A Comprehensive Analysis of H5N1 Evolution: Phylogenetic Insights and Emerging Mutations in Turkey's Avian Influenza Landscape

A Comprehensive Analysis of H5N1 Evolution: Phylogenetic Insights and Emerging Mutations in Turkey's Avian Influenza Landscape | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A Comprehensive Analysis of H5N1 Evolution: Phylogenetic Insights and Emerging Mutations in Turkey's Avian Influenza Landscape Hamza KADI, Ahmet Yilmaz COBAN, Ozge CAGIRGAN, Mehmet NOTUROGLU, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3831007/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Avian influenza (AI) H5N1 viruses, known for their high mortality in humans and poultry, pose a significant public health threat. This study examines seventeen H5N1 isolates from the 2006 outbreak in Turkey and one 2023 isolate for mutations in the hemagglutinin gene's receptor binding domain, which determines host specificity, and the evolutionary changes in the isolates. The hemagglutinin gene region of these isolates was partially amplified using RT-PCR, and critical mutations in the receptor binding domains were examined, alongside the phylogenetic relationships of the isolates in a time- and geography-dependent manner. Our findings showed that key mutations known for altering host selectivity (N224K, G225D, Q226L, S227N, G228S) were absent. However, additional mutations (D110N and S171N), potentially affecting receptor selectivity, were identified. Comprehensive phylogenetic analysis, conducted separately based on geographic regions and temporal distribution, encompassed H5N1 strains isolated from various locations and hosts from 2007 to 2023. This study highlights significant genetic divergence between the 2006 Turkish isolates and the 2023 isolate, highlighting notable evolutionary changes. Distinct clustering of the isolates was observed in both geographic and temporal phylogenetic frameworks, indicating substantial evolutionary shifts within the circulating H5N1 strains in Turkey. Given the global spread potential of H5N1 viruses via migratory birds and the risk of a new pandemic if human-to-human transmission is attained, monitoring H5N1's molecular traits and evolution is crucial for mitigating public health impacts. H5N1 enzyme hemagglutinin mutation receptor Figures Figure 1 Figure 2 Figure 3 Inroduction Influenza A viruses (AIVs), classified under the family Orthomyxoviridae and genus Influenzavirus A, are negative-sense, single-stranded RNA viruses with a global presence, primarily found in wild waterfowl and other aquatic birds. The viral genome of AIVs comprises eight RNA segments that encode at least twelve key proteins, including the basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural (NS) proteins. Notably, AIV subtypes are distinguished based on the antigenic properties of two surface glycoproteins, HA and NA. To date, sixteen HA subtypes (H1–H16) and nine NA subtypes (N1–N9) have been identified in aquatic birds. Additionally, recent discoveries have revealed two novel HA subtypes (H17 and H18) and two novel NA subtypes (N10 and N11) in bats, expanding our understanding of AIV diversity [ 1 – 3 ]. H5N1 viruses, which naturally circulate in aquatic birds, have increasingly become integrated into poultry populations, leading to their endemic presence in domestic birds in some countries [ 4 ]. This significant shift from wild to domestic hosts highlights the ongoing risk of H5N1 viruses adapting to new species, including the potential for increased human infections. Additionally, the sporadic transmission of Avian Influenza Viruses (AIVs) from waterfowl to domestic avian species has been documented, further emphasizing the complexity of H5N1 transmission dynamics [ 1 ]. Human-to-human transmission of influenza viruses can occur through direct contact, indirect contact via fomites (contaminated environmental surfaces) and/or airborne transmission via small aerosols or large respiratory droplets [ 5 ]. The potential for Avian Influenza Viruses (AIV) to enhance transmissibility in mammals depends on the acquisition and selection of mutations during spread. Although numerous experimental mutation studies have been conducted, data from natural infections remain limited [ 4 ]. The first human deaths directly attributable to avian A/H5N1 virus were recorded in Hong Kong in 1997, and it has since become important for humans [ 6 ]. HA proteins bind to sialoside receptors on the host cell surface. Both avian and human influenza A viruses exhibit a preference for binding sialic acids connected to a penultimate galactose through α2,3 or α2,6 linkages, respectively [ 7 ]. Human influenza A viruses recognize the α2,6-galactose (SAα2,6Gal) saccharides situated at the terminus of sialic acid chains. In contrast, avian influenza A viruses identify the α2,3-galactose (SAα2,3Gal) saccharides, which are also present among sialic acid receptors. The transition from SAα2,3Gal to SAα2,6Gal represents a crucial change, suggesting that avian influenza (AI) viruses can effectively replicate and cause pandemics in humans. Identifying mutations in the receptor-binding site of the HA molecule in H5N1 influenza viruses can offer insights into potential future pandemics [ 8 – 10 ]. Although H5N1 viruses have not yet acquired the ability to be transmitted among humans, the absence of immunity in humans to influenza viruses carrying the H5 HA raises the likelihood of an emerging, transmissible H5 HA-carrying virus leading to a pandemic. Consequently, understanding the molecular changes that may occur in H5 HA-carrying viruses is crucial for preparedness in such scenarios. This information can be valuable for monitoring variants with pandemic potential, focusing eradication efforts on viruses already possessing critical molecular changes for mammalian transmission, stockpiling antiviral compounds in regions where such viruses circulate, and initiating vaccine production and large-scale manufacturing ahead of a potential pandemic [ 12 ]. Among the AIV subtypes, H5N1 has garnered significant attention due to its potential for zoonotic transmission and severe impact on avian and human health. Since its initial detection in the late 20th century, H5N1 has evolved through various genetic reassortments and mutations, leading to the emergence of multiple strains with varying pathogenicity and transmissibility. This continuous evolution poses a significant challenge for public health, necessitating ongoing surveillance and research to track its progression and adapt strategies for control and prevention. In Turkey, as in many parts of the world, H5N1 has been a persistent concern, impacting both wild and domestic avian populations. Given the strategic geographical location of Turkey, bridging Asia and Europe, the country serves as a crucial point in the migration routes of wild birds, thereby playing a key role in the dissemination and evolution of H5N1 strains. This study aims to provide a comprehensive analysis of the molecular evolution of H5N1 in Turkey over a span of 17 years, from 2006 to 2023. By examining 18 isolates from different time points, this research focuses on identifying significant mutations in the receptor-binding regions of the hemagglutinin gene, which are crucial for host specificity and viral infectivity. Additionally, the study conducts phylogenetic analyses based on geographical and temporal distribution to understand the evolutionary trajectory of H5N1 in the region. Such insights are vital for informing both national and global strategies in avian influenza surveillance and response. Materials and Methods Ethics committee approval The study was deemed appropriate with the approval of the Samsun Veterinary Control Institute Ethics Committee. (Date: 29.11.2016, Number: 11 − 2). AI H5N1 isolates Seventeen AI H5N1 isolates from poultry during the AI epidemic, which occurred in the Black Sea region of Turkey in the first quarter of 2006 (January–February) and reported to the World Organization for Animal Health (WOAH), were initially used in this study (Fig. 1 ). To supplement these findings, a recent H5N1 isolate obtained from the Aegean region of Turkey in 2023 has been included to provide a current perspective on the evolution of the virus. Both sets of isolates were handled using the same protocol to ensure comparability of the results. To stimulate viruses and increase their amount, the 2006 isolates and the 2023 isolate were inoculated into the chorioallantoic membranes of 9–11 days old specific pathogen-free (SPF) embryo-bearing chicken eggs [ 12 ]. The eggs were checked daily for viability, and chorioallantoic fluid was collected on the third through fifth days post-inoculation. The newly obtained 2023 isolate was processed in the same manner as the 2006 isolates to maintain consistency in the experimental approach. RNA extraction Viral RNA was extracted from the chorioallantoic fluid using a commercial nucleic acid extraction kit (Roche High Pure Viral Nucleic Acid Kit) according to the manufacturer's instructions and stored at -20 ℃ for later use. Amplification of the HA gene receptor-binding site by RT-PCR The HA gene's receptor-binding site, corresponding to the HA1 region [ 13 ], was analyzed for previously isolated Turkish H5N1 strains (GenBank accession nos. DQ407519, KF042152, KF042153, and EF619980) using Bioedit software. Specific primers, HK1F1 (5'-GATCAGATTTGCATTGGTTACC-3') and HK2R1 (5'-TTGAGGGCTATTTCTGAGCCCAG-3'), were designed for RT-PCR amplification using the Qiagen one-step kit. The reaction setup included 24.5 µl of RNase-free water, 10 µl 5X buffer, 2 µl of dNTPs, 3 µl of each primer, 0.5 µl RNase inhibitor, 2 µl enzyme mix, and 5 µl template RNA. Thermal cycling conditions were: 30 min at 50℃ for reverse transcription, 15 min at 95℃ for inactivation of reverse transcriptase/activation of Taq polymerase, followed by 40 cycles of 1 min at 94℃ for denaturation, 1 min at 55℃ for annealing, and 1 min at 72℃ for extension, with a final extension at 72℃ for 10 min. Sanger sequencing and phylogenetic analysis The sequencing of the PCR products was carried out by BM LABOSIS Gene Research and Biotechnology, based in Ankara, Turkey. The nucleotide sequences obtained were edited and assembled using BioEdit software (version 7.0.5) and submitted to GenBank, securing the corresponding accession numbers. Subsequent alignment of the hemagglutinin gene sequences was performed using the ClustalW algorithm within the MEGA X software [ 14 ], in comparison with other sequences sourced from public databases such as the National Center for Biotechnology Information Virus (NCBI Virus). Within the MEGA X environment, the most appropriate nucleotide substitution model was selected based on the Bayesian Information Criterion (BIC), and phylogenetic trees with geographic and temporal structure were constructed using the Maximum Likelihood method, supported by 1000 bootstrap replicates, to analyze the spatiotemporal dynamics of the viral evolution. Lastly, sequence data were examined for significant amino acid changes in the hemagglutinin gene receptor binding site that could substantially influence host selectivity, utilizing the FluSurver database with the A/goose/Guangdong/1/1996 (H5N1) isolate as a reference. ( http://flusurver.bii.a-star.edu.sg ). Results Determination of the receptor-binding site of the HA gene The receptor-binding site of the HA genes that belong to the AIV H5N1 isolates were amplified by RT-PCR. Subsequently, to determine the critical amino acid positions within the receptor-binding region of the HA gene and to elucidate their phylogenetic relationships, further studies were undertaken. These investigations involved a detailed comparative analysis of the HA gene sequences across different AIV H5N1 strains, enabling a comprehensive understanding of the structural variations and their potential impact on the virus's affinity towards host receptors. The sequencing results of the HA receptor-binding site and phylogenetic analysis In this study, none of the 18 isolates from 2006 and 2023 exhibited the significant amino acid substitutions at positions 224, 225, 226, 227, 228 (H3 numbering), which are associated with host selectivity. However, according to the Flusurver database, the mutation D110N was identified in all isolates except A/chicken/Brnvoa/2023, while the mutation S171N was found in eight isolates. Notably, the isolate with the accession number OK103838 possessed the unique S227I mutation (Table-1). Similarly, the newly analyzed A/chicken/Brnvoa/2023 Turkish isolate also presented a mutation at the position, S149A, contributing further data to the evolving pattern of amino acid substitutions observed in these avian influenza viruses. The phylogenetic analysis based on the geographical distribution of the HA gene of H5N1 isolates reveals a cohesive clustering of the Turkish isolates from 2006, indicating a regional homogeneity. In contrast, the isolate from Turkey in 2023 exhibits significant divergence from the 2006 cohort and demonstrates closer phylogenetic relationships with isolates from France (A/Mule-duck/France/21351/2021) and Spain (A/Anser anser/Spain/750-3/2022), isolated in the years 2021 and 2022, respectively (Figure-2). When examining the temporal phylogenetic tree, it is evident that the Turkish isolates from 2006 are closely situated to one another, forming a distinct cluster. The 2023 Turkish isolate, appears to share a closer relationship with an isolate from Russia (A/common teal/Chelyabinsk/1379-1/2021). Furthermore, the genetic differentiation between the Turkish isolates of 2006 and the 2023 Turkish isolate is strikingly evident (Figure-3) The combined analysis of both trees underscores the homogeneous clustering of the 2006 isolates, while the 2023 Turkish isolate (A/chicken/Turkey/Brnvoa/2023) is genetically distinct from this group. These findings suggest a potential evolution or introduction of a new lineage of H5N1 in Turkey since 2006, highlighting the importance of continuous surveillance and genomic characterization of emerging influenza strains. Table 1 receptor binding site of amino acids in the HA gene Virus isolate Sampled location Acces. No Amino acid position 110 149 171 224 * 225 * 226 * 227 * 228 * A/goose/Guangdong/1/1996 (ref seq.) Guandong AF144305 D S S N G Q S G A/chicken/Brnvoa/2023 Afyon OR733698 S A D N G Q R G A/chicken/Turkey/1/2006 Samsun OK092293 N S N N G Q S G A/chicken /Turkey/2/2006 Samsun OK092561 N S D N G Q S G A/chicken/Turkey/3/2006 Sinop OK092562 N S N N G Q S G A/chicken/Turkey/6/2006 Tokat OK092563 N S N N G Q S G A/chicken/Turkey/7/2006 Rize OK103764 N S D N G Q S G A/chicken/Turkey/8/2006 Samsun OK103787 N S D N G Q S G A/chicken/Turkey/10/2006 Samsun OK103790 N S D N G Q S G A/hawk/Turkey/11/2006 Samsun OK103789 N S D N G Q S G A/chicken/Turkey/15/2006 Samsun OK103793 N S D N G Q S G A/chicken/Turkey/17/2006 Samsun OK103792 N S D N G Q S G A/dove/Turkey/18/2006 Amasya OK103795 N S D N G Q S G A/pigeon/Turkey/21/2006 Samsun OK103801 N S N N G Q S G A/chicken/Turkey/23/2006 Sivas OK103802 N S N N G Q S G A/duck/Turkey/24/2006 Tokat OK103838 N S N N G Q I G A/chicken/Turkey/25/2006 Samsun OK103837 N S N N G Q S G A/chicken/ Turkey/26/2006 Samsun OK103845 N S N N G Q S G A/chicken/Turkey/27/2006 Samsun OK103846 N S N N G Q S G I :Isoleucine (Ile), R :Arginine (Arg), Q :Glutamine (Glu), N :Asparagine (Asn), G :Glycine (Gly), S :Serine, D :Aspartate (Asp) *: H3 Numbering Discussion After the AI H5N1 virus was first isolated from a domestic goose in the Guandong region of the People's Republic of China in 1996, there have been many outbreaks in poultry, wild birds, and sporadic human infections in more than 60 countries. The first human infection was reported in Hong Kong in 1997, with 18 cases and six deaths [ 15 – 17 ]. H5N1 HPAI viruses are capable of high morbidity and mortality rates within the human population and can cause a pandemic in the future if they gain the ability of a human to human transmission [ 18 ]. This study's analysis of the receptor-binding site of the HA gene in 18 H5N1 isolates from 2006 and 2023 has provided critical insights into the molecular evolution of this virus. Notably, none of the isolates exhibited significant amino acid substitutions at positions 224, 225, 226, 227, and 228 (H3 numbering), which are essential for host selectivity, indicating a relative conservation in these positions over the years. However, the presence of mutations such as D110N in almost all isolates and S171N in a subset, along with the unique S227I mutation in one isolate and S149A in the 2023 Turkish isolate, underscores the continuous genetic evolution of H5N1. These findings are particularly significant considering the potential impact of such mutations on the virus's affinity towards host receptors and its transmissibility. Furthermore, the phylogenetic analysis based on geographical distribution reveals a clear divergence between the 2006 and 2023 Turkish isolates. While the 2006 isolates show regional homogeneity, the 2023 isolate is genetically distinct, aligning more closely with recent isolates from France and Spain. This suggests not only the geographical spread of H5N1 but also the introduction or evolution of new lineages within Turkey over this period. Such genetic differentiation, especially the closer relationship of the 2023 Turkish isolate with a Russian isolate from 2021, highlights the dynamic nature of H5N1's evolution and the possibility of trans-regional migration or exchange of viral strains. The contrast in the genetic makeup of the 2006 and 2023 isolates points to the ongoing evolution of H5N1, emphasizing the importance of continuous surveillance to monitor these changes and understand their implications for both avian and human health. A study conducted using the Hk213 H5 virus proved that the virus could bind to both human and avian receptors, and the reason for such double-sided binding was due to the amino acid at position 227 of the HA molecule. The presence of asn at amino acid position 227 of the HA molecule causes dual receptor recognition. In addition, the N227S mutation has been reported to reduce its binding affinity to human bronchial mucosa [ 19 ]. In our study, the position of the 227th amino acid in all isolates except A/duck/Turkey/24/2006 was identified to have a serine (S) residue, suggesting a potential weak binding affinity to human epithelial cells. However, the 2023 Turkey isolate exhibits a notable deviation with an arginine (R) at this position. This finding is consistent with previous reports indicating that an arginine (R) at the 227th position enhances binding to avian-type receptors [ 20 ]. The amino acid substitution observed in the 2023 isolate is significant, suggesting a possible shift in receptor preference, which could affect the virus's host range and transmissibility. In another study, established a bird-human reassortant virus following the identification of high genetic compatibility between the H5N1 and the 2009 pandemic H1N1 virus. Here, the HA gene was derived from H5N1, while the other seven genes were from the 2009 pandemic H1N1 virus, forming the H5HA/pdm09. Subsequent mutations introduced in this virus (H5HA-mutant/pdm09), specifically the N224K and Q226L mutations, demonstrated respiratory droplet transmission among ferrets. However, these observations were made using ferret models, and such airborne transmission-related point mutations may not induce the same biological trait changes in other mammalian species, particularly humans [ 11 ]. The role of the A/duck/Turkey/24/2006 isolate in terms of receptor selectivity remains unclear, necessitating further research for a better understanding. The detection of arginine (R) at the 227th position in the 2023 Turkey isolate calls for in-depth studies to determine its influence on the virus's interaction with host cells and its pathogenicity. Research has demonstrated that the occurrence of either a single mutation (Q226L) or a combination of mutations (G228S, Q226L) in the receptor-binding domain of the HA protein in the A/Vietnam/1203/2004 H5N1 isolate results in a pronounced affinity towards human-type receptors [ 21 ]. Furthermore, the study emphasized that the Q226L change specified in the receptor-binding site of the HA gene with another mutation (VN1203mut) generated on this isolate was vital for the virus to gain the ability to bind to human receptors [ 22 ]. Different mutation studies performed with the A/Duck/Egypt/10185SS/2010 and A/Vietnam/1203/2004 isolates stated that the Q226L mutation was one of the key mutations for the virus to adapt from birds to humans and that the virus preferred humans over avian receptors [ 10 , 23 ]. Moreover, a recent study reported that the N224K and Q226L mutations were especially effective in human-type receptor selectivity for the H5N1 virus [ 20 ]. The inclusion of the Q222L and G224S mutations in the HA of A/H5N1 altered the receptor binding preference from α-2,3-linked SA to α-2,6-linked SA, thereby shifting its binding affinity [ 5 ]. This modification in the HA's receptor-binding specificity underscores the evolving nature of the H5N1 virus and its potential zoonotic implications. In our investigation using the FluSurver platform with A/goose/Guandong/1/1996(H5N1) as the reference strain, the S149A mutation identified in the receptor binding domain of the HA gene from the 2023 H5N1 isolate emerged as a critical finding. This substitution is recognized to prompt a shift in host specificity. It has been previously documented that such a mutation increases the virus's binding affinity to alpha2,6-sialic acid receptors predominantly found in humans, thereby suggesting an adaptation towards human host cells. This adaptation is concurrently accompanied by a decreased binding to alpha2,3-sialic acid receptors typical of avian cells potentially indicating a zoonotic potential of the virus [ 24 ]. The sequence analysis of the H5N1 isolates from 2006 has consistently shown a D110N mutation. The D110N mutation has been associated with a modification in host receptor preference, which may have implications for inter-species transmission. The observed enhancement in hemagglutinin's binding to human-type receptors and concurrent decrease in avian receptor affinity could be indicative of an adaptive mechanism, possibly increasing the virus's ability to infect new hosts [ 25 ]. Moreover, the presence of the S171N mutation in eight of the 2006 isolates, raises further questions about the virus's evolutionary trajectory. The S171N mutation is known for increasing binding to human-type receptors without sacrificing binding to avian receptors. This dual affinity suggests that the virus may be maintaining its avian host specificity while potentially expanding its host range to include humans. The implications of this mutation for the zoonotic transfer are particularly compelling, considering that mutations conferring increased binding to human receptors are often seen in the context of pandemic strains [ 26 ]. The collective evidence from these mutations underscores the importance of continued surveillance and analysis of the H5N1 virus. It is crucial to monitor these and other mutations closely, as they provide insight into the virus's potential to adapt to human hosts, with significant ramifications for public health preparedness and response strategies. In this study, both geographical and temporal phylogenetic analyses of H5N1 isolates were conducted. The phylogenetic analyses based on geographical distribution revealed that the H5N1 viruses isolated from Turkey in 2006 displayed a cohesive clustering, indicating regional homogeneity. This finding suggests that the H5N1 isolates emerging in Turkey during 2006 possessed distinct regional characteristics and tended to spread locally. In contrast, the 2023 Turkish isolate significantly differed from the 2006 cohort, showing closer phylogenetic relationships with isolates from waterfowl in France and Spain, isolated in 2021 and 2022, respectively. This suggests that the 2023 Turkish isolate genetically diverged from the strains observed in previous years in the region and potentially originated from a different source. Notably, the isolation of this strain from a chicken in Afyon in Turkey, and Afyon's location on the migratory paths of north-south traveling birds [ 27 ], points to the possibility of the virus being brought to the region by migratory birds. Also, It has been noted that the HPAI A/H5N1 viruses found in wild birds in the regions close to the Chile-Peru border demonstrate genetic similarities with the North American-originated Peruvian isolates and possess unexpected mutations [ 28 ]. Reflecting on these findings, our study in Turkey with the A/Chicken/Bornova/2023 isolate reveals that it is phylogenetically clustered with the South American isolates (including those from Peru, Uruguay, and Venezuela). This placement highlights the significant role of bird migration routes in influencing the genetic diversity and geographic distribution of the HPAI A/H5N1 virus. Such a situation provides new and substantial contributions to the current understanding of the evolution and spreading dynamics of the HPAI A/H5N1 virus The findings align with the migration routes and stopover sites of birds, highlighting a significant role in understanding the geographical spread and genetic changes of H5N1 [ 29 – 30 ]. Particularly, recognizing Western Siberia as the world's largest wetland, along with other significant wetlands in Europe and Asia, is crucial for understanding the sources of H5N1 and other avian influenza viruses. This knowledge is vital for comprehending the geographical and temporal differentiation of these viruses. Recent studies have demonstrated that bird migration networks are more reflective of the observed viral gene sequence data than other networks, playing a key role in the seasonal outbreaks of H5N1 [ 31 – 32 ]. These findings underscore the critical importance of migratory birds in the geographical distribution and transmission of the virus to new areas. This study significantly advances our understanding of the molecular evolution and potential zoonotic threat of the H5N1 virus. By analyzing critical amino acid positions in the HA gene of isolates from 2006 and 2023, it highlights notable findings such as the unique S227I and S149A mutations, which shed light on the virus's evolving affinity for host receptors and its transmissibility. The research notably reveals a significant divergence in the 2023 Turkish isolate compared to earlier strains, indicating the emergence of new lineages and suggesting shifts in the virus's geographical distribution and host preferences. This divergence, particularly evidenced by the genetic proximity of the 2023 isolate to recent strains from Europe, underscores the dynamic nature of H5N1's evolution and the role of migratory bird patterns in its spread. The study's findings on mutations that enhance the virus's binding to human-type receptors, especially in the context of the D110N and S171N mutations, provide crucial insights into the virus's potential adaptation from avian to human hosts. These insights are invaluable for informing public health strategies and underline the necessity for ongoing surveillance to monitor H5N1's evolution, aiding in the preparedness and response to potential future pandemics. In conclusion, this study, through a detailed molecular analysis of H5N1 isolates from 2006 and 2023, has revealed significant changes in the evolutionary patterns and geographical distribution of the H5N1 virus in Turkey, underscoring the critical importance of these findings in understanding the role of bird migration routes in the virus's spread and in developing strategies for the surveillance and control of influenza viruses. Declarations Acknowledgments I would like to Dr. Cüneyt TAMER and Dr. Ahmed Eisa ELHAG (from the Virology Department of the same University) for their valuable help in certain parts of the laboratory stages, evaluation of the results and proofreading of this manuscript, which was a part of my doctoral thesis. Authorship contribution This study summarizes the doctoral thesis of the corresponding author (HK). HK: Methodology, Software, Investigation, Writing - Original Draft. AYC: Supervision, Writing - review & editing. OC and MN: Resource. HA: Data curation. All authors have approved the final article. All authors have read and agreed to the published version of the manuscript. Funding The Ph.D project was funded by Samsun Veterinary Control Institute, Ministry of Agriculture And Forestry, Republic of Turkey. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Conflict of Interest The authors declare that they have no confict of interest. Ethical standards The study was deemed appropriate with the approval of the Samsun Veterinary Control Institute Ethics Committee. (Date: 29.11.2016, Number: 11-2). 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Appl Environ Microbiol 49:914–919 Stallknecht DE, Shane SM (1988) Host range of avian influenza virus in free-living birds. Vet Res Commun 12:125–141 Durand LO, Glew P, Gross D et al (2015) Timing of influenza A(H5N1) in poultry and humans and seasonal influenza activity worldwide, 2004–2013. Emerg Infect Dis 21:202–208 Tian H, Zhou S, Dong L et al (2015) Avian influenza H5N1 viral and bird migration networks in Asia. Proc Natl Acad Sci 112:172–177 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3831007","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":265297728,"identity":"654738c5-648d-45db-a124-45f7f51ceeb4","order_by":0,"name":"Hamza KADI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACAzB5gIGxjb2x8TGYw8zcQKQWnsPNxmA+MyORWhok3NukIXwCWszZTyd/+HHGTrZPgrGtuqDiTzR/O1DLj4ptOLVY9uRuMOy5kWzcJt3YdnvGGYPcGYcZGxh7ztzG7bADuRsSeD4wJ7bJHGy7zdtmkNsA1MLM2IZHy/m3Gw7++VCf2CaR2FYM0jKfoJYbuRubeW4cBmthBmnZQFjL283MMmeOG7fxHGyW5jljnLsRqOUgXr+cz9388c2xatn57e0PP/NUyOXOO3/44IMfFbi1YAcHSFQ/CkbBKBgFowANAADzwGLmts2i8gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-7706-4251","institution":"TC Tarım ve Orman Bakanlığı: Turkiye Cumhuriyeti Tarim ve Orman Bakanligi","correspondingAuthor":true,"prefix":"","firstName":"Hamza","middleName":"","lastName":"KADI","suffix":""},{"id":265297729,"identity":"b3fe00b1-3c09-40f4-81a4-d8cd516eaa18","order_by":1,"name":"Ahmet Yilmaz COBAN","email":"","orcid":"","institution":"Akdeniz Üniversitesi: Akdeniz Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"Yilmaz","lastName":"COBAN","suffix":""},{"id":265297730,"identity":"934106a3-a748-49d3-b6fe-a0b1cd21c828","order_by":2,"name":"Ozge CAGIRGAN","email":"","orcid":"","institution":"TC Tarım ve Orman Bakanlığı: Turkiye Cumhuriyeti Tarim ve Orman Bakanligi","correspondingAuthor":false,"prefix":"","firstName":"Ozge","middleName":"","lastName":"CAGIRGAN","suffix":""},{"id":265297731,"identity":"1393541c-423c-451f-8bfb-786ce66cc29b","order_by":3,"name":"Mehmet NOTUROGLU","email":"","orcid":"","institution":"TC Tarım ve Orman Bakanlığı: Turkiye Cumhuriyeti Tarim ve Orman Bakanligi","correspondingAuthor":false,"prefix":"","firstName":"Mehmet","middleName":"","lastName":"NOTUROGLU","suffix":""},{"id":265297732,"identity":"0ffc9f35-1c2b-4b44-9a19-ee5080740d46","order_by":4,"name":"Harun ALBAYRAK","email":"","orcid":"","institution":"Ondokuz Mayis University: Ondokuz Mayis Universitesi","correspondingAuthor":false,"prefix":"","firstName":"Harun","middleName":"","lastName":"ALBAYRAK","suffix":""}],"badges":[],"createdAt":"2024-01-03 04:37:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3831007/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3831007/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49302002,"identity":"fe3c03d1-5cdc-4152-af4b-49ec503a3fda","added_by":"auto","created_at":"2024-01-08 09:55:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110324,"visible":true,"origin":"","legend":"\u003cp\u003eThe locations of the samples areas painted in green.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3831007/v1/b4e446c4294f36f2bcdb9786.jpeg"},{"id":49302003,"identity":"8024b24b-496d-439b-b47c-fd261177e4c7","added_by":"auto","created_at":"2024-01-08 09:55:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":974431,"visible":true,"origin":"","legend":"\u003cp\u003ePartial Phylogenetic Analysis of the Hemagglutinin Genes of AI H5N1 Isolates Based on Geographic Distribution. The tree was constructed with the Maximum likelihood (ML) method replicates 1000 bootstrap with MEGA X software. The black triangle represent the 2023 Turkey isolates, while the black squares denote the 2006 Turkey isolates.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3831007/v1/40494cc71f9a93fb861446a6.jpeg"},{"id":49302001,"identity":"d15b88ba-6098-4f9d-896d-47846d075a41","added_by":"auto","created_at":"2024-01-08 09:55:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34511,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal Phylogenetic Analysis of Hemagglutinin Genes in AI H5N1 Isolates. The tree was constructed with the Maximum likelihood (ML) method replicates 1000 bootstrap with MEGA X software. Black triangle indicate isolate from Turkey in 2023, illustrating recent developments, while black squares represent the earlier isolates from Turkey in 2006, providing a historical perspective.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3831007/v1/d5dcd147b2740a1ad112f77b.png"},{"id":49950871,"identity":"9c1e1f79-208e-486e-b728-49874b1138e1","added_by":"auto","created_at":"2024-01-22 06:15:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":826174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3831007/v1/81fbb01e-0ceb-4d93-9891-e5a48974ac43.pdf"}],"financialInterests":"","formattedTitle":"A Comprehensive Analysis of H5N1 Evolution: Phylogenetic Insights and Emerging Mutations in Turkey's Avian Influenza Landscape","fulltext":[{"header":"Inroduction","content":"\u003cp\u003eInfluenza A viruses (AIVs), classified under the family \u003cem\u003eOrthomyxoviridae\u003c/em\u003e and genus Influenzavirus A, are negative-sense, single-stranded RNA viruses with a global presence, primarily found in wild waterfowl and other aquatic birds. The viral genome of AIVs comprises eight RNA segments that encode at least twelve key proteins, including the basic polymerase 2 (PB2), basic polymerase 1 (PB1), acidic polymerase (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M), and nonstructural (NS) proteins. Notably, AIV subtypes are distinguished based on the antigenic properties of two surface glycoproteins, HA and NA. To date, sixteen HA subtypes (H1\u0026ndash;H16) and nine NA subtypes (N1\u0026ndash;N9) have been identified in aquatic birds. Additionally, recent discoveries have revealed two novel HA subtypes (H17 and H18) and two novel NA subtypes (N10 and N11) in bats, expanding our understanding of AIV diversity [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eH5N1 viruses, which naturally circulate in aquatic birds, have increasingly become integrated into poultry populations, leading to their endemic presence in domestic birds in some countries [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This significant shift from wild to domestic hosts highlights the ongoing risk of H5N1 viruses adapting to new species, including the potential for increased human infections. Additionally, the sporadic transmission of Avian Influenza Viruses (AIVs) from waterfowl to domestic avian species has been documented, further emphasizing the complexity of H5N1 transmission dynamics [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Human-to-human transmission of influenza viruses can occur through direct contact, indirect contact via fomites (contaminated environmental surfaces) and/or airborne transmission via small aerosols or large respiratory droplets [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The potential for Avian Influenza Viruses (AIV) to enhance transmissibility in mammals depends on the acquisition and selection of mutations during spread. Although numerous experimental mutation studies have been conducted, data from natural infections remain limited [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The first human deaths directly attributable to avian A/H5N1 virus were recorded in Hong Kong in 1997, and it has since become important for humans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHA proteins bind to sialoside receptors on the host cell surface. Both avian and human influenza A viruses exhibit a preference for binding sialic acids connected to a penultimate galactose through α2,3 or α2,6 linkages, respectively [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Human influenza A viruses recognize the α2,6-galactose (SAα2,6Gal) saccharides situated at the terminus of sialic acid chains. In contrast, avian influenza A viruses identify the α2,3-galactose (SAα2,3Gal) saccharides, which are also present among sialic acid receptors. The transition from SAα2,3Gal to SAα2,6Gal represents a crucial change, suggesting that avian influenza (AI) viruses can effectively replicate and cause pandemics in humans. Identifying mutations in the receptor-binding site of the HA molecule in H5N1 influenza viruses can offer insights into potential future pandemics [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although H5N1 viruses have not yet acquired the ability to be transmitted among humans, the absence of immunity in humans to influenza viruses carrying the H5 HA raises the likelihood of an emerging, transmissible H5 HA-carrying virus leading to a pandemic. Consequently, understanding the molecular changes that may occur in H5 HA-carrying viruses is crucial for preparedness in such scenarios. This information can be valuable for monitoring variants with pandemic potential, focusing eradication efforts on viruses already possessing critical molecular changes for mammalian transmission, stockpiling antiviral compounds in regions where such viruses circulate, and initiating vaccine production and large-scale manufacturing ahead of a potential pandemic [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmong the AIV subtypes, H5N1 has garnered significant attention due to its potential for zoonotic transmission and severe impact on avian and human health. Since its initial detection in the late 20th century, H5N1 has evolved through various genetic reassortments and mutations, leading to the emergence of multiple strains with varying pathogenicity and transmissibility. This continuous evolution poses a significant challenge for public health, necessitating ongoing surveillance and research to track its progression and adapt strategies for control and prevention.\u003c/p\u003e \u003cp\u003eIn Turkey, as in many parts of the world, H5N1 has been a persistent concern, impacting both wild and domestic avian populations. Given the strategic geographical location of Turkey, bridging Asia and Europe, the country serves as a crucial point in the migration routes of wild birds, thereby playing a key role in the dissemination and evolution of H5N1 strains. This study aims to provide a comprehensive analysis of the molecular evolution of H5N1 in Turkey over a span of 17 years, from 2006 to 2023. By examining 18 isolates from different time points, this research focuses on identifying significant mutations in the receptor-binding regions of the hemagglutinin gene, which are crucial for host specificity and viral infectivity. Additionally, the study conducts phylogenetic analyses based on geographical and temporal distribution to understand the evolutionary trajectory of H5N1 in the region. Such insights are vital for informing both national and global strategies in avian influenza surveillance and response.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics committee approval\u003c/h2\u003e \u003cp\u003eThe study was deemed appropriate with the approval of the Samsun Veterinary Control Institute Ethics Committee. (Date: 29.11.2016, Number: 11\u0026thinsp;\u0026minus;\u0026thinsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAI H5N1 isolates\u003c/h2\u003e \u003cp\u003eSeventeen AI H5N1 isolates from poultry during the AI epidemic, which occurred in the Black Sea region of Turkey in the first quarter of 2006 (January\u0026ndash;February) and reported to the World Organization for Animal Health (WOAH), were initially used in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To supplement these findings, a recent H5N1 isolate obtained from the Aegean region of Turkey in 2023 has been included to provide a current perspective on the evolution of the virus. Both sets of isolates were handled using the same protocol to ensure comparability of the results.\u003c/p\u003e \u003cp\u003eTo stimulate viruses and increase their amount, the 2006 isolates and the 2023 isolate were inoculated into the chorioallantoic membranes of 9\u0026ndash;11 days old specific pathogen-free (SPF) embryo-bearing chicken eggs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The eggs were checked daily for viability, and chorioallantoic fluid was collected on the third through fifth days post-inoculation. The newly obtained 2023 isolate was processed in the same manner as the 2006 isolates to maintain consistency in the experimental approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction\u003c/h2\u003e \u003cp\u003eViral RNA was extracted from the chorioallantoic fluid using a commercial nucleic acid extraction kit (Roche High Pure Viral Nucleic Acid Kit) according to the manufacturer's instructions and stored at -20 ℃ for later use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAmplification of the HA gene receptor-binding site by RT-PCR\u003c/h2\u003e \u003cp\u003eThe HA gene's receptor-binding site, corresponding to the HA1 region [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], was analyzed for previously isolated Turkish H5N1 strains (GenBank accession nos. DQ407519, KF042152, KF042153, and EF619980) using Bioedit software. Specific primers, HK1F1 (5'-GATCAGATTTGCATTGGTTACC-3') and HK2R1 (5'-TTGAGGGCTATTTCTGAGCCCAG-3'), were designed for RT-PCR amplification using the Qiagen one-step kit. The reaction setup included 24.5 \u0026micro;l of RNase-free water, 10 \u0026micro;l 5X buffer, 2 \u0026micro;l of dNTPs, 3 \u0026micro;l of each primer, 0.5 \u0026micro;l RNase inhibitor, 2 \u0026micro;l enzyme mix, and 5 \u0026micro;l template RNA. Thermal cycling conditions were: 30 min at 50℃ for reverse transcription, 15 min at 95℃ for inactivation of reverse transcriptase/activation of Taq polymerase, followed by 40 cycles of 1 min at 94℃ for denaturation, 1 min at 55℃ for annealing, and 1 min at 72℃ for extension, with a final extension at 72℃ for 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSanger sequencing and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe sequencing of the PCR products was carried out by BM LABOSIS Gene Research and Biotechnology, based in Ankara, Turkey. The nucleotide sequences obtained were edited and assembled using BioEdit software (version 7.0.5) and submitted to GenBank, securing the corresponding accession numbers. Subsequent alignment of the hemagglutinin gene sequences was performed using the ClustalW algorithm within the MEGA X software [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], in comparison with other sequences sourced from public databases such as the National Center for Biotechnology Information Virus (NCBI Virus). Within the MEGA X environment, the most appropriate nucleotide substitution model was selected based on the Bayesian Information Criterion (BIC), and phylogenetic trees with geographic and temporal structure were constructed using the Maximum Likelihood method, supported by 1000 bootstrap replicates, to analyze the spatiotemporal dynamics of the viral evolution. Lastly, sequence data were examined for significant amino acid changes in the hemagglutinin gene receptor binding site that could substantially influence host selectivity, utilizing the FluSurver database with the A/goose/Guangdong/1/1996 (H5N1) isolate as a reference. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://flusurver.bii.a-star.edu.sg\u003c/span\u003e\u003cspan address=\"http://flusurver.bii.a-star.edu.sg\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the receptor-binding site of the HA gene\u003c/h2\u003e \u003cp\u003eThe receptor-binding site of the HA genes that belong to the AIV H5N1 isolates were amplified by RT-PCR. Subsequently, to determine the critical amino acid positions within the receptor-binding region of the HA gene and to elucidate their phylogenetic relationships, further studies were undertaken. These investigations involved a detailed comparative analysis of the HA gene sequences across different AIV H5N1 strains, enabling a comprehensive understanding of the structural variations and their potential impact on the virus's affinity towards host receptors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe sequencing results of the HA receptor-binding site and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eIn this study, none of the 18 isolates from 2006 and 2023 exhibited the significant amino acid substitutions at positions 224, 225, 226, 227, 228 (H3 numbering), which are associated with host selectivity. However, according to the Flusurver database, the mutation D110N was identified in all isolates except A/chicken/Brnvoa/2023, while the mutation S171N was found in eight isolates. Notably, the isolate with the accession number OK103838 possessed the unique S227I mutation (Table-1). Similarly, the newly analyzed A/chicken/Brnvoa/2023 Turkish isolate also presented a mutation at the position, S149A, contributing further data to the evolving pattern of amino acid substitutions observed in these avian influenza viruses.\u003c/p\u003e \u003cp\u003eThe phylogenetic analysis based on the geographical distribution of the HA gene of H5N1 isolates reveals a cohesive clustering of the Turkish isolates from 2006, indicating a regional homogeneity. In contrast, the isolate from Turkey in 2023 exhibits significant divergence from the 2006 cohort and demonstrates closer phylogenetic relationships with isolates from France (A/Mule-duck/France/21351/2021) and Spain (A/Anser anser/Spain/750-3/2022), isolated in the years 2021 and 2022, respectively (Figure-2). When examining the temporal phylogenetic tree, it is evident that the Turkish isolates from 2006 are closely situated to one another, forming a distinct cluster. The 2023 Turkish isolate, appears to share a closer relationship with an isolate from Russia (A/common teal/Chelyabinsk/1379-1/2021). Furthermore, the genetic differentiation between the Turkish isolates of 2006 and the 2023 Turkish isolate is strikingly evident (Figure-3)\u003c/p\u003e \u003cp\u003eThe combined analysis of both trees underscores the homogeneous clustering of the 2006 isolates, while the 2023 Turkish isolate (A/chicken/Turkey/Brnvoa/2023) is genetically distinct from this group. These findings suggest a potential evolution or introduction of a new lineage of H5N1 in Turkey since 2006, highlighting the importance of continuous surveillance and genomic characterization of emerging influenza strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ereceptor binding site of amino acids in the HA gene\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirus isolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSampled location\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAcces. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c11\" namest=\"c4\"\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAmino acid position\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e224\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e225\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e226\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e227\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e228\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/goose/Guangdong/1/1996 (ref seq.)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuandong\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAF144305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eA/chicken/Brnvoa/2023\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAfyon\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOR733698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/1/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK092293\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken /Turkey/2/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK092561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/3/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSinop\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK092562\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/6/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTokat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK092563\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/7/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRize\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103764\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/8/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103787\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e 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align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/hawk/Turkey/11/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103789\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/15/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/17/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/dove/Turkey/18/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmasya\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/pigeon/Turkey/21/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/23/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSivas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103802\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/duck/Turkey/24/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTokat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103838\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/25/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/ Turkey/26/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103845\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA/chicken/Turkey/27/2006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamsun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOK103846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003cb\u003eI\u003c/b\u003e:Isoleucine (Ile), \u003cb\u003eR\u003c/b\u003e:Arginine (Arg), \u003cb\u003eQ\u003c/b\u003e:Glutamine (Glu), \u003cb\u003eN\u003c/b\u003e:Asparagine (Asn), \u003cb\u003eG\u003c/b\u003e:Glycine (Gly), \u003cb\u003eS\u003c/b\u003e:Serine, \u003cb\u003eD\u003c/b\u003e:Aspartate (Asp)\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e*: H3 Numbering\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAfter the AI H5N1 virus was first isolated from a domestic goose in the Guandong region of the People's Republic of China in 1996, there have been many outbreaks in poultry, wild birds, and sporadic human infections in more than 60 countries. The first human infection was reported in Hong Kong in 1997, with 18 cases and six deaths [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. H5N1 HPAI viruses are capable of high morbidity and mortality rates within the human population and can cause a pandemic in the future if they gain the ability of a human to human transmission [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study's analysis of the receptor-binding site of the HA gene in 18 H5N1 isolates from 2006 and 2023 has provided critical insights into the molecular evolution of this virus. Notably, none of the isolates exhibited significant amino acid substitutions at positions 224, 225, 226, 227, and 228 (H3 numbering), which are essential for host selectivity, indicating a relative conservation in these positions over the years. However, the presence of mutations such as D110N in almost all isolates and S171N in a subset, along with the unique S227I mutation in one isolate and S149A in the 2023 Turkish isolate, underscores the continuous genetic evolution of H5N1. These findings are particularly significant considering the potential impact of such mutations on the virus's affinity towards host receptors and its transmissibility.\u003c/p\u003e \u003cp\u003eFurthermore, the phylogenetic analysis based on geographical distribution reveals a clear divergence between the 2006 and 2023 Turkish isolates. While the 2006 isolates show regional homogeneity, the 2023 isolate is genetically distinct, aligning more closely with recent isolates from France and Spain. This suggests not only the geographical spread of H5N1 but also the introduction or evolution of new lineages within Turkey over this period. Such genetic differentiation, especially the closer relationship of the 2023 Turkish isolate with a Russian isolate from 2021, highlights the dynamic nature of H5N1's evolution and the possibility of trans-regional migration or exchange of viral strains. The contrast in the genetic makeup of the 2006 and 2023 isolates points to the ongoing evolution of H5N1, emphasizing the importance of continuous surveillance to monitor these changes and understand their implications for both avian and human health.\u003c/p\u003e \u003cp\u003eA study conducted using the Hk213 H5 virus proved that the virus could bind to both human and avian receptors, and the reason for such double-sided binding was due to the amino acid at position 227 of the HA molecule. The presence of asn at amino acid position 227 of the HA molecule causes dual receptor recognition. In addition, the N227S mutation has been reported to reduce its binding affinity to human bronchial mucosa [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In our study, the position of the 227th amino acid in all isolates except A/duck/Turkey/24/2006 was identified to have a serine (S) residue, suggesting a potential weak binding affinity to human epithelial cells. However, the 2023 Turkey isolate exhibits a notable deviation with an arginine (R) at this position. This finding is consistent with previous reports indicating that an arginine (R) at the 227th position enhances binding to avian-type receptors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The amino acid substitution observed in the 2023 isolate is significant, suggesting a possible shift in receptor preference, which could affect the virus's host range and transmissibility.\u003c/p\u003e \u003cp\u003eIn another study, established a bird-human reassortant virus following the identification of high genetic compatibility between the H5N1 and the 2009 pandemic H1N1 virus. Here, the HA gene was derived from H5N1, while the other seven genes were from the 2009 pandemic H1N1 virus, forming the H5HA/pdm09. Subsequent mutations introduced in this virus (H5HA-mutant/pdm09), specifically the N224K and Q226L mutations, demonstrated respiratory droplet transmission among ferrets. However, these observations were made using ferret models, and such airborne transmission-related point mutations may not induce the same biological trait changes in other mammalian species, particularly humans [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of the A/duck/Turkey/24/2006 isolate in terms of receptor selectivity remains unclear, necessitating further research for a better understanding. The detection of arginine (R) at the 227th position in the 2023 Turkey isolate calls for in-depth studies to determine its influence on the virus's interaction with host cells and its pathogenicity. Research has demonstrated that the occurrence of either a single mutation (Q226L) or a combination of mutations (G228S, Q226L) in the receptor-binding domain of the HA protein in the A/Vietnam/1203/2004 H5N1 isolate results in a pronounced affinity towards human-type receptors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, the study emphasized that the Q226L change specified in the receptor-binding site of the HA gene with another mutation (VN1203mut) generated on this isolate was vital for the virus to gain the ability to bind to human receptors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Different mutation studies performed with the A/Duck/Egypt/10185SS/2010 and A/Vietnam/1203/2004 isolates stated that the Q226L mutation was one of the key mutations for the virus to adapt from birds to humans and that the virus preferred humans over avian receptors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, a recent study reported that the N224K and Q226L mutations were especially effective in human-type receptor selectivity for the H5N1 virus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe inclusion of the Q222L and G224S mutations in the HA of A/H5N1 altered the receptor binding preference from α-2,3-linked SA to α-2,6-linked SA, thereby shifting its binding affinity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This modification in the HA's receptor-binding specificity underscores the evolving nature of the H5N1 virus and its potential zoonotic implications.\u003c/p\u003e \u003cp\u003eIn our investigation using the FluSurver platform with A/goose/Guandong/1/1996(H5N1) as the reference strain, the S149A mutation identified in the receptor binding domain of the HA gene from the 2023 H5N1 isolate emerged as a critical finding. This substitution is recognized to prompt a shift in host specificity. It has been previously documented that such a mutation increases the virus's binding affinity to alpha2,6-sialic acid receptors predominantly found in humans, thereby suggesting an adaptation towards human host cells. This adaptation is concurrently accompanied by a decreased binding to alpha2,3-sialic acid receptors typical of avian cells potentially indicating a zoonotic potential of the virus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe sequence analysis of the H5N1 isolates from 2006 has consistently shown a D110N mutation. The D110N mutation has been associated with a modification in host receptor preference, which may have implications for inter-species transmission. The observed enhancement in hemagglutinin's binding to human-type receptors and concurrent decrease in avian receptor affinity could be indicative of an adaptive mechanism, possibly increasing the virus's ability to infect new hosts [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, the presence of the S171N mutation in eight of the 2006 isolates, raises further questions about the virus's evolutionary trajectory. The S171N mutation is known for increasing binding to human-type receptors without sacrificing binding to avian receptors. This dual affinity suggests that the virus may be maintaining its avian host specificity while potentially expanding its host range to include humans. The implications of this mutation for the zoonotic transfer are particularly compelling, considering that mutations conferring increased binding to human receptors are often seen in the context of pandemic strains [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe collective evidence from these mutations underscores the importance of continued surveillance and analysis of the H5N1 virus. It is crucial to monitor these and other mutations closely, as they provide insight into the virus's potential to adapt to human hosts, with significant ramifications for public health preparedness and response strategies.\u003c/p\u003e \u003cp\u003eIn this study, both geographical and temporal phylogenetic analyses of H5N1 isolates were conducted. The phylogenetic analyses based on geographical distribution revealed that the H5N1 viruses isolated from Turkey in 2006 displayed a cohesive clustering, indicating regional homogeneity. This finding suggests that the H5N1 isolates emerging in Turkey during 2006 possessed distinct regional characteristics and tended to spread locally. In contrast, the 2023 Turkish isolate significantly differed from the 2006 cohort, showing closer phylogenetic relationships with isolates from waterfowl in France and Spain, isolated in 2021 and 2022, respectively. This suggests that the 2023 Turkish isolate genetically diverged from the strains observed in previous years in the region and potentially originated from a different source. Notably, the isolation of this strain from a chicken in Afyon in Turkey, and Afyon's location on the migratory paths of north-south traveling birds [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], points to the possibility of the virus being brought to the region by migratory birds. Also, It has been noted that the HPAI A/H5N1 viruses found in wild birds in the regions close to the Chile-Peru border demonstrate genetic similarities with the North American-originated Peruvian isolates and possess unexpected mutations [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Reflecting on these findings, our study in Turkey with the A/Chicken/Bornova/2023 isolate reveals that it is phylogenetically clustered with the South American isolates (including those from Peru, Uruguay, and Venezuela). This placement highlights the significant role of bird migration routes in influencing the genetic diversity and geographic distribution of the HPAI A/H5N1 virus. Such a situation provides new and substantial contributions to the current understanding of the evolution and spreading dynamics of the HPAI A/H5N1 virus\u003c/p\u003e \u003cp\u003eThe findings align with the migration routes and stopover sites of birds, highlighting a significant role in understanding the geographical spread and genetic changes of H5N1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Particularly, recognizing Western Siberia as the world's largest wetland, along with other significant wetlands in Europe and Asia, is crucial for understanding the sources of H5N1 and other avian influenza viruses. This knowledge is vital for comprehending the geographical and temporal differentiation of these viruses. Recent studies have demonstrated that bird migration networks are more reflective of the observed viral gene sequence data than other networks, playing a key role in the seasonal outbreaks of H5N1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings underscore the critical importance of migratory birds in the geographical distribution and transmission of the virus to new areas. This study significantly advances our understanding of the molecular evolution and potential zoonotic threat of the H5N1 virus. By analyzing critical amino acid positions in the HA gene of isolates from 2006 and 2023, it highlights notable findings such as the unique S227I and S149A mutations, which shed light on the virus's evolving affinity for host receptors and its transmissibility. The research notably reveals a significant divergence in the 2023 Turkish isolate compared to earlier strains, indicating the emergence of new lineages and suggesting shifts in the virus's geographical distribution and host preferences. This divergence, particularly evidenced by the genetic proximity of the 2023 isolate to recent strains from Europe, underscores the dynamic nature of H5N1's evolution and the role of migratory bird patterns in its spread. The study's findings on mutations that enhance the virus's binding to human-type receptors, especially in the context of the D110N and S171N mutations, provide crucial insights into the virus's potential adaptation from avian to human hosts. These insights are invaluable for informing public health strategies and underline the necessity for ongoing surveillance to monitor H5N1's evolution, aiding in the preparedness and response to potential future pandemics.\u003c/p\u003e \u003cp\u003eIn conclusion, this study, through a detailed molecular analysis of H5N1 isolates from 2006 and 2023, has revealed significant changes in the evolutionary patterns and geographical distribution of the H5N1 virus in Turkey, underscoring the critical importance of these findings in understanding the role of bird migration routes in the virus's spread and in developing strategies for the surveillance and control of influenza viruses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI would like to Dr. C\u0026uuml;neyt TAMER and Dr. Ahmed Eisa ELHAG (from the Virology Department of the same University) for their valuable help in certain parts of the laboratory stages, evaluation of the results and proofreading of this manuscript, which was a part of my doctoral thesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study summarizes the doctoral thesis of the corresponding author (HK). HK: Methodology, Software, Investigation, Writing - Original Draft. AYC: Supervision, Writing - review \u0026amp; editing. OC and MN: Resource. HA: Data curation.\u0026nbsp;All authors have approved the final article. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ph.D project was funded by Samsun Veterinary Control Institute, Ministry of Agriculture And Forestry, Republic of Turkey.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no confict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical standards\u0026nbsp;\u003c/strong\u003eThe study was deemed appropriate with the approval of the Samsun Veterinary Control Institute Ethics Committee. \u003cstrong\u003e(Date: 29.11.2016, Number: 11-2).\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSuttie A, Karlsson EA, Deng YM et al (2019) Avian influenza in the Greater Mekong Subregion, 2003\u0026ndash;2018. 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Emerg Microbes Infect 12:2220569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalvorson DA, Kelleher CJ, Senne DA (1985) Epizootiology of avian influenza: effect of season on incidence in sentinel ducks and domestic turkeys in Minnesota. Appl Environ Microbiol 49:914\u0026ndash;919\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStallknecht DE, Shane SM (1988) Host range of avian influenza virus in free-living birds. Vet Res Commun 12:125\u0026ndash;141\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDurand LO, Glew P, Gross D et al (2015) Timing of influenza A(H5N1) in poultry and humans and seasonal influenza activity worldwide, 2004\u0026ndash;2013. Emerg Infect Dis 21:202\u0026ndash;208\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian H, Zhou S, Dong L et al (2015) Avian influenza H5N1 viral and bird migration networks in Asia. Proc Natl Acad Sci 112:172\u0026ndash;177\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"H5N1, enzyme, hemagglutinin, mutation, receptor","lastPublishedDoi":"10.21203/rs.3.rs-3831007/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3831007/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAvian influenza (AI) H5N1 viruses, known for their high mortality in humans and poultry, pose a significant public health threat. This study examines seventeen H5N1 isolates from the 2006 outbreak in Turkey and one 2023 isolate for mutations in the hemagglutinin gene's receptor binding domain, which determines host specificity, and the evolutionary changes in the isolates. The hemagglutinin gene region of these isolates was partially amplified using RT-PCR, and critical mutations in the receptor binding domains were examined, alongside the phylogenetic relationships of the isolates in a time- and geography-dependent manner. Our findings showed that key mutations known for altering host selectivity (N224K, G225D, Q226L, S227N, G228S) were absent. However, additional mutations (D110N and S171N), potentially affecting receptor selectivity, were identified. Comprehensive phylogenetic analysis, conducted separately based on geographic regions and temporal distribution, encompassed H5N1 strains isolated from various locations and hosts from 2007 to 2023. This study highlights significant genetic divergence between the 2006 Turkish isolates and the 2023 isolate, highlighting notable evolutionary changes. Distinct clustering of the isolates was observed in both geographic and temporal phylogenetic frameworks, indicating substantial evolutionary shifts within the circulating H5N1 strains in Turkey. Given the global spread potential of H5N1 viruses via migratory birds and the risk of a new pandemic if human-to-human transmission is attained, monitoring H5N1's molecular traits and evolution is crucial for mitigating public health impacts.\u003c/p\u003e","manuscriptTitle":"A Comprehensive Analysis of H5N1 Evolution: Phylogenetic Insights and Emerging Mutations in Turkey's Avian Influenza Landscape","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 09:55:22","doi":"10.21203/rs.3.rs-3831007/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b855d4df-6ed2-42c0-add0-4ad1174ca2a8","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-22T06:07:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-08 09:55:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3831007","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3831007","identity":"rs-3831007","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}