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| Credit NIAID |
#18,178
Just as we often see with growing antibiotic resistance, over time viral evolution can degrade the ability of antivirals to work. Not quite 20 years ago, we lost our frontline influenza anti-viral drug - Amantadine - after it suddenly lost effectiveness.
Amantadine would probably have eventually been retired, but its use as an additive to chicken feed in China to combat bird flu is widely believed to have led to its sudden demise (see Nature News China's chicken farmers under fire for antiviral abuse).
Luckily, we had an (admittedly more expensive) alternative - oseltamivir (aka `Tamiflu') which had been approved in the late 1990s. Although occasional instances of Oseltamivir resistance had been observed, in nearly every case, it developed after a person was placed on the drug (i.e. `spontaneous mutations’).
Wile an obvious concern to the patient receiving treatment, it occurred in only about 1% of treated cases, and studies suggested that these resistant strains were `less biologically fit’, and were therefore believed unlikely to spread from human-to-human.
That assumption was dispelled in 2008, when in the course of a less than a year the H1N1 virus defied all expectations and went from being 99% susceptible to oseltamivir to 99% resistant (see CIDRAP article With H1N1 resistance, CDC changes advice on flu drugs).
Although an influenza antiviral crisis seemed unavoidable, in an unlikely Deus Ex Machina moment a new swine-origin H1N1 virus - one that happened to retain its sensitivity to Tamiflu - swooped in as a pandemic strain in the spring of 2009, supplanting the older resistant H1N1 virus.
This antiviral resistance was primarily due to an H275Y mutation - where a single amino acid substitution (histidine (H) to tyrosine (Y)) occurs at the neuraminidase position 275 (Note: some scientists use 'N2 numbering' (H274Y)).
Since 2009 flu surveillance centers around the world have been looking for any signs of growing resistance to NAI inhibitors. For the most part, we've seen the same 1% incidence of spontaneous mutations in people receiving the antiviral, although we've seen a few `clusters' of cases.
- Four years ago, in EID Journal: Cluster of Oseltamivir-Resistant & Antigenically Drifted Influenza A(H1N1)pdm09 Viruses, Texas, USA, January 2020, we looked at a cluster of resistant seasonal flu viruses which emerged just before COVID shut down influenza around the globe.
- In 2016, in Eurosurveilance: A(H1N1)pdm09 Virus With Cross-Resistance To Oseltamivir & Peramivir - Japan, March 2016 we looked at an elevated number of NAI resistant viruses with `permissive mutations' circulating in Japan.
- In 2014's Eurosurveillance: Community Cluster Of Antiviral Resistant pH1N1 in Japan, we looked at a cluster of six genetically similar resistant viruses in Sapporo, Japan - but without epidemiological links.
Four months ago, however, we saw a worrisome report in The Lancet - Global Emergence of Neuraminidase Inhibitor-Resistant Influenza A(H1N1)pdm09 Viruses with I223V and S247N Mutations - which reported a much higher incidence of oseltamivir resistance among samples tested in Hong Kong in 2023 (along with a concurrent rise in GISAID sequences deposited since last summer).
Instead of the H275Y mutation which caused so much trouble in 2008, these viruses carried dual I223V/S247N mutations.
While oseltamivir isn't our only antiviral option (see FDA Approved Xofluza : A New Class Of Influenza Antiviral), it is the most popular antiviral, and has been stockpiled by countries the most.
Three weeks ago we took a cursory look at a preprint published on bioRxiv by researchers at the Robert Koch-Institute that reported seeing an uptick in permissive secondary mutations (NA-V241I and NA-N369K) in the NA or circulating H1N1 viruses that they believe may enable resistant strains to transmit more efficiently.
Today the full peer-reviewed study was published by the journal viruses (see below), and while the actual number of resistant strains detected thus far remains limited, the authors warn`. . . The accumulation of secondary synergistic substitutions in the NA of A(H1N1)pdm09 viruses increases the probability of the emergence of antiviral-resistant viruses'.
Due to its length, I've only posted the abstract and some excerpts, so follow the link to read the study in its entirety. I'll have a brief postscript after the break.
Increase of Synergistic Secondary Antiviral Mutations in the Evolution of A(H1N1)pdm09 Influenza Virus Neuraminidases
Susanne C. Duwe *, Jeanette Milde, Alla Heider, Marianne Wedde, Brunhilde Schweiger and Ralf Dürrwald
Abstract
The unexpected emergence of oseltamivir-resistant A(H1N1) viruses in 2008 was facilitated in part by the establishment of permissive secondary neuraminidase (NA) substitutions that compensated for the fitness loss due to the NA-H275Y resistance substitution. These viruses were replaced in 2009 by oseltamivir-susceptible A(H1N1)pdm09 influenza viruses.
Genetic analysis and screening of A(H1N1)pdm09 viruses circulating in Germany between 2009 and 2024 were conducted to identify any potentially synergistic or resistance-associated NA substitutions. Selected viruses were then subjected to further characterization in vitro. In the NA gene of circulating A(H1N1)pdm09 viruses, two secondary substitutions, NA-V241I and NA-N369K, were identified. These substitutions demonstrated a stable lineage in phylogenetic analysis since the 2010–2011 influenza season.
The data indicate a slight increase in viral NA bearing two additional potentially synergistic substitutions, NA-I223V and NA-S247N, in the 2023–2024 season, which both result in a slight reduction in susceptibility to NA inhibitors. The accumulation of secondary synergistic substitutions in the NA of A(H1N1)pdm09 viruses increases the probability of the emergence of antiviral-resistant viruses. Therefore, it is crucial to closely monitor the evolution of circulating influenza viruses and to develop additional antiviral drugs against different target proteins.
(SNIP)
4. Discussion
Neuraminidase inhibitors, primarily oral oseltamivir, remain the treatment of choice for influenza, especially in people who cannot be vaccinated and who are at risk of severe disease progression due to other medical conditions [7].
In 2009, the pandemic A(H1N1)pdm09 viruses replaced the previously oseltamivir-resistant seasonal A(H1N1) viruses and have been circulating worldwide as seasonal viruses ever since [29]. These 2009 pandemic A(H1N1) viruses remain largely oseltamivir-sensitive due to a reassortment event [13]. The introduction of NA-H275Y in A/California/4/2009 NA also resulted in a sharp decrease in total surface-expressed activity in these A(H1N1)pdm09 viruses, as in the former seasonal A(H1N1) viruses [9]. However, the evolutionary development of the A(H1N1)pdm09 viruses led to the establishment of the secondary NA substitutions NA-V241I and NA-N369K. In Germany, the prevalence of viruses carrying these substitutions was about 60% in the 2010–2011 season. Since the influenza season of 2011–2012, all A(H1N1)pdm09 viruses analyzed showed the valine-isoleucine substitution at NA-241 and the asparagine were substituted by lysine at the NA-369 position.
Using reverse-engineered viruses, these two substitutions, NA-V241I and NA-N369K, were shown to confer robust fitness to A(H1N1)pdm09 viruses carrying NA-H275Y by increasing the surface expression and enzymatic activity of the enzyme [19]. Nevertheless, the prevalence of resistant viruses in Germany and worldwide remained low (<1%) [17,30,31,32]. In contrast to previous seasonal viruses, these permissive secondary neuraminidase substitutions did not lead to the emergence and rapid spread of resistant viruses. A possible reason for this could be the structure of pandemic N1pdm09 NA, which generally differs from that of group N1 and group N2 neuraminidases. For example, the NA of H1N1pdm09 lacks the 150-cavity that is typical for group 1 NA, and additionally the salt bridge between Asp-147 and His-150 that is typical for group 2 NA [33,34]. These structural features may require several permissive mutations to occur in the NA in a series of gradual steps to support the emergence of a resistant variant and establish it as a stable lineage.
Since the 2011–2012 influenza season, the prevalence of the NA-V241I and NA-369K substitutions is 100% in circulating A(H1N1)pdm09 viruses. There is evidence of two additional secondary substitutions, namely NA-I223V and NA-S247N. Our data show that the NA substitution from isoleucine to valine at position 223 and the change of serine by asparagine at position 247 have only a marginal effect on the susceptibility of A(H1N1)pdm09 viruses to NA inhibitors. However, NAs bearing both substitutions demonstrated a more than 10-fold reduction in susceptibility to oseltamivir and almost a four-fold reduction in susceptibility to zanamivir compared to wild-type virus [35]. Enzymatic analysis has proven that the NA-I223V substitution can offset the loss of affinity of NA for its substrate resulting from the NA-H275Y resistance substitution, while simultaneously boosting resistance [36]. Thermodynamic and structural analyses showed that the combination of NA-H275Y with NA-I223V or NA-S247N leads to an extreme reduction in the inhibitory potential of oseltamivir [21]. However, the influence of the NA-223V and NA-247N substitutions on the surface expression of NA in the presence and absence of NA-275Y is not yet known and should be analyzed in further studies on their effect on viral fitness.
Prior to the COVID-19 pandemic, A(H1N1)pdm09 NA-S247N viruses had only been detected in Germany during the 2010–2011 season. It is interesting to note that in the first few months of 2011, the prevalence of this virus variant in community samples was more than 10% in Singapore and more than 30% in northern Australia [37]. Viruses bearing the NA-I223V/R substitution were detected sporadically in community samples in 2009 in Portugal, in the 2015–2016 influenza season in Iran and 2010 in a child after prolonged oseltamivir treatment [38,39,40].
After a period of quiescence during the COVID-19 pandemic, the incidence of influenza has increased following the relaxation of non-pharmaceutical measures [4]. In Germany, A(H1N1)pdm09 viruses circulated with very low prevalence in the 2021–2022 and 2022–2023 influenza seasons, while the 2023–2024 season was dominated by A(H1N1)pdm09 (in preparation). In this season, a very small increase in the secondary substitutions NA-I223V and NA-S247N was observed compared to the pre-COVID-19 pandemic influenza seasons.
The recent analysis of collected sequence data available through GISAID indicates an increased prevalence of these two substitutions in globally circulating A(H1N1)pdm09 viruses, with highest incidences of NA-I223V in August and October 2023 and of NA-S247N in September [35].
During these months, influenza viruses circulate primarily in the Southern Hemisphere and are considered precursors to those circulating in the northern hemisphere during the winter (October–April). A stronger spread of A(H1N1)pdm09 viruses with secondary mutations could therefore be expected for the coming influenza seasons. Based on the experience with the unexpected emergence and subsequent strong spread of oseltamivir-resistant earlier seasonal A(H1N1) viruses, it is necessary to closely monitor the evolution of pandemic A(H1N1)pdm09 viruses.
It seems likely that the viruses have reached the next stage in the evolution of prerequisite viruses that enable the emergence and spread of stable lineages of resistant viruses, in which the substitutions NA-I223V and NA-S247N may have been added in 2023–2024 after the appearance of the two permissive substitutions NA-V241I and NA-N369K in 2011. If synergistic amino acid changes such as NA-I223V and NA-S247N spread globally, there is the risk that other NA mutations which may have previously caused only slight or moderate reductions in susceptibility could instead cumulatively decrease NAI susceptibility to levels that may be clinically significant and affect treatment efficacy [37]
Continued monitoring of these mutations is essential to ensure preparedness for the potential emergence of neuraminidase-resistant viruses. In parallel, the advancement of alternative antiviral agents, including polymerase inhibitors, is of great urgency.
5. Conclusions
Although a relationship between permissive secondary NA mutations and the emergence and spread of oseltamivir-resistant influenza viruses has been described in the past, little is known about the importance of permissive mutations in molecular evolution. In the absence of treatment selection pressure, synergistic or permissive secondary mutations could support selection for resistant variants by decreasing antiviral susceptibility and/or compensating for their reduced viral fitness. As a result, the spread of antiviral-resistant influenza viruses throughout the community seems likely.
Our data showed that the prevalence of secondary antiviral mutations in the A(H1N1)pdm09 neuraminidase increased during the 2023-2024 influenza season. It will be crucial to determine whether this increase persists into the next season. Therefore, closely monitoring the evolution of circulating influenza viruses is important.
Whether H1N1pdm09 will continue on this trajectory, or veer off in another direction, remains to be seen. The trends, however, are worrisome.
But it would greatly complicate treating influenza-like illnesses, since most rapid tests (used by clinics) don't differentiate between subtypes.
Switching to Baloxavir (Xofluza) is a possibility, but it has shown some signs of creeping resistance of its own (see Eurosurveillance: A community Cluster of Influenza A(H3N2) Virus infection with Reduced Susceptibility to Baloxavir - Japan 2023).
A reminder that evolution never stops, and our pharmacological victories over bacteria, fungi, and viruses - while they can be lifesaving - are often fleeting.
