A classic serial passage experiment showing Host adaptation
#17,942
Yesterday's EFSA systematic review of The Role of Mammals in Avian Influenza looked at the known (and feared) mammalian adaptations that avian flu viruses may accrue as they continue to spill over into non-avian species. Adaptations that might increase their transmissibility, pathogenicity, or both.
In addition to HPAI H5 and H7 viruses, this study also looked at LPAI H10, H3N8, and H9N2, all of which have shown some ability to infect, and sicken, humans.
H9N2 gets a lot of our attention because 1) it is ubiquitous in poultry - particularly in Asia and the Middle East, 2) it readily reassorts with other, often more dangerous viruses, 3) it has been poorly controlled by poultry vaccination 4) and while it usually only produces mild illness in humans, seroprevalence studies suggest it infects people more often than case counts would suggest.
Globally, a bit over 100 cases have been confirmed (see FluTrackers list), mostly from China. The CDC has designated 2 different lineages (A(H9N2) G1 and A(H9N2) Y280) for their short list of influenza viruses with zoonotic potential (see CDC IRAT SCORE), and several candidate vaccines have been developed.
Due to H9N2's relatively mild presentation in humans, we tend to worry primarily about its role as a possible co-conspirator in a reassortment scenario (with seasonal flu, avian, canine, or swine flu viruses).
All of which brings us to a new study, published in the journal Infectious Medicine, that looks at the pathogenicity (in mice) of more than 40 H9N2 viruses collected from humans in China between 2013 and 2021.
While most of these H9N2 viruses showed little or no pathogenicity in mice, three strains (A/Hubei-songzi/1631/2019 (HB-1631), A/Hunan/11173/2020 (HN-11173), A/Guangdong/00470/2021 (GD-00470) produced significant weight loss in inoculated mice.
An analysis of these 3 outliers led investigators to identify a single amino acid change - L226Q (H3 numbering) in the HA protein sequence - as responsible for increased replication and pathogenicity in mice.
The researchers then selected three viruses (A/Hunan/11173/2020, A/Hunan/34179/2018, and A/Guangdong/12902/2021) - all containing HA 226L - for serial passage through mice (see graphic at top of blog). They reported:
The sequencing results showed that the proportion of HA 226Q reads exceeded 98% in the lung tissues of all of the infected mice in both the P3 and P4 generations (Table S3). This result indicated that the HA L226Q mutation rapidly occurred during viral replication in the lungs of mice, and was commonly observed in these human H9N2 isolates.
L226Q indicates the switch from leucine to glutamine substitution at residue 226 in the HA. Due to its length, I've only posted the abstract and some excerpts. Follow the link to read the study in its entirety.
I'll have a brief postscript after the break.
Rapid adaptive substitution of L226Q in HA protein increases the pathogenicity of H9N2 viruses in mice
Min Tan a, Ye Zhang a, Hong Bo a, Xiyan Li a, Shumei Zou a, Lei Yang a, Jia Liu a, Qi Chen a b, Xiaohao Xu a b, Wenfei Zhu a, Dayan Wang aShow more
https://doi.org/10.1016/j.imj.2024.100090Get rights and content
Under a Creative Commons license
Abstract
Background
Since the first human infection with H9N2 virus was reported in 1998, the number of cases of H9N2 infection has exceeded one hundred by 2021. However, there is no systematic description of the biological characteristics of H9N2 viruses isolated from humans.
Methods
Therefore, this study analyzed the pathogenicity in mice of all available H9N2 viruses isolated from human cases in China from 2013 to 2021.
Results
Although most of the H9N2 viruses analyzed showed low or no pathogenicity in mice, the leucine to glutamine substitution at residue 226 (L226Q) in the hemagglutinin (HA) protein rapidly emerged during the adaptation of H9N2 viruses, and was responsible for severe infections and even fatalities. HA amino acid 226Q conferred a remarkable competitive advantage on H9N2 viruses in mice relative to viruses containing 226L, increasing their virulence, infectivity, and replication.
Conclusion
Thus, our study demonstrates that the adaptive substitution HA L226Q rapidly acquired by H9N2 viruses during the course of infection in mice contributed to their high pathogenicity.
4. Discussion
The key findings of the present study are as follows. Forty-three strains of human-infecting H9N2 viruses were screened by inoculation in mice and 3 strains possessed higher pathogenicity.
Sequence analysis showed that the rapid amino acid substitution L226Q in HA mediated the increased pathogenicity of H9N2. HA L226Q also increased the virulence and replication of H9N2 and the inflammatory damage in mice. Although strains such as HN-34179 developed L226Q after multiple mouse lung tissue passages, the original strain with HA 226L had low pathogenicity in mice after initial infection with high viral titers, indicating the significance of this specific mutation in determining pathogenicity.
Avian influenza has undergone adaptive amino acid mutations during its adaptation to mammals, leading to pathological changes in the virus [21]. For example, mutations at E627K [22] and D701N [23] in PB2 of the H9N2 viruses increase their virulence and replication capacities. Combined mutations in HA (L226Q) and PB2 (M147L, V250G, or E627K) or HA (L226Q) and M1 (R210K) significantly increase the virulence of the H9N2 chicken isolate in mice, whereas the single mutation HA L226Q only increases viral replication [24]. In the present study, we confirmed the substitution of L226Q in H9N2 viruses from human cases caused phenotypic changes and increased the pathogenicity of the virus in mice. Our animal experiments also showed that viruses with HA 226Q had a competitive advantage in mice over viruses with HA 226L. The proportion of HA 226Q exceeded 84% in the P2 generation and was nearly 100% in the P3 generation of mice inoculated with RG HB-1631 virus.
Notably, the L226Q substitution in HA destroyed the binding preference of the virus for the α2,6-SA receptor, allowing it dual-receptor binding ability, binding to both α2,3-SA and α2,6-SA receptors.
(SNIP)
In summary, we have identified a wt H9N2 virus from human cases with a HA L226Q mutation. We have demonstrated that this mutation arises in a proportion of viruses during replication in a mouse model, alters the viral phenotype, and induces lethal infections in mice. These findings provide valuable insight into the mechanisms underlying the pathogenicity of H9N2 virus in humans, potentially informing the development of effective prevention and control strategies.
While this study is narrowly focused on H9N2's passage through and adaptation in mice, it is illustrative of our general concerns whenever we see a novel flu virus spillover - and sometimes spread - in pigs, mink, seals, or other mammalian species.
Each event is an opportunity for the virus to pick up host adaptations that, over time, could increase its pandemic potential.
If it were easy for an avian virus to fully adapt to humans, we'd be hip-deep in pandemic viruses all of the time. Luckily, it isn't. But it happens often enough that we can't afford to ignore the changes we are seeing in the spread and evolution of novel flu viruses around the globe.
