Highly Pathogenic Avian Influenza Viruses at the Wild–Domestic Bird Interface in Europe: Future Directions for Research and Surveillance

Highly pathogenic avian influenza (HPAI) outbreaks in wild birds and poultry are no longer a rare phenomenon in Europe. In the past 15 years, HPAI outbreaks—in particular those caused by H5 viruses derived from the A/Goose/Guangdong/1/1996 lineage that emerged in southeast Asia in 1996—have been occuring with increasing frequency in Europe. Between 2005 and 2020, at least ten HPAI H5 incursions were identified in Europe resulting in mass mortalities among poultry and wild birds. Until 2009, the HPAI H5 virus outbreaks in Europe were caused by HPAI H5N1 clade 2.2 viruses, while from 2014 onwards HPAI H5 clade 2.3.4.4 viruses dominated outbreaks, with abundant genetic reassortments yielding subtypes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6 and H5N8. The majority of HPAI H5 virus detections in wild and domestic birds within Europe coincide with southwest/westward fall migration and large local waterbird aggregations during wintering. In this review we provide an overview of HPAI H5 virus epidemiology, ecology and evolution at the interface between poultry and wild birds based on 15 years of avian influenza virus surveillance in Europe, and assess future directions for HPAI virus research and surveillance, including the integration of whole genome sequencing, host identification and avian ecology into risk-based surveillance and analyses.     

Highly Pathogenic Avian Influenza Clade 2.3.4.4b Subtype H5N8 Virus Isolated from Mandarin Duck in South Korea, 2020

In October 2020, a highly pathogenic avian influenza (HPAI) subtype H5N8 virus was identified from a fecal sample of a wild mandarin duck (Aix galericulata) in South Korea. We sequenced all eight genome segments of the virus, designated as A/Mandarin duck/Korea/K20-551-4/2020(H5N8), and conducted genetic characterization and comparative phylogenetic analysis to track its origin. Genome sequencing and phylogenetic analysis show that the hemagglutinin gene belongs to H5 clade 2.3.4.4 subgroup B. All genes share high levels of nucleotide identity with H5N8 HPAI viruses identified from Europe during early 2020. Enhanced active surveillance in wild and domestic birds is needed to monitor the introduction and spread of HPAI via wild birds and to inform the design of improved prevention and control strategies.     

Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.     

Subclinical Infection and Transmission of Clade 2.3.4.4 H5N6 Highly Pathogenic Avian Influenza Virus in Mandarin Duck (Aix galericulata) and Domestic Pigeon (Columbia livia domestica)

Since 2014, H5Nx clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIV) have caused outbreaks in wild birds and poultry in multiple continents, including Asia, Europe, Africa, and North America. Wild birds were suspected to be the sources of the local and global spreads of HPAIV. This study evaluated the infectivity, pathogenicity, and transmissibility of clade 2.3.4.4 H5N6 HPAIV in mandarin ducks (Aix galericulata) and domestic pigeons (Columbia livia domestica). None of the birds used in this study, 20 mandarin ducks or 8 pigeons, showed clinical signs or mortality due to H5N6 HPAI infection. Two genotypes of H5N6 HPAIV showed replication and transmission by direct and indirect contact between mandarin ducks. H5N6 HPAIV replicated and transmitted by direct contact between pigeons, although the viral shedding titer and duration were relatively lower and shorter than those in mandarin ducks. Influenza virus antigen was detected in various internal organs of infected mandarin ducks and pigeons, indicating systemic infection. Therefore, our results indicate mandarin ducks and pigeons can be subclinically infected with clade 2.3.4.4 H5N6 HPAIV and transfer the virus to adjacent birds. The role of mandarin ducks and pigeons in the spread and prevalence of clade 2.3.4.4 H5N6 viruses should be carefully monitored.     

Characterization of the In Vitro and In Vivo Efficacy of Baloxavir Marboxil against H5 Highly Pathogenic Avian Influenza Virus Infection

Human infections caused by the H5 highly pathogenic avian influenza virus (HPAIV) sporadically threaten public health. The susceptibility of HPAIVs to baloxavir acid (BXA), a new class of inhibitors for the influenza virus cap-dependent endonuclease, has been confirmed in vitro, but it has not yet been fully characterized. Here, the efficacy of BXA against HPAIVs, including recent H5N8 variants, was assessed in vitro. The antiviral efficacy of baloxavir marboxil (BXM) in H5N1 virus-infected mice was also investigated. BXA exhibited similar in vitro activities against H5N1, H5N6, and H5N8 variants tested in comparison with seasonal and other zoonotic strains. Compared with oseltamivir phosphate (OSP), BXM monotherapy in mice infected with the H5N1 HPAIV clinical isolate, the A/Hong Kong/483/1997 strain, also caused a significant reduction in viral titers in the lungs, brains, and kidneys, thereby preventing acute lung inflammation and reducing mortality. Furthermore, compared with BXM or OSP monotherapy, combination treatments with BXM and OSP using a 48-h delayed treatment model showed a more potent effect on viral replication in the organs, accompanied by improved survival. In conclusion, BXM has a potent antiviral efficacy against H5 HPAIV infections.     

Multiple Reassortants of H5N8 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Viruses Detected in South Korea during the Winter of 2020–2021

During October 2020–January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.     

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas: A Species of Conservation Concern in Great Britain

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance.     

Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses

Highly pathogenic avian influenza viruses (HPAIVs) cause fatal systemic infections in chickens, which are associated with endotheliotropism. HPAIV infections in wild birds are generally milder and not endotheliotropic. Here, we aimed to elucidate the species-specific endotheliotropism of HPAIVs using primary chicken and duck aortic endothelial cells (chAEC and dAEC respectively). Viral replication kinetics and host responses were assessed in chAEC and dAEC upon inoculation with HPAIV H5N1 and compared to embryonic fibroblasts. Although dAEC were susceptible to HPAIV upon inoculation at high multiplicity of infection, HPAIV replicated to lower levels in dAEC than chAEC during multi-cycle replication. The susceptibility of duck embryonic endothelial cells to HPAIV was confirmed in embryos. Innate immune responses upon HPAIV inoculation differed between chAEC, dAEC, and embryonic fibroblasts. Expression of the pro-inflammatory cytokine IL8 increased in chicken cells but decreased in dAEC. Contrastingly, the induction of antiviral responses was stronger in dAEC than in chAEC, and chicken and duck fibroblasts. Taken together, these data demonstrate that although duck endothelial cells are permissive to HPAIV infection, they display markedly different innate immune responses than chAEC and embryonic fibroblasts. These differences may contribute to the species-dependent differences in endotheliotropism and consequently HPAIV pathogenesis.     

Characterization of Clade 2.3.2.1 H5N1 Highly Pathogenic Avian Influenza Viruses Isolated from Wild Birds (Mandarin Duck and Eurasian Eagle Owl) in 2010 in Korea

Starting in late November 2010, the H5N1 highly pathogenic avian influenza (HPAI) virus was isolated from many types of wild ducks and raptors and was subsequently isolated from poultry in Korea. We assessed the genetic and pathogenic properties of the HPAI viruses isolated from a fecal sample from a mandarin duck and a dead Eurasian eagle owl, the most affected wild bird species during the 2010/2011 HPAI outbreak in Korea. These viruses have similar genetic backgrounds and exhibited the highest genetic similarity with recent Eurasian clade 2.3.2.1 HPAI viruses. In animal inoculation experiments, regardless of their originating hosts, the two Korean isolates produced highly pathogenic characteristics in chickens, ducks and mice without pre-adaptation. These results raise concerns about veterinary and public health. Surveillance of wild birds could provide a good early warning signal for possible HPAI infection in poultry as well as in humans.     

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis

A vast diversity of 16 influenza hemagglutinin (HA) subtypes are found in birds. Interestingly, viruses from only two subtypes, H5 and H7, have so far evolved into highly pathogenic avian influenza viruses (HPAIVs) following insertions or substitutions at the HA cleavage site by the viral polymerase. The mechanisms underlying this striking subtype specificity are still unknown. Here, we compiled a comprehensive dataset of 20,488 avian influenza virus HA sequences to investigate differences in nucleotide and amino acid usage at the HA cleavage site between subtypes and how these might impact the genesis of HPAIVs by polymerase stuttering and realignment. We found that sequences of the H5 and H7 subtypes stand out by their high purine content at the HA cleavage site. In addition, fewer substitutions were necessary in H5 and H7 HAs than in HAs from other subtypes to acquire an insertion-prone HA cleavage site sequence, as defined based on in vitro and in vivo data from the literature. Codon usage was more favorable for HPAIV genesis in sequences of viruses isolated from species or geographical regions in which HPAIV genesis is more frequently observed in nature. The results of the present analyses suggest that the subtype restriction of HPAIV genesis to H5 and H7 influenza viruses might be due to the particular codon usage at the HA cleavage site in these subtypes.     

Molecular Characterization of Highly Pathogenic Avian Influenza Viruses H5N6 Detected in Denmark in 2018–2019

Beginning in late 2017, highly pathogenic avian influenza (HPAI) H5N6 viruses caused outbreaks in wild birds and poultry in several European countries. H5N6 viruses were detected in 43 wild birds found dead throughout Denmark. Most of the Danish virus-positive dead birds were found in the period from February to April 2018. However, unlike the rest of Europe, sporadic HPAI H5N6-positive dead wild birds were detected in Denmark in July, August, September, and December 2018, with the last positive bird being found in January 2019. HPAI viruses were not detected in active surveillance of apparently healthy wild birds. In this study, we use full genome sequencing and phylogenetic analysis to investigate the wild bird HPAI H5N6 viruses found in Denmark. The Danish viruses were found to be closely related to those of contemporary HPAI H5N6 viruses detected in Europe. Their sequences formed two clusters indicating that at least two or more introductions of H5N6 into Denmark occurred. Notably, all viruses detected in the latter half of 2018 and in 2019 grouped into the same cluster. The H5N6 viruses appeared to have been maintained undetected in the autumn 2018.     

Genotype Uniformity,Wild Bird-to-Poultry Transmissions,and Farm-to-Farm Carryover during the Spread of the Highly Pathogenic Avian Influenza H5N8 in the Czech Republic in 2021

In 2020–2021, the second massive dissemination of a highly pathogenic avian influenza of the H5Nx subtype occurred in Europe. During this period, the virus caused numerous outbreaks in poultry, including in the Czech Republic. In the present study, we provide an insight into the genetic variability of the Czech/2021 (CZE/2021) H5N8 viruses to determine the relationships between strains from wild and domestic poultry and to infer transmission routes between the affected flocks of commercial poultry. For this purpose, whole genome sequencing and phylogenetic analysis of 70 H5N8 genomes representing 79.7% of the cases were performed. All CZE/2021 H5N8 viruses belonged to the 2.3.4.4b H5 lineage and circulated without reassortment, retaining the A/chicken/Iraq/1/2020 H5N8-like genotype constellation. Phylogenetic analysis suggested the frequent local transmission of H5N8 from wild birds to backyard poultry and extensive spread among commercial poultry farms. In addition, the analysis suggested one cross-border transmission event. Indirect transmission via contaminated materials was considered the most likely source of infection. Improved biosecurity and increased collaboration between field veterinarians and the laboratory are essential to limit the local spread of the virus and to reveal and interrupt critical routes of infection.     

Emergence of a Reassortant 2.3.4.4b Highly Pathogenic H5N1 Avian Influenza Virus Containing H9N2 PA Gene in Burkina Faso,West Africa,in 2021

Since 2006, the poultry population in Burkina Faso has been seriously hit by different waves of Highly Pathogenic Avian Influenza (HPAI) H5N1 epizootics. In December 2021, three distinct regions of Burkina Faso, namely, Gomboussougou, Bonyollo, and Koubri, detected HPAI H5N1 viruses in poultry. Whole genome characterization and statistical phylogenetic approaches were applied to shed light on the potential origin of these viruses and estimate the time of virus emergence. Our results revealed that the HPAI H5N1 viruses reported in the three affected regions of Burkina Faso cluster together within clade 2.3.4.4b, and are closely related to HPAI H5N1 viruses identified in Nigeria and Niger in the period 2021–2022, except for the PA gene, which clusters with H9N2 viruses of the zoonotic G1 lineage collected in West Africa between 2017 and 2020. These reassortant viruses possess several mutations that may be associated with an increased zoonotic potential. Although it is difficult to ascertain where and when the reassortment event occurred, the emergence of a H5N1/H9N2 reassortant virus in a vulnerable region, such as West Africa, raises concerns about its possible impact on animal and human health. These findings also highlight the risk that West Africa may become a new hotspot for the emergence of new genotypes of HPAI viruses.     

Connect to Protect: Dynamics and Genetic Connections of Highly Pathogenic Avian Influenza Outbreaks in Poultry from 2016 to 2021 in Germany

During autumn/winter in 2016–2017 and 2020–2021, highly pathogenic avian influenza viruses (HPAIV) caused severe outbreaks in Germany and Europe. Multiple clade 2.3.4.4b H5 HPAI subtypes were responsible for increased mortality in wild birds and high mortality and massive losses in the poultry sector. To clarify putative entry sources and delineate interconnections between outbreaks in poultry holdings and wild birds, we applied whole-genome sequencing and phylodynamic analyses combined with the results of epidemiological outbreak investigations. Varying outbreak dynamics of the distinct reassortants allowed for the identification of individual, putatively wild bird-mediated entries into backyard holdings, several clusters comprising poultry holdings, local virus circulation for several weeks, direct farm-to-farm transmission and potential reassortment within a turkey holding with subsequent spill-over of the novel reassorted virus into the wild bird population. Whole-genome sequencing allowed for a unique high-resolution molecular epidemiology analysis of HPAIV H5Nx outbreaks and is recommended to be used as a standard tool. The presented detailed account of the genetic, temporal, and geographical characteristics of the recent German HPAI H5Nx situation emphasizes the role of poultry holdings as an important source of novel genetic variants and reassortants.     

Molecular Analysis of the Avian H7 Influenza Viruses Circulating in South Korea during 2018–2019: Evolutionary Significance and Associated Zoonotic Threats

Avian influenza virus (AIV) subtypes H5 and H7, possessing the ability to mutate spontaneously from low pathogenic (LP) to highly pathogenic (HP) variants, are major concerns for enormous socio-economic losses in the poultry industry, as well as for fatal human infections. Through antigenic drift and shift, genetic reassortments of the genotypes pose serious threats of increased virulence and pathogenicity leading to potential pandemics. In this study, we isolated the H7-subtype AIVs circulating in the Republic of Korea during 2018–2019, and perform detailed molecular analysis to study their circulation, evolution, and possible emergence as a zoonotic threat. Phylogenetic and nucleotide sequence analyses of these isolates revealed their distribution into two distinct clusters, with the HA gene sharing the highest nucleotide identity with either the A/common teal/Shanghai/CM1216/2017, isolated from wild birds in Shanghai, China, or the A/duck/Shimane/2014, isolated from Japan. Mutations were found in HA (S138A (H3 numbering)), M1 (N30D and T215A), NS1 (P42S), PB2 (L89V), and PA (H266R and F277S) proteins—the mutations had previously been reported to be related to mammalian adaptation and changes in the virulence of AIVs. Taken together, the results firmly put forth the demand for routine surveillance of AIVs in wild birds to prevent possible pandemics arising from reassortant AIVs.     

Evolutionary Dynamics of H5 Highly Pathogenic Avian Influenza Viruses (Clade 2.3.4.4B) Circulating in Bulgaria in 2019–2021

The first detection of a Highly Pathogenic Avian Influenza (HPAI) H5N8 virus in Bulgaria dates back to December 2016. Since then, many outbreaks caused by HPAI H5 viruses from clade 2.3.4.4B have been reported in both domestic and wild birds in different regions of the country. In this study, we characterized the complete genome of sixteen H5 viruses collected in Bulgaria between 2019 and 2021. Phylogenetic analyses revealed a persistent circulation of the H5N8 strain for four consecutive years (December 2016–June 2020) and the emergence in 2020 of a novel reassortant H5N2 subtype, likely in a duck farm. Estimation of the time to the most recent common ancestor indicates that this reassortment event may have occurred between May 2019 and January 2020. At the beginning of 2021, Bulgaria experienced a new virus introduction in the poultry sector, namely a HPAI H5N8 that had been circulating in Europe since October 2020. The periodical identification in domestic birds of H5 viruses related to the 2016 epidemic as well as a reassortant strain might indicate undetected circulation of the virus in resident wild birds or in the poultry sector. To avoid the concealed circulation and evolution of viruses, and the risk of emergence of strains with pandemic potential, the implementation of control measures is of utmost importance, particularly in duck farms where birds display no clinical signs.     

Comprehensive Characterization of Serum MicroRNA Profile in Response to the Emerging Avian Influenza A (H7N9) Virus Infection in Humans

A novel avian-origin influenza A (H7N9) virus recently occurred in China and caused 137 human infection cases with a 32.8% mortality rate. Although various detection procedures have been developed, the pathogenesis of this emerging virus in humans remains largely unknown. In this study, we characterized serum microRNA (miRNA) profile in response to H7N9 virus infection using TaqMan Low Density Arrays. Upon infection, a total of 395 miRNAs were expressed in the serum pool of patients, far beyond the 221 in healthy controls. Among the 187 commonly expressed miRNAs, 146 were up-regulated and only 7 down-regulated in patients. Further analysis by quantitative RT-PCR revealed that the serum levels of miR-17, miR-20a, miR-106a and miR-376c were significantly elevated in patients compared with healthy individuals (p < 0.05). Receiver operating characteristic (ROC) curves were constructed to show that each miRNA could discriminate H7N9 patients from controls with area under the curve (AUC) values ranging from 0.622 to 0.898, whereas a combination of miR-17, miR-20a, miR-106a and miR-376c obtained a higher discriminating ability with an AUC value of 0.96. Our findings unravel the significant alterations in serum miRNA expression following virus infection and manifest great potential of circulating miRNAs for the diagnosis of viral diseases.     

Epidemiological Features of the Highly Pathogenic Avian Influenza Virus H5N1 in a Densely Populated Area of Lombardy (Italy) during the Epidemic Season 2021–2022

In the last two years, there have been three major epidemic seasons in the territory of the European Union and the HPAI epizootic in 2021–2022 is the most severe in recent history. In Italy, the disease was introduced to dense poultry areas with serious economic consequences for the entire sector. In Lombardy, the analysis of the risk factors was carried out, also taking into account the density of domestic birds. In the most affected areas, 66.7% of the outbreaks occurred in the areas with the highest poultry density and the likelihood of an outbreak occurring increased with an increase in the density of birds per km². In cells 10 × 10 km with a density greater than 10,000 birds/km², the probability of outbreak occurrence was over 66.7%. The provinces involved in the last epidemic were the same involved in previous epidemics and, given the risk factors present in the area, it is plausible that the risk remains high also for future epidemic seasons. Therefore, to avoid the repetition of similar events, certain control measures shall be strengthened and vaccination considered as a complementary tool for the control of HPAI virus in risk areas.     

Molecular Characterization and Pathogenesis of H6N6 Low Pathogenic Avian Influenza Viruses Isolated from Mallard Ducks (Anas platyrhynchos) in South Korea

The subtype H6N6 has been identified worldwide following the increasing frequency of avian influenza viruses (AIVs). These AIVs also have the ability to bind to human-like receptors, thereby increasing the risk of animal-human transmission. In September 2019, an H6N6 avian influenza virus—KNU2019-48 (A/Mallard (Anas platyrhynchos)/South Korea/KNU 2019-48/2019(H6N6))—was isolated from Anas platyrhynchos in South Korea. Phylogenetic analysis results revealed that the hemagglutinin (HA) gene of this strain belongs to the Korean lineage, whereas the neuraminidase (NA) and polymerase basic protein 1 (PB1) genes belong to the Chinese lineage. Outstanding internal proteins such as PB2, polymerase acidic protein, nucleoprotein, matrix protein, and non-structural protein belong to the Vietnamese lineage. Additionally, a monobasic amino acid (PRIETR↓GLF) at the HA cleavage site; non-deletion of the stalk region (residue 59–69) in the NA gene; and E627 in the PB2 gene indicate that the KNU2019-48 isolate is a typical low-pathogenic avian influenza (LPAI) virus. The nucleotide sequence similarity analysis of HA revealed that the highest homology (97.18%) of this isolate is to that of A/duck/Jiangxi/01.14 NCJD125-P/2015(H6N6), and the amino acid sequence of NA (97.38%) is closely related to that of A/duck/Fujian/10.11_FZHX1045-C/2016 (H6N6). An in vitro analysis of the KNU2019-48 virus shows a virus titer of not more than 2.8 Log10 TCID ₅₀/mL until 72 h post-infection, whereas in the lungs, the virus is detected at 3 dpi (days post-infection). The isolated KNU2019-48 (H6N6) strain is the first reported AIV in Korea, and the H6 subtype virus has co-circulated in China, Vietnam, and Korea for half a decade. Overall, our study demonstrates that Korean H6N6 strain PB1-S375N, PA-A404S, and S409N mutations are infectious in humans and might contribute to the enhanced pathogenicity of this strain. Therefore, we emphasize the importance of continuous and intensive surveillance of the H6N6 virus not only in Korea but also worldwide.     

Rapid Detection and Differentiation of Swine-Origin Influenza A Virus (H1N1/2009) from Other Seasonal Influenza A Viruses

We previously developed a rapid and simple gold nanoparticle(NP)-based genomic microarray assay for identification of the avian H5N1 virus and its discrimination from other influenza A virus strains (H1N1, H3N2). In this study, we expanded the platform to detect the 2009 swine-origin influenza A virus (H1N1/2009). Multiple specific capture and intermediate oligonucleotides were designed for the matrix (M), hemagglutinin (HA), and neuraminidase (NA) genes of the H1N1/2009 virus. The H1N1/2009 microarrays were printed in the same format as those of the seasonal influenza H1N1 and H3N2 for the HA, NA, and M genes. Viral RNA was tested using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining. The signal from the 4 capture-target-intermediates of the HA and NA genes was specific for H1N1/2009 virus and showed no cross hybridization with viral RNA from other influenza strains H1N1, H3N2, and H5N1. All of the 3 M gene captures showed strong affinity with H1N1/2009 viral RNA, with 2 out of the 3 M gene captures showing cross hybridization with the H1N1, H3N2, and H5N1 samples tested. The current assay was able to detect H1N1/2009 and distinguish it from other influenza A viruses. This new method may be useful for simultaneous detection and subtyping of influenza A viruses and can be rapidly modified to detect other emerging influenza strains in public health settings.