US study uncovers antiviral resistance in swine-origin influenza, urging enhanced pandemic surveillance

Swine-origin influenza viruses show mutations that resist antiviral drugs, posing a pandemic risk and highlighting the urgent need for ongoing surveillance and updated treatment strategies.

In a recent study published in the journal Emerging Infectious Diseases, researchers at the Centers for Disease Control and Prevention (CDC) in the United States of America (US) analyzed genome sequences of swine-origin influenza A viruses. They conducted phenotypic assays to establish subtype-specific susceptibility baselines for antiviral drugs. The findings contribute significantly to ongoing pandemic preparedness efforts, highlighting the need for vigilant monitoring of swine-origin influenza A viruses, which pose a continuous threat of reassortment and the potential for new pandemic strains.

They found that nearly all viruses had the S31N M2 resistance marker, two showed baloxavir resistance-related substitution mutations, and only the I38M substitution significantly reduced baloxavir susceptibility by 27-fold. These results underscore the importance of understanding antiviral resistance patterns in swine-origin viruses, as genetic mutations and reassortments could reduce the effectiveness of current antiviral treatments.

Background

Swine-origin influenza viruses (S-OIVs) are enzootic in pig populations worldwide, making pigs a key "mixing vessel" where human, bird, and pig influenza viruses can combine, increasing pandemic risks.

Swine influenza A viruses, consisting of H1N1, H1N2, and H3N2 subtypes, are enzootic globally and can mix with human and avian viruses, leading to reassortments with pandemic potential.

Since the late 1990s, North American swine viruses have acquired a triple-reassortment internal gene (TRIG) cassette from human, avian, and classical swine viruses. The 2009 pandemic A(H1N1)pdm09 virus emerged from such reassortment and has since been reintroduced into pigs, increasing viral diversity.

The continuing introduction of human pandemic strains back into swine populations creates opportunities for further reassortment, which complicates public health preparedness efforts and highlights the zoonotic threat posed by these viruses.

Variant swine-origin viruses are monitored under the World Health Organization’s (WHO’s) International Health Regulations due to their potential for generating new pandemic strains. Influenza antiviral drugs face challenges from genetic mutations, reassortments, and antiviral resistance, especially for matrix-2 (M2) blockers. Resistance to neuraminidase inhibitors and the continuing threat of antiviral-resistant strains highlight the need for ongoing surveillance and risk assessment of swine-origin influenza viruses. In particular, the study provides critical data on subtype-specific baselines for drug susceptibility, a key step in refining global antiviral susceptibility testing methodologies.

About the study

Swine flu symptoms in humans resemble typical influenza, including fever, cough, and muscle pains, but swine influenza viruses have a higher potential for reassortment due to genetic diversity.

All the procedures were conducted in enhanced Biosafety Level 2 laboratories. Various reagents and methodologies were employed to test variant influenza viruses. Neuraminidase inhibitors (NAIs) such as oseltamivir, zanamivir, peramivir, and laninamivir were dissolved in sterile distilled water, while baloxavir acid was prepared in dimethyl sulfoxide.

Broadly cross-reactive monoclonal antibodies FI6v3 and CR9114 were used for neutralization assessments, and antiserum from ferrets was treated with a receptor-destroying enzyme prior to use. The study revealed a wide range of neutralization activity for these antibodies across subtypes, with some variant viruses showing low or no neutralization even at high antibody concentrations. This variability underscores the challenges in developing monoclonal antibody therapies against swine-origin influenza viruses, as antigenic differences outside known epitopes may reduce their effectiveness.

Control experiments utilized CDC antiviral susceptibility reference virus panels. For genetic analysis, whole-genome sequences were generated and analyzed.

Results and Discussion

From January 2013 to April 2024, 167 human infections caused by variant influenza viruses were reported across 22 US states, including A(H1N1)v, A(H1N2)v, and A(H3N2)v cases.

Genetic analysis of 147 variant viruses identified molecular markers related to antiviral resistance. All except one variant had a resistance-conferring substitution (S31N) in the M2 protein, while no known markers for neuraminidase inhibitors (NAIs) were detected. The study provides critical insights into antiviral resistance mechanisms, emphasizing the importance of molecular surveillance in identifying markers that could affect treatment outcomes.

However, A/Iowa/02/2021 (H1N1)v had the S247N substitution, which reduces oseltamivir inhibition for A(H5N1). A few substitutions linked to reduced baloxavir susceptibility were also found in the polymerase acidic protein.

Susceptibility assessments showed that all variant viruses retained susceptibility to NAIs, with most displaying potent inhibition by oseltamivir, zanamivir, peramivir, and laninamivir. A(H1N2)v exhibited higher half-maximal inhibitory concentration (IC50) values compared to the other subtypes.

For baloxavir, A/Iowa/33/2017 (H1N1)v showed 27-fold lower susceptibility due to the I38M substitution, while other viruses did not show any effect. This significant reduction in baloxavir susceptibility highlights the potential for resistance mutations to compromise antiviral efficacy, particularly in swine-origin viruses.

Antigenic analysis indicated that recent variant viruses showed reduced reactivity to candidate vaccine viruses, pointing to antigenic evolution in swine-origin influenza viruses. Neutralization assays using human monoclonal antibodies targeting hemagglutinin (HA) revealed varying neutralization efficacy across subtypes, with CR9114 being slightly more effective against A(H1)v. However, the broad variability in neutralization efficacy suggests that current vaccine candidates may require updating to keep pace with the antigenic changes observed in swine-origin viruses.

Overall, the study highlights the need for continuous monitoring of antiviral susceptibility in variant influenza viruses to ensure that therapeutic strategies remain effective against emerging strains.

While the study improves our understanding of the epidemiology, susceptibility, and potential threat of variant influenza viruses, it is limited by incomplete virus testing, challenges in interpreting phenotypic data, and assay-dependent variability in antiviral susceptibility results. The study notes that phenotypic data interpretation is particularly challenging due to the lack of standardized correlates for clinically relevant resistance and assay variability. The findings underscore the importance of establishing subtype-specific baselines and harmonizing laboratory methodologies to improve the accuracy of antiviral susceptibility assessments.

Conclusion

In conclusion, the study provides an approach to standardizing testing methodologies for swine-origin influenza viruses and improving the interpretation of laboratory results, ultimately enhancing our understanding of viruses that present a potential pandemic threat. By refining antiviral susceptibility testing and tracking antigenic evolution in swine-origin viruses, the research offers vital insights into public health preparedness and pandemic risk assessment.

Journal reference:

• Gao R, Pascua PNQ, Chesnokov A, Nguyen HT, Uyeki TM, Mishin VP, et al. Antiviral susceptibility of swine-origin influenza A viruses isolated from humans, United States. Emerg Infect Dis. 2024 Nov DOI: 10.3201/eid3011.240892, https://wwwnc.cdc.gov/eid/article/30/11/24-0892_article