Avian Influenza in 2025: Low Pathogenic Strains, Global Distribution, and Notifiable Disease Status

Introduction

Avian influenza virus (AIV) remains a persistent challenge to global poultry health and wild bird conservation. The virus, an enveloped, negative-sense, single-stranded RNA orthomyxovirus, is classified into subtypes based on the antigenicity of its surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). To date, 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) have been identified in avian reservoirs, with H17 and H18 identified in bats. AIV is further categorized by pathotype: highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). This distinction is primarily determined by the presence of multiple basic amino acids at the HA cleavage site, a feature that confers systemic cleavability by ubiquitous host proteases [1]. LPAI strains possess a monobasic HA cleavage site, restricting replication to tissues expressing trypsin-like proteases, such as the respiratory and intestinal tracts [1]. In 2025, the global landscape of LPAI is characterized by the sustained circulation of several subtypes, most notably H9N2 [2], H3N8 [3], H6N6 [4], and H5N2 [5], each with distinct epidemiological and molecular features.

Molecular Determinants of Low Pathogenicity

The molecular basis of low pathogenicity is fundamentally linked to the structure of the HA glycoprotein. The HA0 precursor protein must be cleaved into HA1 and HA2 subunits for the virus to become infectious. In LPAI viruses, the cleavage site contains a single arginine residue, which is recognized only by extracellular, trypsin-like proteases present in the respiratory and gastrointestinal epithelium [1]. This restriction limits viral replication to these mucosal surfaces, typically resulting in mild or subclinical disease in gallinaceous poultry. In contrast, HPAI viruses possess a polybasic cleavage site (e.g., R-X-R/K-R) that is cleaved by ubiquitous intracellular furin-like proteases, enabling systemic replication and high mortality.

Recent research has elucidated additional molecular mechanisms that modulate LPAI virulence. Lee et al. demonstrated a synergistic relationship between the HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for LPAI [1]. The NA glycoprotein of certain LPAI strains can bind and sequester host plasminogen, which, upon conversion to plasmin, can cleave the monobasic HA site, thereby expanding tissue tropism [1]. This mechanism provides a pathway for LPAI strains to acquire enhanced pathogenicity without acquiring a polybasic cleavage site [1]. The PB2 gene segment, particularly residue 627, is a critical determinant of mammalian host adaptation and pathogenicity. Zhao et al. showed that PB2 627V and HA 217 sites synergistically affect the lethality of H9N2 in murine models, highlighting that LPAI strains can possess genetic markers associated with increased virulence in non-avian hosts.

Global Distribution of Key LPAI Subtypes in 2025

H9N2: The Most Prevalent LPAI Subtype

The H9N2 subtype is the most widespread LPAI virus in global poultry populations, having established enzootic circulation across Asia, Africa, and the Middle East [2, 3, 6]. In 2025, H9N2 continues to evolve, with new clades and reassortants emerging. Li et al. reported a new clade of H9N2 circulating in Laos, underscoring the ongoing genetic diversification in Southeast Asia [2]. In China, Zhang et al. characterized H9N2 viruses from Shandong Province (2021-2023), revealing sustained evolution and antigenic drift [3]. Similarly, Zhang et al. analyzed the molecular characteristics and genetic evolution of H9N2 in the eastern monsoon zone of China, identifying multiple genotypes. In Africa, H9N2 is now endemic in several countries. Kadja et al. detected H9N2 in laying hen and broiler farms in Senegal, and Jallow et al. characterized H9N2 viruses from live bird markets (LBMs) in the same country [6]. Mosaad et al. isolated and characterized H9N2 viruses from poultry in Egypt between 2019 and 2023, confirming the continued circulation of this subtype in North Africa. The virus has also been detected in atypical hosts. Garcia-Glaessner et al. reported an outbreak of H9N2 in lesser rhea in Peru, demonstrating the expanding host range of this LPAI subtype [7]. H9N2 has been isolated from swine in China, indicating its capacity for cross-species transmission into mammalian populations [8].

H3 Subtype Viruses

H3 subtype AIVs, including H3N8 and H3N1, are commonly isolated from wild waterfowl and occasionally spill over into poultry. Miao et al. conducted surveillance and biological characterization of H3 subtype AIVs in Eastern China, identifying these viruses as important components of the LPAI ecology [4]. Zhao et al. detected an H3N8 virus in wild birds in Beijing, China, in September 2024, providing evidence for its ongoing circulation in migratory flyways [9]. The H3N8 subtype is of particular interest due to its ability to reassort and its documented mammalian infections. Nazki et al. investigated how infectious bursal disease virus (IBDV) infection influences H3N8 challenge in chickens, demonstrating that immunosuppression can exacerbate LPAI pathogenesis [10]. Śmietanka et al. compared the pathogenicity of a virulent field isolate and an NA S122N genetically modified H3N1 virus in chickens, providing insights into the role of NA mutations in virulence modulation [11].

H6 and Other LPAI Subtypes

H6 subtype viruses, particularly H6N6, are frequently isolated from domestic ducks and have been associated with the induction of pyroptosis in macrophages. Zhu et al. demonstrated that H6N6 AIV infection induced pyroptosis of M1 macrophages by activating caspase-1, a finding that has implications for understanding LPAI immunopathogenesis [12]. H5N2 LPAI viruses also circulate in wild bird populations. Magalhães et al. reported the first detection of an H5N2 subtype in Charadrius collaris from the Brazilian Pantanal, highlighting the role of South American shorebirds as reservoirs [5]. H1N1 LPAI viruses, such as the A/Mallard/South Korea/KNU2019-34/2019 (H1N1) strain, have been shown to possess the potential to increase mammalian pathogenicity, as demonstrated by Kim et al.. Kan et al. characterized three duck-original H1N1 influenza A viruses isolated in China, further documenting the diversity of LPAI in waterfowl [13].

Diagnostic Approaches for LPAI

The diagnosis of LPAI relies on a combination of virus isolation, molecular detection, and serological assays. Given the often subclinical nature of LPAI infections, surveillance programs are critical for early detection.

Molecular Detection

Real-time reverse transcription polymerase chain reaction (RT-PCR) targeting the matrix (M) gene is the standard screening method for influenza A virus detection [14]. Subtype-specific RT-PCR assays are then employed to determine the HA and NA subtypes. An et al. developed a multiplex and universal RT-PCR assay for HA and NA subtyping of AIV, which improves throughput and reduces turnaround time [14]. Xue et al. validated an H5 single-plex assay for detection across multiple platforms, enhancing diagnostic preparedness for H5N1, a principle applicable to LPAI H5 detection. High-throughput sequencing technologies, including those using improved sequencing chemistries, have become increasingly important for genomic characterization and surveillance. Ratcliff et al. demonstrated improved resolution of AIV using Oxford Nanopore R10 sequencing chemistry, enabling rapid, field-deployable genomic surveillance.

Serological Assays

Serological detection of antibodies against AIV is performed using hemagglutination inhibition (HI) tests and enzyme-linked immunosorbent assays (ELISAs). The HI test is subtype-specific and is the gold standard for serotyping [15, 16]. Commercial ELISA kits, which detect antibodies against the nucleoprotein (NP) or other conserved viral proteins, are used for flock-level screening [16]. Differentiating infected from vaccinated animals (DIVA) strategies are important for LPAI control programs. Zhang et al. developed an inactivated H9N2 subtype serological DIVA vaccine using a chimeric A/B NA epitope approach, allowing serological distinction between vaccinated and naturally infected birds [15].

Emerging Diagnostic Technologies

Surface-enhanced Raman spectroscopy (SERS) has been explored as a novel diagnostic tool for AIV detection. Qadir and Yang reviewed the role of SERS in poultry health monitoring, highlighting its potential for rapid, label-free detection of viral antigens [17]. Nanobody-based detection systems have also been developed. Ye et al. developed and applied a VNAR-based detection nanobody for H9N2 AIV, offering a highly specific and stable diagnostic reagent [18]. The use of chicken lung organoids for in vitro modeling of AIV-host cell interaction, as described by Nicholson et al., provides a platform for studying viral replication dynamics and testing antiviral compounds without the need for live animal experiments [19].

flowchart TD
 A["Clinical Sample: Oropharyngeal/Cloacal Swab"] --> B{RNA Extraction & RT-PCR for Influenza A Matrix Gene}
 B -- Positive --> C{Subtype Identification}
 C --> D[HA Subtype RT-PCR]
 C --> E[NA Subtype RT-PCR]
 D --> F[HA Sequencing for Pathotype Determination]
 F --> G{Cleavage Site Analysis}
 G -- Monobasic --> H[LPAI Confirmed]
 G -- Polybasic --> I[HPAI Confirmed]
 H --> J[Report to National Veterinary Authority]
 I --> J
 B -- Negative --> K[No AIV Detected]
 K --> L["Consider Differential Diagnoses: Infectious Coryza, Newcastle Disease, etc."]

Notifiable Disease Status and Regulatory Framework

LPAI is classified as a notifiable disease by the World Organisation for Animal Health (WOAH). Under the WOAH Terrestrial Animal Health Code, infection with LPAI viruses of the H5 and H7 subtypes is notifiable due to the potential for these subtypes to mutate into HPAI. This mutation can occur through the introduction of basic amino acids at the HA cleavage site, either by nucleotide substitution or by recombination with host cell RNA. Therefore, the detection of any H5 or H7 LPAI virus triggers mandatory reporting and implementation of control measures, including stamping out, movement restrictions, and enhanced surveillance.

The notifiable status of LPAI H5 and H7 is a cornerstone of global avian influenza control. Surveillance programs are designed to detect these viruses in both domestic poultry and wild bird populations. Blin et al. analyzed surveillance data for HPAI in wild birds in France from 2016 to 2022, providing insights into the utility of mortality data for early warning systems [20]. Trogu et al. conducted surveillance for AIV in wild birds in the Lombardy region of Italy from 2022 to 2024, detecting both LPAI and HPAI strains. The detection of LPAI in wild birds, such as the H5N2 in Brazilian shorebirds and H3N8 in Chinese wild birds, underscores the importance of these surveillance efforts [5, 9]. Non-H5/H7 LPAI subtypes, such as H9N2 and H6N6, are generally not subject to mandatory stamping-out policies, but their circulation is monitored due to their economic impact and zoonotic potential [2, 3, 12].

Pathogenesis and Host Interactions

LPAI viruses primarily infect epithelial cells of the respiratory and intestinal tracts. The virus attaches to host cell sialic acid receptors via the HA glycoprotein. Avian influenza viruses preferentially bind to alpha-2,3-linked sialic acid receptors, which are abundant in the avian intestinal tract. Upon attachment, the virus is internalized via receptor-mediated endocytosis, and the low pH of the endosome triggers HA-mediated membrane fusion, releasing the viral genome into the cytoplasm. Replication occurs in the nucleus, and new virions are assembled at the plasma membrane, where NA cleaves sialic acid to facilitate viral release. In LPAI infections, the host immune response is typically sufficient to clear the virus, but co-infections with other pathogens can exacerbate disease. Regragui et al. documented the coinfection dynamics of infectious bronchitis virus (IBV) and H9N2 in Moroccan broiler farms, demonstrating that coinfection can lead to more severe respiratory disease. Similarly, the interaction between IBDV and H3N8, as studied by Nazki et al., shows that immunosuppression can increase the severity of LPAI infection [10]. Transcriptomic studies have provided insights into the early host response to LPAI. Kosonsiriluk et al. investigated the early transcriptomic responses of the uterovaginal junction and vagina to AIV infection in turkey breeder hens, revealing tissue-specific immune gene expression patterns [21].

Vaccination Strategies for LPAI

Vaccination is a key tool for controlling LPAI, particularly for H9N2 in endemic regions. Both inactivated whole-virus vaccines and recombinant vectored vaccines are used. Hossain et al. evaluated the protective efficacy of bivalent inactivated H9N2 and Newcastle disease vaccines commercially available in Bangladesh, finding variable levels of protection [16]. Abdelhalim et al. demonstrated enhanced protection through genotype-matched bivalent H9N2-Newcastle disease virus vaccination, emphasizing the importance of vaccine strain matching [22]. Novel vaccine platforms are under development. Yang et al. developed a recombinant chimeric Newcastle disease virus-vectored vaccine conferring single-dose, triple protection against genotype VII NDV, IBDV, and H9N2 AIV [23]. Luqman et al. explored the use of semi-purified LPAI H9N2 virus-like particles (VLPs) for broiler immunity, showing promise as an alternative to traditional vaccines. Lai et al. constructed a replication-defective recombinant virus and cell-based vaccine for H9N2, representing a next-generation approach.

Conclusion

Low pathogenic avian influenza remains a significant challenge to global poultry health in 2025. The sustained circulation of H9N2 across Asia and Africa [2, 3], the detection of H3 and H6 subtypes in wild birds [4, 12], and the emergence of novel reassortants underscore the need for continuous surveillance and molecular characterization. The notifiable status of H5 and H7 LPAI subtypes, as mandated by WOAH, remains a critical regulatory framework for preventing the emergence of HPAI. Advances in molecular diagnostics, including multiplex RT-PCR [14], high-throughput sequencing, and nanobody-based detection [18], have improved the capacity for rapid and accurate LPAI detection. Vaccination strategies, particularly for H9N2, continue to evolve, with a focus on genotype matching and DIVA compatibility [15, 22]. The integration of molecular virology, field epidemiology, and computational biology will be essential for managing the complex and dynamic landscape of LPAI in the years ahead.

References

[1] Lee HM, Sutton K, Harvey W, et al. Synergy between HA cleavage site sequence and NA-mediated plasminogen recruitment as a virulence mechanism for low-pathogenic avian influenza. mBio. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41744684/

[2] Li J, Zhang C, Li R, et al. A new clade of H9N2 avian influenza virus circulating in Laos. Emerg Microbes Infect. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42243068/

[3] Zhang W, Sun L, Wang Q, et al. Molecular characterization and pathogenicity of H9N2 avian influenza viruses in poultry in Shandong Province, China, 2021-2023. Poult Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42184734/

[4] Miao X, Zhao X, Zhang N, et al. Surveillance and biological characterization of H3 subtype avian influenza viruses in Eastern China. Virulence. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42154626/

[5] Magalhães TBS, da Rosa Bueno E, de Assis Pereira N, et al. First detection of an H5N2 subtype of Influenza A virus detected in Charadrius collaris from the Brazilian Pantanal. Sci Rep. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41866361/

[6] Kadja MC, Bako ABI, Onidje E, et al. Molecular Detection and Genetic Characterization of H9N2 Avian Influenza Virus in Laying Hen and Broiler Farms in Dakar and Thiès Regions, Senegal. Vet Ital. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41077876/

[7] Garcia-Glaessner A, Crespo-Bellido A, Muñoz-Saavedra B, et al. Outbreak of H9N2 avian influenza viruses in lesser rhea in Peru, June-July 2025. bioRxiv. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42182300/

[8] Zhao Y, Liu C, Li Y, et al. Genetic and biological characterization of H9N2 avian influenza viruses isolated from swine in China. BMC Vet Res. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42104325/

[9] Zhao J, Liu L, Li L, et al. Phylogenetic and Molecular Characteristics of An H3N8 Avian Influenza Virus Detected in Wild Birds - Beijing, China, September 2024. China CDC Wkly. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41256044/

[10] Nazki S, Tennakoon C, Reddy VRAP, et al. Evaluating how infectious bursal disease virus (IBDV) infection influences influenza H3N8 challenge in chickens. J Gen Virol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41805654/

[11] Śmietanka K, Tarasiuk K, Kycko A, et al. Comparative pathogenicity of a virulent field isolate and an NA S122N genetically modified H3N1 avian influenza virus in chickens. Avian Pathol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41568430/

[12] Zhu H, He D, Liu S, et al. H6N6 Avian Influenza Virus Infection Induced Pyroptosis of M1 Macrophages by Activating Caspase-1. Viruses. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41305513/

[13] Kan Q, Yin X, Guo X, et al. Genetic characteristics of three duck-original H1N1 influenza A viruses isolated in China. Vet Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41270509/

[14] An SH, Heo GB, Kang YM, et al. Development of a multiplex and universal RT-PCR assay for hemagglutinin and neuraminidase subtyping of avian influenza virus. J Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41045234/

[15] Zhang J, Fan Z, Pan Q, et al. Development of an inactivated H9N2 subtype avian influenza serological DIVA vaccine using the chimeric A/B NA epitope approach. Vet Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41653783/

[16] Hossain MR, Tasnim S, Akter MS, et al. Protective efficacy of bivalent inactivated avian influenza subtype H9N2 and Newcastle disease vaccines commercially available in Bangladesh. J Adv Vet Anim Res. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41328232/

[17] Qadir MF, Yang Y. Overcoming Challenges in Avian Influenza Diagnosis: The Role of Surface-Enhanced Raman Spectroscopy in Poultry Health Monitoring. Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41295690/

[18] Ye H, Dong Q, Qian M, et al. Development and application of a VNAR-based detection nanobody for avian influenza virus H9N2. Front Immunol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41953018/

[19] Nicholson HF, Zdyrski C, Leyson CM, et al. Development and characterization of chicken lung organoids for in vitro modeling of avian influenza virus-host cell interaction. Sci Rep. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41519874/

[20] Blin L, Calenge C, Decors A, et al. Surveillance of Highly Pathogenic Avian Influenza in Wild Birds in France, 2016-22: What Did We Learn from Mortality Data? J Wildl Dis. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41759901/

[21] Kosonsiriluk S, Santativongchai P, Reed KM, et al. Transcriptomic insights into early responses of the uterovaginal junction and vagina to avian influenza virus infection in turkey breeder hens. Front Physiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41584795/

[22] Abdelhalim A, Yahia N, Elharrak M, et al. Enhanced protection through genotype-matched bivalent H9N2-Newcastle disease virus vaccination: Comparative efficacy against contemporary field strains in specific-pathogen-free chickens. Vet Microbiol. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41045702/

[23] Yang P, Xiang M, Tian K, et al. Development of a recombinant chimeric Newcastle disease virus-vectored vaccine conferring single-dose, triple protection against genotype VII NDV, IBDV, and H9N2 AIV. Poult Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42056828/

[24] Xu S, Zhang Q, Xie X, et al. A neutralizing nanobody targeting a conserved lateral patch on HA1 confers protection against multiple H7 avian influenza viruses. J Virol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42274212/

[25] Cordero-Ortiz M, Solís-Hernández M, Cayetano-Mondragón M, et al. Antibody Recognition of Highly and Low-Pathogenic A/H5Nx Influenza Viruses in Sera of Mexican Donors. Pathogens. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42075679/

[26] Cargnin Faccin F, Gay LC, Regmi D, et al. Experimental infection and viral pathogenesis of a human isolate of H5N1 highly pathogenic avian influenza strain in Jersey cows. npj Vet Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/42006910/

[27] Porto-Fett ACS, Alvarez-Narvaez S, Vinayamohan PG, et al. Inactivation of Avian Influenza Virus in Raw Milk Kefir. Food Environ Virol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41801534/

[28] Son DH, Balupuri A, Nam JH, et al. Analyses of receptor binding specificity of highly pathogenic avian influenza A (H5N1) viruses isolated from felines in South Korea, 2023. Virulence. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41725526/

[29] Shosha EAE, Mohamd MK, Shehata MAE, et al. Genomic and evolutionary characterization of newly emerged highly pathogenic avian influenza H5N1 clade (2023-2025). Vet World. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41716167/

[30] Yan S, Hu C, Li H, et al. Genetic evolution and pathogenicity analysis of two natural recombinant isolates of avian influenza. Poult Sci. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41713014/

[31] Si YJ, Lee SH, Lee HE, et al. Early detection and genetic characterization of clade 2.3.4.4b H5N1 and H5N9 highly pathogenic avian influenza viruses at the onset of fall migration in wild birds during october 2025 in South Korea. Front Cell Infect Microbiol. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41660429/

[32] Daniel K, Ullrich L, Ruchnewitz D, et al. Pre-existing neutralizing antibodies against cattle-transmitted influenza A virus H5N1 are detectable in unexposed individuals. Immunity. 2026. URL: https://pubmed.ncbi.nlm.nih.gov/41619731/

[33] Meng L, Tang X, Gu W, et al. The phylogenetic, pathogenicity and transmission capacity analysis of one H9N2 strain. Front Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41509518/

[34] Adaszek Ł, Domańska-Blicharz K, Jańczak D, et al. Cats infected with H5N1 avian influenza - a new infectious disease in Poland. Pol J Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41416603/

[35] Al-Dalawi L, Yang J, Blanchard AM, et al. Research note: Use of in vitro and in ovo approaches to characterise avian influenza viruses and assess species specific responses to infection. Poult Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41135468/

[36] Hawman DW, Griffin A, McCarthy N, et al. A replicating RNA vaccine protects cynomolgus macaques against lethal clade 2.3.4.4b influenza A H5N1 virus challenge. Sci Transl Med. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41061043/

[37] Rondón-Espinoza J, Castro-Sanguinetti G, Apaza-Chiara A, et al. Immunogenicity of a Recombinant Avian Influenza H2 Protein Using an Abdominal Inoculation Model in Chickens. Vaccines (Basel). 2025. URL: https://pubmed.ncbi.nlm.nih.gov/41012129/

[38] Mariacher A, Di Nicola MR, Senese M, et al. Detection of avian influenza virus in the alien invasive African sacred ibis (Threskiornis aethiopicus) in Italy. Front Vet Sci. 2025. URL: https://pubmed.ncbi.nlm.nih.gov/40989950/ 39 *** Disclaimer: This article is for educational and informational purposes only. It is not intended to substitute for professional veterinary advice, diagnosis, treatment, or regulatory guidance. Always consult a licensed veterinarian or qualified specialist regarding animal health, disease diagnosis, and therapeutic decisions.