Antiviral susceptibility of clade 2.3.4.4b highly pathogenic avian influenza A(H5N1) viruses from humans - study

Sep 29, 2026

Since October 2024, 55 human cases of influenza A(H5N1) of clade 2.3.4.4b were reported in the United States. Sequencing of 46 viruses revealed genotypes B3.13, D1.1, and D1.3, with viral genomes analyzed for mutations linked to reduced antiviral susceptibility. Most viruses were assessed susceptible based on in vitro testing using M2 blockers, neuraminidase inhibitors, and baloxavir. However, an M2-S31N variant conferred cross-resistance to M2 blockers, and a PA-I38M substitution caused 17-fold reduced baloxavir susceptibility. In mice, baloxavir was less effective against the PA-I38M virus, reflected by survival, time-to-death, weight loss, and organ viral replication. Replication was mildly attenuated in vivo but not in cell culture, while animal-derived samples showed low-frequency resistance-associated mutations including NA-H275Y.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages12

Reading Time30 min

AccessFree to download

Abstract

Introduction

Clade 2.3.4.4b outbreaks and transmissions

Human cases and clinical severity

Antiviral treatment options and resistance concern

Emerging Microbes & Infections 2026, VOL. 15, 2601372 (12 pages) [https://doi.org/10.1080/22221751.2025.2601372](https://doi.org/10.1080/22221751.2025.2601372) Antiviral susceptibility of clade [2.3.4.4](2.3.4.4) b highly pathogenic avian influenza A(H5N1) viruses from humans in the United States, October 2024 to February 2025 Philippe Noriel Q. Pascua*, Anton P. Chesnokov*, Ha T. Nguyen, Chloe Champion, Rongyuan Gao, Juan A. De La Cruz, Yunho Jang, Yasuko Hatta, Zhu Guo, Timothy M. Uyeki, Han Di, James Stevens, Charles T. Davis and Larisa V. Gubareva Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA, USA ABSTRACT Since October 2024, 55 human cases of influenza A(H5N1), clade [2.3.4.4](2.3.4.4)b, were reported in the US. Sequencing of 46 viruses identified genotypes B3.13, D1.1, and D1.3 . Virus genomes were analyzed for mutations associated with reduced antiviral susceptibility. Except for two, viruses from human cases were assessed as susceptible to antiviralsand confirmed by in vitro susceptibility testing of representative viruses with M2 blockers, neuraminidase (NA) inhibitors, and the polymerase acidic (PA) inhibitor baloxavir. One D1.1 virus had an M2-S31N substitution, which conferred cross-resistance to M2 blockers. One B3.13 virus had a PA-I38M substitution and displayed 17-fold reduced baloxavir susceptibility in cell culture. In mice, baloxavir, given orally at 5, 15, and 45 mg/kg bid for 5 days starting at 2 h post-infection, was notably less effective against the PA-I38M virus compared to the closely matched control virus, as shown by survival rate, time-to-death, weight loss, and viral replication in various organs. Replication of the PA-I38M virus was mildly attenuated in mice but not in cell culture. Viruses collected from animals also contained mutations associated with reduced antiviral susceptibility, including M2-S31N, PA-I38 T/M, and NA-H275Y, albeit at low frequency. Regardless of genotype, most mutations were found in NA. Using recombinant NA proteins generated from B3.13 and D1.1 backgrounds, we showed that among mutations which reduced inhibition by NA inhibitors, only NA-H275Y had no effect on enzyme function. Spontaneous emergence of drug-resistant influenza viruses, especially those that maintain replicative fitness, pose persistent threat to human health and must be closely monitored. ARTICLE HISTORY Received 11 September 2025; Revised 22 November 2025; Accepted 4 December 2025 KEYWORDS A(H5N1) viruses; neuraminidase; neuraminidase inhibitors; bovine flu; drug resistance; baloxavir Introduction Since late 2021, clade [2.3.4.4](2.3.4.4)b highly pathogenic avian influenza (HPAI) A(H5N1) viruses have caused widespread outbreaks in wild birds and poultry in North America. These viruses have reassorted with low pathogenicity avian influenza A viruses that led to co-circulation of genetically diverse HPAI A(H5) viruses ([https://github.com/USDA-VS/GenoFLU](https://github.com/USDA-VS/GenoFLU)) [1]. To date, over 100 distinct genotypes of clade [2.3.4.4](2.3.4.4)b viruses have been identified in North American birds and other animals. In early 2024, a genotype B3.13 virus spilled over to dairy cows in the United States (U.S.) causing unprecedented multi-state outbreaks [2–4] . The interspecies transmission of clade [2.3.4.4](2.3.4.4)b viruses also led to sporadic human cases, mostly among individuals working closely with infected cows or poultry. Thus, the ongoing clade [2.3.4.4](2.3.4.4)b virus outbreaks in animals are a concern to public health. Between 28 March 2024, and 12 February 2025, a total of 70 human cases of A(H5N1) in the U.S. were confirmed by the Centers for Disease Control and Prevention (CDC) [5,6] . The only other confirmed case occurred in 2022 [7] . Forty-one of the A(H5N1) cases were exposed to sick cows and 26 worked with infected poultry; the source of exposure in three cases was not determined [6] . While most of the human infections

FAQs

Which antiviral resistance-associated mutations were identified in the clade 2.3.4.4b human viruses?

The study identified M2-S31N, PA-I38M, and NA-H275Y as mutations associated with reduced antiviral susceptibility, with most resistance-related changes detected in neuraminidase.

How did the PA-I38M mutation affect baloxavir susceptibility in vitro and in mice?

PA-I38M showed 17-fold reduced baloxavir susceptibility in cell culture. In mice, baloxavir was notably less effective against the PA-I38M virus, based on survival outcomes, time-to-death, weight loss, and viral replication in organs.

Were most viruses from human cases susceptible to antivirals?

Yes. Except for two, viruses from human cases were assessed as susceptible to antivirals and were confirmed using in vitro susceptibility testing with M2 blockers, neuraminidase inhibitors, and the polymerase acidic inhibitor baloxavir.

Sep 29, 2026

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