EID Journal Dispatch: Replication Efficiency of Contemporary Highly Pathogenic Avian Influenza A(H5N1) Virus Isolates in Human Nasal Epithelium Model

#19,135

In the past, H5N1 transmission in humans has been thought to be hampered by the generally hostile environment found in the human upper airway; cooler temperatures, the virus's preference for avian (α2,3-linked SA) receptors, and the cytokine signaling defenses of the innate immune system.

While the evidence continues to suggest that HPAI H5N1 viruses are not transmitting efficiently (or often) among humans, it is equally clear that some mild (or asymptomatic) infections have flown under the radar (see MMWR: Serologic Evidence of Recent Infection with HPAI A(H5) Virus Among Dairy Workers).

Over the past year, we've seen growing evidence that contemporary H5 viruses may be evolving towards overcoming some of these obstacles. Some recent blogs include: 

• Last month, in EID Journal: Tropism and Replication Competence of Cattle Influenza A(H5N1) Genotype B3.13 Virus in Human Bronchus and Lung Tissue, we saw a report that the `Bovine' H5N1 virus showed partial adaptation to human respiratory tissue, and replicated in the lung tissue on par with the 2009 H1N1pdm virus. 

• Last January, in Preprint: Bovine-derived Influenza A virus (H5N1) Shows Efficient Replication in Well-differentiated Human Nasal Epithelial Cells Without Requiring Genetic Adaptation, we saw another study - this time on the upper airway - which found H5N1Tex/24 crucially replicated effectively at 33 degrees Celsius, whereas earlier avian H5N1 strains require 37 degrees Celsius.

• And six months ago, in J.I.D.: Avian influenza virus A(H5N1) genotype D1.1 is better adapted to human nasal and airway organoids than genotype B3.13, researchers demonstrated that D1.1 genotype replicates better in lab-grown nasal and lung tissues than the bovine B3.13 strain, and it binds more tightly to human‑type (α2,6-linked SA) receptors.  

Yesterday the CDC's EID Journal published another in this growing list of studies suggesting that recently emerged strains of HPAI H5N1 clade 2.3.4.4b are becoming better adapted to the human upper respiratory system than older strains.  

This NIAID study compares several HPAI and seasonal flu strains and finds that some strains of HPAI H5 (B3.13 & D1.1) have become better adapted to  human physiology; including tolerance to lower temperatures and the ability to evade some of our innate immune responses. 

While these are incremental changes, and the virus still has a ways to go, this is a worrying trajectory.  Due to its technical nature, I've only posted some excerpts from the dispatch.  Follow the link to read it in its entirety. 

Replication Efficiency of Contemporary Highly Pathogenic Avian Influenza A(H5N1) Virus Isolates in Human Nasal Epithelium Model

Meaghan Flagg1, Christopher J. Winski1, Bridget G. Brackney, Tessa R. Lutterman, Johan A. Ortiz-Morales2, Brandi N. Williamson, and Emmie de Wit 

Abstract

Replication of influenza A virus in human nasal epithelium affects transmissibility and disease. We compared virus replication and immune responses in human nasal epithelium infected with seasonal and highly pathogenic avian influenza A(H5N1) viruses. Contemporary H5N1 viruses replicated better than the historical isolate; however, interferon response to B3.13 genotype viruses was dampened.

Since March 2024, a total of 70 human cases of highly pathogenic avian influenza (HPAI) A(H5N1) have been reported in the United States as a result of sporadic spillover events from poultry and dairy cattle (1). HPAI H5N1 clade 2.3.4.4b genotype B3.13 was responsible for many of the early cases (2).

On January 31, 2025, HPAI H5N1 clade 2.3.4.4b genotype D1.1 was detected in dairy cattle (https://www.aphis.usda.gov/news/program-update/aphis-confirms-d11-genotype-dairy-cattle-nevada-0External Link); D1.1 was later identified in humans (1). Those spillover events sparked global health concerns about the potential for large-scale spread of clade 2.3.4.4b HPAI H5N1 viruses and their risk to human and animal health.

Seasonal influenza A and HPAI H5N1 viruses both cause severe respiratory disease despite different tissue tropisms. Seasonal influenza A viruses primarily infect the upper respiratory tract (URT), whereas HPAI H5N1 viruses preferentially replicate in the lower respiratory tract (LRT). This contrast in tissue affinity is explained by differences in receptor specificity and has been implicated in transmission efficiency (3). Specifically, the URT predominantly expresses sialic acids linked to galactose by an α-2,6 linkage; the LRT expresses sialic acid linked to galactose via α-2,3. 

Despite the inefficient human-to-human transmission of HPAI H5N1 viruses, recent emergence and circulation of new genotypes in mammals emphasize the need to characterize these novel viruses in relevant respiratory tract models.

Here, we compare the replication kinetics and host innate immune responses in human nasal epithelium of several seasonal influenza A virus isolates and historical and contemporary HPAI H5N1 virus isolates of 3 different genotypes.

(SNIP)

HPAI H5N1 isolate A/Texas/37/2024 (B3.13 genotype) replicated most efficiently in nasal tissue even when compared with seasonal isolates (Figure 1). Although we noted differences in replication kinetics between viruses of the same genotype, the B3.13 and D1.1 genotype isolates replicated more efficiently than the historical HPAI H5N1 isolate A/Vietnam/1203/2004. The B3.6 HPAI H5N1 isolate A/mountain lion/MT/1/2024 replicated least efficiently (Figure 1). Presence of known mammalian adaptations of polymerase basic (PB) 2 E627K, PB2 D701N, and PB2 M631L was associated with more efficient virus replication (Table; Figure 1).

(SNIP)

HPAI H5N1 virus replication can be affected by the physiologic temperature of the human nasal mucosa, for which the reference is 33°C (11,12). To address the potential effect of temperature on virus replication, we quantified virus replication kinetics at 37°C and 33°C in MDCK cells.

At 8 hours postinoculation, the titers of HPAI H5N1 virus isolates were lower at 33°C than at 37°C (Appendix Figure 2). However, all viruses reached the same maximum titer at 33°C and 37°C. In addition, the relative difference observed between viruses at 37°C were similar at 33°C, suggesting that adjusting the temperature in the nasal epithelial cultures to 33°C would not have substantially altered our results.

Conclusions

Our results reveal that contemporary HPAI H5N1 isolates with known mammalian adaptations replicate more efficiently than historical HPAI H5N1 virus used. Despite high levels of virus replication, ISG induction was limited in response to B3.13 genotype virus infection. Additional studies are needed to further understand how virus replication efficiency and innate immune responses affect mammalian transmission efficiency. Existing immunity to other influenza A viruses might protect against contemporary H5N1 infection and onward transmission.

       (Continue . . . )

 
While it will likely take more than just adapting to the human upper airway to make H5N1 a pandemic contender, increasingly breaching our first line of defense is an important milestone.