y este-oeste). Se recolectaron un total de 21,511 muestras de 105 especies de aves silvestres que representan a 27 familias y 11 órdenes. Se aislaron ochenta y dos virus de influenza aviar de baja patogenicidad (incluido un virus H5N2 de baja patogenicidad del 2010) de aves silvestres con un total de 23 combinaciones antigénicas de hemaglutininas (HA) y neuraminidasas (NA). Se aislaron quince de los 16 subtipos de HA aviar conocidos. Dos virus de alta patogenicidad H5N8 y dos virus H5N2 de baja patogenicidad se aislaron de aves silvestres vivas y de muestras ambientales (heces de aves frescas) durante la vigilancia antes del brote en avicultura. Los virus ucranianos de alta patogenicidad H5N1, H5N8 y de baja patogenicidad H5N2 pertenecen a diferentes grupos filogenéticos de H5. Estos resultados demuestran la gran diversidad de virus de la influenza aviar en aves silvestres en Ucrania, así como la importancia de esta región para estudiar la ecología de la influenza aviar.

Smartphone-Based Point-of-Care Microfluidic Platform Fabricated with a ZnO Nanorod Template for Colorimetric Virus Detection.

Viruses pose serious infectious disease threats to humans and animals. To significantly decrease the mortality and morbidity caused by virus infections, there is an urgent need of sensitive and rapid point-of-care platforms for virus detection, especially in low-resource settings. Herein, we developed a smartphone-based point-of-care platform for highly sensitive and selective detection of the avian influenza virus based on nanomaterial-enabled colorimetric detection. The 3D nanostructures, which serve as a scaffold for antibody conjugation to capture the avian influenza virus, are made on PDMS herringbone structures with a ZnO nanorod template. After virus capture, the on-chip gold nanoparticle-based colorimetric reaction allows virus detection by naked eyes with a detection limit of 2.7 × 104 EID50/mL, which is one order of magnitude better than that of conventional fluorescence-based ELISA. Furthermore, a smartphone imaging system with data processing capability further improves the detection limit, reaching down to 8 × 103 EID50/mL. The entire virus capture and detection process can be completed in 1.5 h. We envision that this point-of-care microfluidic system integrated with smartphone imaging and colorimetric detection would provide a fast, cheap, sensitive, and user-friendly platform for virus detection in low-resource settings.

Airborne transmission may have played a role in the spread of 2015 highly pathogenic avian influenza outbreaks in the United States.

The unprecedented 2015 outbreaks of highly pathogenic avian influenza (HPAI) H5N2 in the U.S. devastated its poultry industry and resulted in over $3 billion economic impacts. Today HPAI continues eroding poultry operations and disrupting animal protein supply chains around the world. Anecdotal evidence in 2015 suggested that in some cases the AI virus was aerially introduced into poultry houses, as abnormal bird mortality started near air inlets of the infected houses. This study modeled air movement trajectories and virus concentrations that were used to assess the probability or risk of airborne transmission for the 77 HPAI cases in Iowa. The results show that majority of the positive cases in Iowa might have received airborne virus, carried by fine particulate matter, from infected farms within the state (i.e., intrastate) and infected farms from the neighboring states (i.e., interstate). The modeled airborne virus concentrations at the Iowa recipient sites never exceeded the minimal infective doses for poultry; however, the continuous exposure might have increased airborne infection risks. In the worst-case scenario (i.e., maximum virus shedding rate, highest emission rate, and longest half-life), 33 Iowa cases had > 10% (three cases > 50%) infection probability, indicating a medium to high risk of airborne transmission for these cases. Probability of airborne HPAI infection could be affected by farm type, flock size, and distance to previously infected farms; and more importantly, it can be markedly reduced by swift depopulation and inlet air filtration. The research results provide insights into the risk of airborne transmission of HPAI virus via fine dust particles and the importance of preventative and containment strategies such as air filtration and quick depopulation of infected flocks.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

Highly Pathogenic and Low Pathogenic Avian Influenza H5 Subtype Viruses in Wild Birds in Ukraine.

There have been three waves of highly pathogenic avian influenza (HPAI) outbreaks in commercial, backyard poultry, and wild birds in Ukraine. The first (2005-2006) and second (2008) waves were caused by H5N1 HPAI virus, with 45 outbreaks among commercial poultry (chickens) and backyard fowl (chickens, ducks, and geese) in four regions of Ukraine (AR Crimea, Kherson, Odesa, and Sumy Oblast). H5N1 HPAI viruses were isolated from dead wild birds: cormorants (Phalacrocorax carbo) and great crested grebes (Podiceps cristatus) in 2006 and 2008. The third HPAI wave consisted of nine outbreaks of H5N8 HPAI in wild and domestic birds, beginning in November 2016 in the central and south regions (Kherson, Odesa, Chernivtsi, Ternopil, and Mykolaiv Oblast). H5N8 HPAI virus was detected in dead mute swans (Cygnus olor), peacocks (Pavo cristatus) (in zoo), ruddy shelducks (Tadorna ferruginea), white-fronted geese (Anser albifrons), and from environmental samples in 2016 and 2017. Wide wild bird surveillance for avian influenza (AI) virus was conducted from 2006 to 2016 in Ukraine regions suspected of being intercontinental (north-south and east-west) flyways. A total of 21 511 samples were collected from 105 species of wild birds representing 27 families and 11 orders. Ninety-five avian influenza (AI) viruses were isolated (including one H5N2 LPAI virus in 2010) from wild birds with a total of 26 antigenic hemagglutinin (HA) and neuraminidase (NA) combinations. Fifteen of 16 known avian HA subtypes were isolated. Two H5N8 HPAI viruses (2016-2017) and two H5N2 LPAI viruses (2016) were isolated from wild birds and environmental samples (fresh bird feces) during surveillance before the outbreak in poultry in 2016-2017. The Ukrainian H5N1, H5N8 HPAI, and H5N2 LPAI viruses belong to different H5 phylogenetic groups. Our results demonstrate the great diversity of AI viruses in wild birds in Ukraine, as well as the importance of this region for studying the ecology of avian influenza.

Virus de influenza aviar del subtipo H5 altamente patógenos y de baja patogenicidad en aves silvestres en Ucrania. Ha habido tres oleadas de brotes de influenza aviar altamente patógena en aves comerciales, de traspatio y en aves silvestres en Ucrania. La primera (2005-2006) y la segunda (2008) fueron causadas por el virus de influenza aviar de alta patogenicidad H5N1, con 45 brotes en aves comerciales (pollos) y aves de traspatio (pollos, patos y gansos) en cuatro regiones de Ucrania (AR Crimea, Kherson, Odesa y Sumy Oblast). Los virus de alta patogenicidad H5N1se aislaron de aves silvestres muertas: cormoranes (Phalacrocorax carbo) y de somormujos lavanco (Podiceps cristatus) en 2006 y 2008. La tercera ola del virus de influenza aviar de alta patogenicidad consistió en nueve brotes del virus de alta patogenicidad subtipo H5N8 en aves silvestres y domésticas, a partir de noviembre de 2016 en las regiones central y sur (Kherson, Odesa, Chernivtsi, Ternopil y Mykolaiv Oblast). Se detectó el virus al patogenicidad H5N8 en cisnes blancos muertos (Cygnus olor), pavos reales (Pavo cristatus) (en zoológicos), tarros canelos (Tadorna ferruginea), gansos caretos (Anser albifrons) y en muestras ambientales en 2016 y 2017. Una vigilancia más amplia de aves silvestres para detectar el virus de la influenza aviar se realizó entre 2006 y 2016 en las regiones de Ucrania sospechosas de ser rutas migratorias intercontinentales (norte-sur y este-oeste). Se recolectaron un total de 21,511 muestras de 105 especies de aves silvestres que representan a 27 familias y 11 órdenes. Se aislaron ochenta y dos virus de influenza aviar de baja patogenicidad (incluido un virus H5N2 de baja patogenicidad del 2010) de aves silvestres con un total de 23 combinaciones antigénicas de hemaglutininas (HA) y neuraminidasas (NA). Se aislaron quince de los 16 subtipos de HA aviar conocidos. Dos virus de alta patogenicidad H5N8 y dos virus H5N2 de baja patogenicidad se aislaron de aves silvestres vivas y de muestras ambientales (heces de aves frescas) durante la vigilancia antes del brote en avicultura. Los virus ucranianos de alta patogenicidad H5N1, H5N8 y de baja patogenicidad H5N2 pertenecen a diferentes grupos filogenéticos de H5. Estos resultados demuestran la gran diversidad de virus de la influenza aviar en aves silvestres en Ucrania, así como la importancia de esta región para estudiar la ecología de la influenza aviar.

Specific detection of avian influenza H5N2 whole virus particles on lateral flow strips using a pair of sandwich-type aptamers.

We report a selection of a cognate pair of aptamers for whole avian influenza virus particles of H5N2 by using graphene-oxide based systemic evolution of ligands by exponential enrichment (GO-SELEX), and the application of a pair of sandwich-type binding aptamers on the lateral flow strips. The aptamers were characterized by GO-FRET assay, and Kd values of the selected aptamers were estimated to be from 6.913 × 105 to 1.27 × 106 EID50/ml (EID50/ml: 50% egg infective dose). Based on the evidence from confocal laser scanning microscope (CLSM), surface plasmon resonance (SPR), and circular dichroism (CD) spectrum analysis, the aptamers, J3APT and JH4APT, were found to be working as a cognate pair that binds to the target virus at the different sites simultaneously. This cognate pair of aptamers then was successfully applied on the lateral flow strips, clearly showing sandwich-type binding images with the presence of the certain numbers of H5N2 virus particles. On the newly developed lateral flow strips, the target virus was detectable down to 6 × 105 EID50/ml in the buffer and 1.2 × 106 EID50/ml in the duck's feces, respectively, by the naked eye. By using the ImageJ software, the LOD was found to be 1.27 × 105 EID50/ml in the buffer and 2.09 × 105 EID50/ml in the duck's feces, respectively. Interestingly, on the lateral flow strips, enhanced specificity towards the target virus (H5N2) appeared over other subtypes of H5Nx. To the best of our knowledge, this is the first report about the application of the cognate pair of aptamers for the detection of influenza virus on the lateral flow strips. This study shows the promising perspective of a cognate pair of aptamers for the on-site detection system which could be useful for rapid detection of avian influenza viruses for preventing the pandemic influenza viruses from spreading.

Shedding of clade 2.3.4.4 H5N8 and H5N2 highly pathogenic avian influenza viruses in peridomestic wild birds in the U.S.

European starlings (Sturnus vulgaris), house sparrows (Passer domesticus) and rock pigeons (Columba livia) are all wild birds commonly found in large numbers in and around human dwellings and domestic livestock operations. This study evaluated the susceptibility of these species to three strains of highly pathogenic avian influenza virus (HP AIV) clade 2.3.4.4 isolated in the U.S.. Experimental infection of European starlings and rock pigeons did not result in any overt signs attributable to AIV infection and no virus shedding was detected from the oral and cloacal routes. House sparrows shed by the oral route and exhibited limited mortality. Individuals from all three species seroconverted following infection. These data suggest that none of these birds are a likely potential bridge host for future HP AIV outbreaks but that their seroconversion may be a useful surveillance tool for detection of circulating H5 HP AIV.

Multiplex one-step real-time PCR assay for rapid simultaneous detection of velogenic and mesogenic Newcastle disease virus and H5-subtype avian influenza virus.

H5 avian influenza virus (AIV) and velogenic Newcastle disease virus (v-NDV) are pathogens listed in the OIE Terrestrial Animal Health Code and are considered key pathogens to be eliminated in poultry production. Molecular techniques for rapid detection of H5 AIV and v-NDV are required to investigate their transmission characteristics and to guide prevention. Traditional virus isolation, using embryonated chicken eggs, is time-consuming and cannot be used as a rapid diagnostic technology. In this study, a multiplex real-time RT-PCR (RRT-PCR) detection method for six H5 AIV clades, three v-NDV subtypes, and one mesogenic NDV subtype was successfully established. The detection limit of our multiplex NDV and H5 AIV RRT-PCR was five copies per reaction for each pathogen, with good linearity and efficiency (y = -3.194x + 38.427 for H5 AIV and y = -3.32x + 38.042 for NDV). Multiplex PCR showed good intra- and inter-assay reproducibility, with coefficient of variance (CV) less than 1%. Furthermore, using the RRT-PCR method, H5 AIV and NDV detection rates in clinical samples were higher overall than those obtained using the traditional virus isolation method. Therefore, our method provides a promising technique for surveillance of various H5 AIV clades and multiple velogenic and mesogenic NDV subtypes in live-poultry markets.

Genetic and biological characterization of two reassortant H5N2 avian influenza A viruses isolated from waterfowl in China in 2016.

Two reassortant H5N2 viruses in which hemagglutinin (HA) was clustered into clade 2.3.4.4, were isolated from apparently healthy waterfowl in live poultry markets in Eastern China in 2016. We used specific pathogen-free chickens, mallard ducks, and BALB/c mice to evaluate the isolates' biological characteristics in different animal models. The newly isolated reassortant H5N2 viruses were able to cause severe disease in chickens and effective contact transmission, only at high doses. Our pathogenicity studies in ducks yielded an interesting result: the intravenous pathogenicity index (IVPI) indicated that isolate A/goose/Eastern China/1106/2016(1106) was low pathogenic and the other isolate A/duck/Eastern China/YD1516/2016(YD1516) was of highly pathogenicity in ducks. However, our 50% duck lethal dose (DLD50) experiment demonstrated that these viruses were all of low pathogenicity (DLD50 > 107.0 EID50) in ducks. Additionally, despite the fact that reassortant H5N2 were of low pathogenicity in mice, they could bind to both avian-type (SAα-2,3 Gal) and human-type (SAα-2,6 Gal) receptors, suggesting that these isolates still present a high risk for human infection. Therefore, it is of great importance to implement continual surveillance of avian influenza virus (AIV) to protect both veterinary and public health.

Spatial transmission of H5N2 highly pathogenic avian influenza between Minnesota poultry premises during the 2015 outbreak.

The spatial spread of highly pathogenic avian influenza (HPAI) H5N2 during the 2015 outbreak in the U.S. state of Minnesota was analyzed through the estimation of a spatial transmission kernel, which quantifies the infection hazard an infectious premises poses to an uninfected premises some given distance away. Parameters were estimated using a maximum likelihood method for the entire outbreak as well as for two phases defined by the daily number of newly detected HPAI-positive premises. The results indicate both a strong dependence of the likelihood of transmission on distance and a significant distance-independent component of outbreak spread for the overall outbreak. The results further suggest that HPAI spread differed during the later phase of the outbreak. The estimated spatial transmission kernel was used to compare the Minnesota outbreak with previous HPAI outbreaks in the Netherlands and Italy to contextualize the Minnesota transmission kernel results and make additional inferences about HPAI transmission during the Minnesota outbreak. Lastly, the spatial transmission kernel was used to identify high risk areas for HPAI spread in Minnesota. Risk maps were also used to evaluate the potential impact of an early marketing strategy implemented by poultry producers in a county in Minnesota during the outbreak, with results providing evidence that the strategy was successful in reducing the potential for HPAI spread.