Summary of amino acid mixtures identified in hemagglutinin (HA) of patient clinical samples.

Overall, the hemagglutinin (HA) sequences from the two clinical samples were closely related to HA sequences detected in other viruses of genotype D1.1, including viruses sequenced from samples collected in November and December 2024 from wild birds and poultry were collected in Louisiana. The HA genes of these viruses were also closely related to the candidate vaccine virus (CVV) A/Ezo Red Fox/Hokkaido/1/2022, with 2 or 3 amino acid changes detected. These viruses have an average of 3 or 4 amino acid changes in the HA in direct comparison with the CVV sequence A/Astrakhan/3212/2020. These data suggest that the viruses detected in respiratory samples from this patient are closely related to existing HPAI A(H5N1) CVVs that are already available to manufacturers and could be used to produce vaccines if necessary.

Some differences were noted between the NP/OP and NP samples. Despite the very high similarity of the D1.1 sequences from the Louisiana human case to avian viruses, deep sequencing analysis of the HA gene segment from the combined NP/OP sample detected low-abundance mixed nucleotides corresponding to notable amino acid residues (using mature HA). consecutive numbering):

• A134A/V (alanine 88%, valine 12%);
• N182N/K (asparagine 65%, lysine 35%); And
• E186E/D (glutamic acid 92%, aspartic acid 8%).

Of note, the NP sample did not exhibit these low-frequency changes, suggesting that they may have been detected through swabs of the patient’s oropharynx. While these low-frequency changes are rare in humans, they have been reported in previous cases of A(H5N1) in other countries and most commonly in severe disease2345. For example, the E186E/D mixture was also identified in a sample taken from the severe human case in British Columbia, Canada67.

This summary analysis focuses on the detection of mixed nucleotides at residues A134V, N182K, and E186D, as these changes may lead to increased virus binding to α2-6 cell receptors in the human upper respiratory tract. It is important to note that these changes represent a small proportion of the total virus population identified in the sample analyzed (i.e. the virus still retains a majority of the “avian” amino acids at the residues associated with receptor binding). The observed changes were likely caused by replication of this virus in patients with advanced disease and were not primarily transmitted at the time of infection. Comparison of influenza A(H5) sequence data from viruses identified in wild birds and poultry in Louisiana, including poultry identified on the patient’s property, and other regions of the United States not Identify these changes. Of note, virus sequences from poultry collected from the patient’s property were nearly identical to the patient’s virus sequences, but did not have the mixed nucleotides identified in the patient’s clinical sample, strongly suggesting that the Changes occurred during infection as the virus replicated in the patient. Although worrying and a reminder that A(H5N1) viruses can develop changes in the clinical course of infection in humans, these changes would be more worrisome if they occurred in animal hosts or in early stages of infection (e.g. within a few days would occur after the onset of symptoms) if these changes were more likely to facilitate spread to close contacts. Notably, no transmission from the patient in Louisiana to other individuals was identified in this case. The Louisiana Department of Public Health and CDC are collaborating to generate additional sequencing data from consecutive patient samples to facilitate further genetic and virological analysis.

Additional genomic analysis

The genetic sequences of the A(H5N1) viruses from the Louisiana patient did not have the PB2-E627K change or other changes in polymerase genes associated with mammalian adaptation, and there was no evidence of low-frequency changes at critical positions. And as with other genotype D1.1 viruses found in birds, the sequences lack PB2 M631L, which is associated with viral adaptation to mammalian hosts and has been detected in >99% of sequences from dairy cows but is only sporadic in birds. Analysis of the N1 genes neuraminidase (NA), matrix (M) and polymerase acid (PA) from the samples revealed no changes associated with known or suspected markers of reduced susceptibility to antiviral drugs. The remaining genetic sequences from A/Louisiana/12/2024 were closely related to sequences detected in genotype D1.1 viruses from wild birds and poultry, including poultry identified on the patient’s property, providing further evidence suggests that humans were most likely infected after contact with birds infected with the D1.1 genotype virus.

Follow-up actions

Overall, the CDC believes that the risk to the public associated with the ongoing HPAI A(H5N1) outbreak in the United States has not changed and remains low. The discovery of a severe human case with genetic alterations in a clinical sample underscores the importance of continued genomic surveillance in humans and animals, containment of avian influenza A(H5) outbreaks in dairy cows and poultry, and prevention measures in humans exposed to infected humans animals or environments.