Researchers from St. Jude Children's Research Hospital CEIRR (SJCEIRR) responded to the influenza H5N1 outbreak in cattle by testing the virus' susceptibility to antivirals. Read about their results below or in PDF format (updated May 30, 2024).
Aim of Study: Antiviral drugs can play an important role in the control of influenza virus infection. Two classes of direct-acting drugs are approved by Food and Drug Administration (FDA) in the United States for treating influenza: neuraminidase (NA) inhibitors (NAIs) (oseltamivir, zanamivir, and peramivir) and a cap-dependent endonuclease inhibitor (CENI) (baloxavir marboxil)1. However, emergence of drug-resistant variants can limit therapeutic options, and a critical component of risk assessment of emerging influenza A viruses is determining their susceptibility to existing antivirals. Here we evaluated the antiviral susceptibility of highly pathogenic avian influenza (HPAI) A(H5N1) viruses of dairy cattle by genotypic and phenotypic approaches.
Influenza Viruses, Cells, and Compounds: Experiments were conducted under biosafety level 3-enhanced conditions in compliance with applicable laws and guidance. A(H5N1) viruses isolated from bovine swab, milk, and urine samples at two U.S. dairy farms, one in Texas and one in Ohio were propagated in allantoic cavities of 10-day-old embryonated chicken eggs for 40 h, 35°C. Madin-Darby canine kidney (MDCK) cells were obtained from ATCC. NAIs (oseltamivir carboxylate, zanamivir, and peramivir) and baloxavir acid (active compound of baloxavir marboxil) were purchased from MedChem Express.
NAI Susceptibility Determinations: NAI susceptibility was assessed in a fluorescence-based assay2, and the fluorescent NA-cleaved substrate was measured with a BioTek Multimode Plate Reader (Agilent) at Ex/Em 360 nm and 460 nm, and dose–response curves were prepared. The half-maximal inhibitory concentration (IC50) of the NAI was then calculated with GraphPad Prism (v.9.5), using a sigmoidal dose–response (variable slope) equation.
CENI Susceptibility Determination: CENI susceptibility was assessed in MDCK cells by an Influenza Replication Inhibition Neuraminidase-based Assay (IRINA), with the virus inoculum normalized to 1.9 nM/well of standard3. NA activity was determined as in the NAI assays, and the baloxavir acid half-maximal effective concentration (EC50) was calculated from the dose–response curve with GraphPad Prism (v.9.5).
Sequence-based Analysis: Sequence data for 202 highly pathogenic influenza A(H5N1) viruses related to an outbreak in dairy cattle in 2024 were kindly provided by USDA. The analysis was based on NA (NAI target) and PA (CENI target) markers reported previously and summarized by the WHO Expert Working Group on Antiviral Susceptibility4.
RESULTS:
Genotypic analysis of 202 NA and PA gene sequences revealed one virus contained NA-T438I substitution (N1 numbering) that has been previously shown to confer reduced inhibition (RI) by zanamivir and peramivir5. None of the established markers of CENI RI were identified. Of the four isolates from Texas that were available for phenotypic testing, A/bovine/Texas/38161/2024 carried the NA-G147E substitution that has not been previously reported to cause NAI RI.
Phenotypic testing confirmed all tested viruses were susceptible to NAIs (oseltamivir, zanamivir and peramivir) and CENI baloxavir at sub-nanomolar concentrations (Table 1). The results did not vary significantly between Texas and Ohio locations. Oseltamivir IC50s of bovine A(H5N1) viruses were slightly lower than those of contemporary clade 2.3.4.4.b viruses circulating in 2022-2023 in birds5. Oseltamivir IC50 against A/bovine/Texas/38161/2024 (NA-G147E) was 3.1 nM corresponding to a 2- and 4-fold increase over the median values for bovine and avian A(H5N1) viruses, respectively, thus failing to meet RI criteria. A minor (2-fold) reduction of baloxavir EC50 was observed for A/bovine/Texas/42041/2024, which is below the > 3-fold arbitrary threshold designating reduced CENI susceptibility.
Table 1. Phenotypic NAI and CENI susceptibility of North American HPAI A(H5N1) viruses isolated from dairy cattle in 2024.
| Influenza A virus | CENI baloxavir EC50 ± SD [nM] (fold change)* | NAI, IC50 ± SD [nM] (fold change)* | ||
Oseltamivir | Zanamivir | Peramivir | ||
| A/bovine/Texas/38161/2024 | 0.33 ± 0.11 (1) | 3.08 ± 0.08 (2) | 0.29 ± 0.03 (1) | 0.08 ± 0.00 (1) |
| A/bovine/Texas/97794/2024 | 0.26 ± 0.03 (1) | 0.70 ± 0.07 (1) | 0.14 ± 0.01 (1) | 0.07 ± 0.00 (1) |
| A/bovine/Texas/43134/2024 | 0.41 ± 0.05 (1) | 0.71 ± 0.07 (1) | 0.14 ± 0.01 (1) | 0.07 ± 0.01 (1) |
| A/bovine/Texas/42041/2024 | 0.62 ± 0.45 (2) | 0.91 ± 0.05 (1) | 0.15 ± 0.00 (1) | 0.07 ± 0.01 (1) |
| Bovine A(H5N1) Texas median (n = 4) | 0.37 ± 0.08 (1) | 0.81 ± 0.07 (1) | 0.14 ± 0.01 (1) | 0.07 ± 0.00 (1) |
| A/bovine/Ohio/B24OSU-302/2024 | 0.25 ± 0.06 (1) | 0.80 ± 0.13 (1) | 0.16 ± 0.02 (1) | 0.08 ± 0.01 (1) |
| A/bovine/Ohio/B24OSU-358/2024 | 0.38 ± 0.00 (1) | 0.84 ± 0.10 (1) | 0.20 ± 0.04 (1) | 0.09 ± 0.00 (1) |
| A/bovine/Ohio/B24OSU-439/2024 | 0.40 ± 0.01 (1) | 0.78 ± 0.05 (1) | 0.15 ± 0.00 (1) | 0.15 ± 0.06 (1) |
| A/bovine/Ohio/B24OSU-541/2024 | 0.18 ± 0.03 (1) | 0.98 ± 0.12 (1) | 0.19 ± 0.01 (1) | 0.09 ± 0.00 (1) |
| Bovine A(H5N1) Ohio median (n = 4) | 0.32 ± 0.02 (1) | 0.82 ± 0.11 (1) | 0.18 ± 0.02 (1) | 0.09 ± 0.01 (1) |
| HPAI (H5N1) clade 2.3.4.4b median (n = 12) | 0.29 ± 0.06 | 1.46 ± 0.18 | 0.24 ± 0.08 | 0.09 ± 0.01 |
Mean values from two independent experiments are provided ± the standard deviation (SD).
*Relative to the baseline susceptibility of North American HPAI A(H5N1) clade 2.3.4.4b viruses5.
CONCLUSIONS:
- Sequence-based analysis of NA and PA genes of bovine influenza A(H5N1) viruses (n = 202) isolated in 2024 showed:
- A single influenza A(H5N1) virus possesses NA-T438I substitution (N1 numbering). We have previously shown that this NA substitution caused reduced inhibition by NAIs zanamivir and peramivir but not oseltamivir5. Virus was not available for phenotypic testing at St. Jude Children’s Research Hospital.
- PA substitutions associated with RI by CENI baloxavir marboxil have not been identified among bovine influenza A(H5N1) viruses.
- Phenotypic testing of 8 bovine A(H5N1) viruses revealed susceptibility to NAIs and CENI.
Overall, highly pathogenic influenza A(H5N1) viruses isolated from dairy cattle in the United States retain susceptibility to FDA-approved antiviral drugs.
REFERENCES:
4. World Health Organization. Laboratory methodologies for testing the antiviral susceptibility of influenza viruses. Accessed May 2024.