Point Mutations and Reassortment
The genetic code of viruses can consist of DNA or RNA. Influenza viruses have RNA. This code has an alphabet of four different letters called nucleotides, namely adenine (A), guanine (G), cytosine (C), and uracil (U). A sequence of three of those letters defines the amino acid to be built. A change of one of those letters is a point mutation, which may or may not change an amino acid, since there are 64 possible combinations resulting in 20 different amino acids. However, only changes of amino acids have an impact on how the virus functions.
Influenza viruses have eight separate RNA segments, each encoding different viral proteins. During a co-infection of a host cell with two or more influenza viruses, reassortment can occur. Reassortment means that segments of different viruses mix and match, leading to the production of new viruses with novel combinations of RNA segments. Reassortment events are relatively rare but can have significant consequences. This is why influenza viruses as a whole are dangerous, not just H5N1. With widespread "seasonal flu" infections, every H5N1 infection comes with the risk of H5N1 obtaining all the needed adaptions to mammals in just one reassortment event.
A large number of known mutations can be found on the CDC website. While this may look straightforward, it is important to keep in mind that amino acids are physical objects and not just some kind of computer code. The physical interactions between them can in some cases alter the properties of the virus.
Our results suggest that the 1918 pandemic virus originated shortly before 1918 when a human H1 virus, which we infer emerged before ∼1907, acquired avian N1 neuraminidase and internal protein genes. We find that the resulting pandemic virus jumped directly to swine but was likely displaced in humans by ∼1922 by a reassortant with an antigenically distinct H1 HA. Hence, although the swine lineage was a direct descendent of the pandemic virus, the post-1918 seasonal H1N1 lineage evidently was not, at least for HA. These findings help resolve several seemingly disparate observations from 20th century influenza epidemiology, seroarcheology, and immunology. The phylogenetic results, combined with these other lines of evidence, suggest that the high mortality in 1918 among adults aged ∼20 to ∼40 y may have been due primarily to their childhood exposure to a doubly heterosubtypic putative H3N8 virus, which we estimate circulated from ∼1889–1900. All other age groups (except immunologically naive infants) were likely partially protected by childhood exposure to N1 and/or H1-related antigens.
This study found that the M segment of the Spanish flu virus is a recombinant chimera originating from avian influenza virus and human influenza virus. The unique mosaic M segment might confer the virus high replication capacity, showing that the recombination might play an important role in inducing high pathogenicity of the virus. In addition, this study also suggested that the NA and NS segments of the virus were generated by reassortment between mammalian and avian viruses. Direct phylogenetic evidence was also provided for its avian origin.
Each of these viral factors is determined not only by the presence or absence of specific amino acids at specific sites but also by biophysical properties arising from the interaction of many sites within and between proteins. To illustrate this point, Tharakaraman et al. engineered the receptor binding site mutations that led to aerosol transmission of the HPAI H5N1 viruses A/Vietnam/1203/04 and A/Indonesia/5/05 into the HA of contemporary circulating H5N1 strains and found that they did not quantitatively switch receptor binding preference.
The Focus of Attention: Segments PB2 and HA
The eight segments are PB2, PB1, PA, NP, HA, NA, M, and NS.
PB2 controls a process called polymerase, which is essential for replication in host cells. HA and NA form the surface proteins, that we use for naming the virus subtypes like H5N1 or H7N9. HA (short for hemagglutinin) is responsible for binding the virus to the cell that is being infected. The main challenge for H5N1 are the different sialic acid (SA) receptors in birds and mammals. The various PB2 mutations develop fairly often after infection of a mammal, but there may be an increasing number of those mutations circulating in birds and the food supply, as the double mutation PB2-E627K/PB2-K526R in all Polish cats probably infected by cat food suggests. However, a relevant PB2 mutation has been detected very rarely in birds so far.
Additionally, a PB2 mutation has been found to inhibit the innate immune response.
We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk.
Among the mutations
in the HA protein which have been previously demonstrated to increase the binding to
human–type receptor, some of them (ie. S137A, S158N, T160A, S128P and R496K) have
been identified in the majority of the A(H5N1) viruses circulating in Europe since October
2022, while others (ie. T192I, S159N, Q196R, V214I) have been sporadically observed.
About half of the characterized viruses contain at least one of the adaptive markers
associated with an increased virulence and replication in mammals in the PB2 protein
(E627K, D701N or T271A) (Suttie et al., 2019). These mutations have never (T271A) or
rarely (E627K, D701N) been identified in the HPAI A(H5) viruses of clade 2.3.4.4b collected
in birds in Europe since October 2020 (<0>
Among the detected mutations, it is worth mentioning the
detection of the mutation PB2-E627K, an adaptive marker associated with an increased
virulence and replication in mammals, in two A(H5N1) viruses, one collected from a
domestic bird in Belgium in December 2022 and one in a wild bird in Sweden in January
2023.
It was surprising to observe that the virus characterised in this study, detected in hens, differed from all other HPAI A(H5N1) clade 2.3.4.4b viruses circulating in poultry and in birds by a mutation in the PB2 protein, T271A, which is a marker of virus adaptation to mammalian species; it has previously been shown to be associated with increased polymerase activity in mammalian cells and is present in the 2009 pandemic A(H1N1) virus. It should be noted that this mutation has never been observed in H5Nx viruses of clade 2.3.4.4b collected from birds in Europe since 2020. In contrast, it has been detected in ca 7% of clade 2.3.4.4b viruses identified in mammals in Europe, including the virus responsible for the outbreak on a mink farm in Spain. This molecular finding suggests that virus spread from mammals to birds cannot be excluded.
Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. (...) Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex.
Clade 2 had the most sequences available (10,734); of those, 738 contained PB2 627K and 16 contained a 627V. Clade 2 had 6.9% of total sequences with 627K; of those, 608 were avian and 130 were mammalian/human. (...) The 2.3.4.4b viruses are responsible for the current outbreak in the U.S. and have been reported in many countries. Of the 5,311 sequences analyzed from 2003 to the present, 53 had PB2 627K making the percentage only 1.0%. However, 48 of the 53 sequences were from 2021 to the present. Bird sequences containing 627K accounted for 23 of the 53 sequences and included chickens/turkeys, ducks, ratites, and common terns.
In our current study, we identify PB2-M535I as a newly emerging mammalian adaptation of clade 2.3.4.4b viruses. The close epidemiological link between the infected gulls and foxes and the genetic similarity of the viral sequences allows the observed differences between the avian and mammalian sequences to be assessed with high confidence. Further in vivo/in vitro studies are needed to clearly define the effect of this mutation on the biological characteristics of the virus.
Clade 2.2 sequences ranged from 1997 to 2017 in the dataset (Table 3, top). Clade 2.2 contributed to 83% (614/738) of the total clade 2 PB2 627K population (Table 2). Clade 2.3 (2003–present) has the largest total number of sequences available (9,797), although only 105 of them had PB2 627K.
The H5N1 HPAIVs from South Korea contained amino acids in HA with binding affinity for avian α-2,3-linked sialic acid receptors (T118, V210, Q222, and G224) (H5 numbering). They also had 2 HA amino acid substitutions, S113A and T156A, associated with increased binding affinity to human α-2,6-linked sialic acid receptors. All 5 isolates had amino acid substitutions that included A515T in PA, known to increase polymerase activity in mammal cells, and N30D, I43M, T215A in MP1 and L89V in PB2, known to increase virulence in mice.
It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place.
On the basis of viral RNA yields, we selected brain samples from 4 sea lions, 1 fur seal, and 1 tern for full-genome sequencing. Specifically, we found Q591K and D701N mutations in polymerase basic 2 associated with increased pathogenicity to mammals. The virus we detected in the South American tern also has those mutations, but they were absent from previously reported HPAI H5N1 viruses from avian hosts in South America (except for A/sanderling/Arica y Parinacota/240265/2023, which has the D701N mutation). That finding further supports the hypothesis that HPAI H5N1 viruses from sea lions from Peru and Chile acquired mammalian adaptation mutations that improved their ability to infect pinnipeds while possibly retaining the ability to infect avian hosts.
Gain-of-function Experiments and Airborne Transmission
The famous -and controversial- gain-of-function experiment of Ron Fouchier with ferrets, that resulted in airborne virus transmission, required one PB2 mutation and three HA mutations. Currently the PB2 mutation and one of the HA mutations are fairly common. In an oversimplified model, that transfers results from ferrets directly to humans, we may be two HA mutations away from a pandemic. There is also the gain-of-function experiment of Yoshihiro Kawaoka, which achieved airborne virus transmission between ferrets. This study used a HA segment from H5N1 with four mutations and replaced the other seven segments with H1N1 segments, simulating reassortment. Another experiment has been conducted with chickens and all mutations required for airborne mammal-to-mammal transmission.
Precise estimates of the probability of evolving the remaining mutations for the virus to become a respiratory droplet transmissible A/H5N1 virus cannot be accurately calculated at this time because of gaps in knowledge of the factors described above. However, the analyses here, using current best estimates, indicate that the remaining mutations could evolve within a single mammalian host, making the possibility of a respiratory droplet transmissible A/H5N1 virus evolving in nature a potentially serious threat.
The remaining substitutions, N154D and T156A in the HA glycosylation sequon, and E627K in PB2 however are common and occur in 942/3,392, 1,803/3,392 and 432/1,612 sequences respectively. Fig. S1 and Tab. (...)For viruses where both HA and PB2 have been sequenced 338/1,533 have lost the 154–156 glycosylation sequon and have E627K in PB2. These viruses have been collected in at least 28 countries in Europe, the Middle East, Africa, and Asia.
The HA glycosylation sequon substitutions, N154D and T156A, have drifted in and out of the avian virus population over time suggesting that they may be under little selective pressure in birds. The other substitutions, which are rare in birds, particularly those that change the sialic acid linkage preference, are likely to be negatively selected in birds.
Because the mutant virus harboring the E627K mutation in PB2 and Q222L and G224S in HA did not transmit in experiment 2, we designed an experiment to force the virus to adapt to replication in the mammalian respiratory tract and to select virus variants by repeated passage(...)
The only amino acid substitution detected upon repeated passage of both A/H5N1wildtype and A/H5N1HA Q222L,G224S PB2 E627K
was T156A (T, Thr; A, Ala) in HA. This substitution removes a potential
N-linked glycosylation site (Asn-X-Thr/Ser; X, any amino acid) in HA
and was detected in 99.6% of the A/H5N1wildtype sequences after 10 passages. T156A was detected in 89% of the A/H5N1HA Q222L,G224S PB2 E627K
sequences after 10 passages, and the other 11% of sequences possessed
the substitution N154K, which removes the same potential N-linked
glycosylation site in HA.
All six samples still harbored substitutions Q222L, G224S, and E627K that had been introduced by reverse genetics. Surprisingly, only two additional amino acid substitutions, both in HA, were consistently detected in all six airborne-transmissible viruses: (i) H103Y (H, His; Y, Tyr), which forms part of the HA trimer interface, and (ii) T156A, which is proximal but not immediately adjacent to the RBS. Although we observed several other mutations, their occurrence was not consistent among the airborne viruses, indicating that of the heterogeneous virus populations generated by passaging in ferrets, viruses with different genotypes were transmissible.
The potential for airborne-transmissible avian-origin influenza viruses to evolve in a mammalian host has been described using mathematical modelling predicting that airborne substitutions could evolve within a single mammalian host, especially in an immunocompromised host. However, the likelihood of such viruses to emerge in their original hosts, i.e. poultry species, has yet to be determined. Exposure to poultry is the most likely route for humans to acquire an infection with avian influenza viruses and has been the source of many documented human cases of infection.
The same concern applies to PA M861I and NS1 D26K, which are present in mammalian samples from Peru and in the human case from Chile.
In 2012, it was shown for the first time that an avian A/H5N1 influenza virus could become transmissible via the air between ferrets after changing three phenotypic viral properties. Only a handful of amino acid substitutions resulted in the following: (a) a shift in receptor-binding specificity of the hemagglutinin (HA) protein from the avian-type α-2,3-linked sialic acid (SA) to the mammalian-type α-2,6-linked SA receptor, (b) an increased acid stability of HA, and (c) an increase in polymerase activity in mammalian cells. Although this airborne-transmissible A/H5N1 virus was generated in the lab, two years later, the same phenotypic changes emerged under natural conditions during the 2014 avian-origin A/H10N7 virus outbreak in seals, which resulted in the death of 10% of the seal population in the North Sea. Not unexpectedly, this A/H10N7 seal virus was also found to be transmissible via the air in the ferret model, confirming the adaptive viral requirements for transmission. Although we understand the biological mechanisms regarding the receptor-binding specificity and polymerase adaptations that favor the transmissibility of influenza viruses, the contribution of HA acid stability to mammalian adaptation and transmission is less well understood.
To render influenza A viruses transmissible via air, three phenotypic viral properties must change, of which receptor-binding specificity and polymerase activity have been well studied. However, the third adaptive property, hemagglutinin (HA) acid stability, is less understood. Recent studies show that there may be a correlation between HA acid stability and virus survival in the air, suggesting that a premature conformational change of HA, triggered by low pH in the airways or droplets, may render viruses noninfectious before they can reach a new host.
The fusion pH has been measured for many influenza virus HA proteins, showing differences across subtypes and host species. For example, human influenza viruses have a lower fusion pH (pH 5.0–5.5) as compared with many avian (pH 5.6–6.0) and swine (pH 5.5–5.9) influenza viruses, suggesting that a reduction in fusion pH of avian and swine influenza virus HAs is required to become human-adapted. Indeed, such a decrease in fusion pH of HA was observed after the adaptation of swine A/H1N1 to humans at the beginning of the 2009 pandemic, where the average fusion pH of the gamma-clade swine viruses — that later contributed the HA gene to 2009 A/pH1N1 — was reduced from pH 5.7 to 5.5 in the human A/pH1N1 pandemic strain. This adaptation even continued in the first years after the pandemic with an HA fusion pH between 5.2 and 5.4 for later human virus isolates. Similarly, it was shown that the avian-to-human adaptation of the 1968 A/H3N2 pandemic virus HA was also accompanied with a decrease in HA fusion pH.
Just One Mutation?
While the gain-of-function experiment of Ron Fouchier et al is a popular reference, there is also a very concerning study about the 1918 H1N1 pandemic virus. It shows that just one mutation can enable the virus to change the sialic acid receptor binding specifity from birds to both birds and mammals. Such a mutation in H5N1 would drastically increase the chances of a pandemic, although further mutations would still be required.
And recently a dangerous mutation which increases the sialic acid receptor binding specifity to mammals, even without reducing the preference for birds significantly, has been discovered in birds.
Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the α2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the α2,6 and the α2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.
Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA193D virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA193N, and all rCT/W811-HA193D direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA193D has enhanced growth kinetics compared with the rCT/W811-HA193N, eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA193D exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.
Further, it is noteworthy that the H7N9 viruses isolated during the first wave of infection in China in 2013 maintained significant binding affinity for the avian (SAα2,3Gal) receptor, but the continuous circulation of H7N9 until 5th waves lead to more amino acid substitutions within the RBD, resulting in a complete switch to SAα2,6Gal specificity. Similarly, our competition experiment demonstrated that when equal amounts of virus were used to infect ferrets, the rCT/W811-HA193D virus exhibited increased replication, becoming more than 90% dominant as early as 3 dpi demonstrating replication advantages in the upper airway of ferrets. Eventually, only rCT/W811-HA193D could transmit to naïve contact animals in these studies. These results suggest that even though several variants may circulate in nature, strains with enhanced replicative fitness advantages could be selected in a novel host leading to competitive transmission advantages.
Further, sequence analysis of contemporary HPAI H5Nx viruses isolated from humans revealed that the HA-193D mutation is present in 12.9% of H5N6 human isolates. (...) This mutation may be one of the critical amino acid changes which could result in strong mammalian adaptability among emerging novel HPAI H5Nx viruses and the presence of such mutation implies a higher risk of avian-to-human infection from these viruses.
In this study, we confirmed the ability of the rCT/W811-HA193D to replicate efficiently in ferrets and human bronchial epithelial (HBE) cells, showing replication efficiency similar to that of the 2009 pandemic CA/04 H1N1 influenza virus, suggesting that these viruses pose a potential threat to human infection.
In this study, we confirmed the ability of the rCT/W811-HA193D to replicate efficiently in ferrets and human bronchial epithelial (HBE) cells, showing replication efficiency similar to that of the 2009 pandemic CA/04 H1N1 influenza virus, suggesting that these viruses pose a potential threat to human infection.
Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk.
It is not only the terminal SA linkage that affects the interplay between influenza virus HA and its receptor. Additional structural features of glycans, such as modifications with other sugar moieties (such as fucose and N-acetylgalactosamine) or other functional groups (such as sulfation), and the length of the SA-presenting glycan chain all affect the interaction. Such detailed glycan structural features are not resolvable by lectin staining and require mass-spectrometry-based glycomic analysis. To date, glycomic analysis has been carried out on human, ferret, pig and mouse respiratory tissue, all of which express a complex set of protein N-linked and O-linked glycans as well as glycolipids. As expected from the knowledge of CMAH genetics, humans and ferrets express only Neu5Ac SA, whereas pig and mouse express both Neu5Ac and Neu5Gc SA. All the tissues analysed express both α2-3 SA and α2-6 SA, with humans expressing the highest proportion of α2-6 SA and the mice expressing the highest proportion of α2-3 SA. However, different glycan structural features can be observed in individual species. For example, uniquely in ferret respiratory tract tissue, the Sda epitope blocks access of HA to α2-3 SAs, potentially restricting their ability to support the transmission of AIVs41.
Natural Bird Flu Defenses
A very recent study has discovered human BTN3A3 (butyrophilin subfamily 3 member A3) as a potent inhibitor of avian influenza. However, a mutation in the NP segment, NP-313F, enables evasion of this mechanism and seems to be fairly common.
This is not to be confused with the basic virus defense mechanism regulated by Mx1, which is not limited to humans, and where genetic improvements made mice resistant to influenza A viruses.
A unique feature of the NS segment of H5N1, NS-92E, confers resistance to interferons and tumor necrosis factor α, basic defense mechanisms against all kinds of threats. Not only may this contribute to the high case fatality rate of H5N1, but it has been proven that other influenza viruses can become more dangerous should they acquire the H5N1 NS segment in a reassortment event.
Vaccination against seasonal influenza (H1N1/H3N2) had increased the antibody response against H5N1 only by a tiny amount. Antibody response to NA of the 2009 pandemic H1N1 virus however correlates strongly with an antibody response to NA of H5N1. This is due to a higher similarity of the NA segments. It is unknown how much actual protection this confers.
Prof Palmarini said "a bit more than 50%" of virus samples from birds and "all seven" cases detected in people this year had resistance to BTN3A3.
Mx genes exist in almost all vertebrate genomes and serve as a defense against RNA viruses. Mx proteins are evolutionarily conserved in vertebrates, suggesting that they are critical for antiviral defense across species. The interferon-induced GTP-binding protein, Mx1, is one such antiviral protein that restricts influenza viruses in humans and mice, although the effect depends on the virus strain.
The H5N1 influenza viruses transmitted to humans in 1997 were highly virulent, but the mechanism of their virulence in humans is largely unknown. Here we show that lethal H5N1 influenza viruses, unlike other human, avian and swine influenza viruses, are resistant to the antiviral effects of interferons and tumor necrosis factor α. The nonstructural (NS) gene of H5N1 viruses is associated with this resistance. Pigs infected with recombinant human H1N1 influenza virus that carried the H5N1 NS gene experienced significantly greater and more prolonged viremia, fever and weight loss than did pigs infected with wild-type human H1N1 influenza virus. These effects required the presence of glutamic acid at position 92 of the NS1 molecule. These findings may explain the mechanism of the high virulence of H5N1 influenza viruses in humans.
Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.
We next determined whether the Mx1+/+ mice would also exhibit enhanced resistance to highly pathogenic H5N1 FLUAV. In a first experiment, groups of Mx1+/+ and Mx1−/− mice were challenged with 10 LD50 for BALB/c mice of A/Vietnam/1203/04 (VN1203), a virus isolated in 2004 from a fatal human case in Vietnam. Weight loss in the Mx1+/+ mice was moderate and all animals survived, whereas all Mx1−/− control mice developed severe disease and succumbed to infection between days 4 and 7 postinfection.
Our results demonstrate that the KWM/Hym mice are resistant to influenza A virus infection. Further, these mice can be used as a model organism to understand the mechanism of influenza A virus susceptibility.
“That is the last barrier,” Beer says. Although MxA’s detection skills appear very weak in ferrets and some other animals, it is more sensitive in humans—and in pigs. “If an H5 virus is spreading in pigs then it really is code red,” Beer says.
In an unpublished experiment Beer and his colleagues infected pigs with H5N1. Even when high doses were used, the virus barely replicated in the animals.
All these findings may be explained by hypothesizing that cross-reactive immunity is targeting the N1 NA antigen. However, whether cross-reactive antibodies to NA and CD4 T cells would be protective against illness and death, especially from influenza (H5N1) infection is not known. Further studies will be necessary to elucidate this point.
In conclusion, we demonstrated that vaccination against seasonal influenza may boost a cross-reactive immunity against an unrelated strain responsible for deadly infections in humans, i.e., the avian influenza (H5N1) strain A/Hong Kong/156/97. These data, together with previous experimental results from mice studies and epidemiologic reports, indicate that cross-type immunity should be considered an important component of the immune response against novel influenza A infections.
In summary, we detected high titers of cross-reactive NAI antibodies against influenza A(H5N1) clade 2.3.4.4b virus in serum samples collected from healthy adults in 2020 but not detected in serum samples collected in 2009. Further studies are needed to confirm whether cross-reactive NAI antibodies confer protection against H5N1 infection or modulate disease severity, but our results suggest that the antibodies against H5N1) and H6N1 viruses might derive from exposure to the conserved epitopes shared between the avian-origin pH1N1 virus and avian N1 proteins.
Two NA Receptor Binding Mutations
It seems that the receptor binding mutations in HA require mutations in NA like NA-I396M and NA-S369I to reestablish some kind of equilibrium. The NA mutations disrupt the second sialic acid-binding site of influenza A virus neuraminidase, 2SBS, which preferentially binds to α2,3-linked sialic acid receptors most common in birds. This host range determinant has a prevalence of 3% of the H5N1 A/Herring_gull/France/22P015977/2022-like genotype.
In addition to the distinction between alpha 2,3-linked sialic acid receptors and alpha 2,6-linked sialic acid receptors, there are Neu5AC and Neu5Gc sialic acids. However, humans share Neu5AC sialic acids with susceptible species like ferrets and seals.
Phylogenetic analysis using Nextstrain indicated that the I396M substitution, seldom observed in other H5N1 viruses, was acquired in an avian host immediately before emergence of the H5N1 virus in mink. The analysis moreover showed that substitution S369I, which is at the position of a sialic acid contact residue in the 2SBS, was obtained immediately before the acquisition of I396M. (...)This preceded the acquisition of mutations in HA that decreased binding to avian-type alpha 2,3-linked sialic acid receptors. It is possible that this concerted evolution of NA and HA is driven by the need to preserve an optimal HA-NA balance. (...)Disruption of the 2SBS in H5N1 viruses in minks is thus far an unreported feature that they have in common with human-adapted influenza A viruses. Loss of the 2SBS may drive selection for changes in the receptor binding properties of HA, possibly resulting in increased binding to human-type receptors. Such changes in HA are expected to promote replication and transmission in mammalian hosts, including humans. Preservation or loss of the 2SBS is likely a viral host range determinant and could be an early adaptation signal that should be included in analysis of the pandemic potential of emerging IAVs.
Moreover, about 3% of the European viruses belonging to the H5N1
A/Herring_gull/France/22P015977/2022-like genotype show mutations in the NA protein
which cause disruption of the second sialic acid binding site (2SBS), a feature typical of
human-adapted influenza A viruses (de Vries and de Haan, 2023).
In mammals, the Neu5Gc modification is created via hydroxylation of Neu5Ac by the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) enzyme(...). This enzyme is expressed through the CMAH gene, which is present in many mammalian species but is non-functional in certain species such as dogs, ferrets, seals, hedgehogs, over a 100 different monkey species, and humans(...).
Amantadine and Rimantadine
Due to widespread resistance mutations in the M segment, mostly M2-S31N/L26I, the M2 inhibitors amantadine and rimantadine are no longer used. However, scientists try to find ways to inhibit the resistances to the inhibitors - an arms race between scientists and the virus.
H5N1 from 2002 to have been found to be resistant to M2 and NA inhibitors (Govorkova et al., 2013). In the case of human and avian origin H5N1 isolated between 1996 and 2005 in Vietnam, Cambodia, Malaysia and Thailand, more than 90% contain the double mutant M2-S31N/L26I channel (...)
Several advancements in the structural characterization of M2 helped the development of not only mono-specific V27A and S31N inhibitors, but also dual inhibitors that target either both WT and V27A (Wang et al., 2011a) or both WT and S31N channels simultaneously (Wang et al., 2013a; Wu et al., 2014). However, no M2 channel blockers have been developed against drug-resistant double mutants such as the M2-S31N/L26I and the M2-S31N/V27A, which are the predominant mutations found in H5N1 viruses.
Resistant mutants to the Matrix-2 (M2) inhibitors such as adamantanes (amantadine and rimantadine) have been detected globally. All currently circulating influenza viruses are resistant to M2 inhibitors
The percentage of influenza A virus (...) that were adamantane-resistant increased from 0.4% during 1994–1995 to 12.3% during 2003–2004 [137]. During the 2005–06 influenza season, CDC determined that 193 (92%) of 209 influenza A (H3N2) viruses isolated from patients in 26 states demonstrated a change at amino acid 31 in the M2 gene that confers resistance to adamantanes
Other Resistance Mutations
Mutations of concern are NA-H275Y and NA-I223R. NA-H275Y offers resistance to oseltamivir(Tamiflu), while NA-I223R offers resistance to zanamivir(Relenza). As this study shows, they can emerge quickly under favorable conditions, like prolonged infections in immunocompromised patients. Both mutations amplify each other. While peramivir gets often mentioned in that context, this study indicates therapeutic success despite the NA-H275Y mutation. However, this study reports a highly reduced susceptibility, although 3 to 4 times less reduced than against oseltamivir.
In 2007/2008, when a H1N1 was a significant seasonal influenza strain, H1N1 surprisingly acquired increased resistance to oseltamivir a due to HA-H275Y to a high degree and worldwide. This situation subsided with the next global outbreak of H1N1 as part of the seasonal influenza in 2013/2014. The widespread occurrence of resistances can manifest itself in a short time span.
The latest data available shows elevated levels of baloxivir resistance in Japan at 4.5% and otherwise little reason for concern. But this may change quickly. Weekly CDC seasonal influenza data including resistance mutations can be found here.
All
the mutations associated to antiviral resistance were identified only sporadically in the
circulating viruses.
Analyses of available avian influenza viruses circulating worldwide suggest that most viruses are susceptible to oseltamivir, peramivir, and zanamivir. However, some evidence of antiviral resistance has been reported in HPAI Asian lineage avian influenza A(H5N1) viruses (“Asian H5N1 viruses”) and Asian lineage avian influenza A(H7N9) viruses (“Asian H7N9 viruses”).
During the 2013- 2014 season, 98.2% of the 2009 H1N1 viruses tested for surveillance were susceptible to oseltamivir, and 100% of the 2009 H1N1 viruses tested were susceptible to zanamivir.
Seemingly from one influenza season to the next, we have lost the use of our leading antiviral influenza drug because of resistance. This winter, the circulating strain of seasonal influenza A virus (H1N1) is resistant to the neuraminidase inhibitor oseltamivir. Moreover, rather than emerging under selective pressure of drug use, as many antibiotic-resistant bacteria do and as has been the concern for influenza, this resistant strain seems to be a natural, spontaneously arising variant.
Three sets of mutations have been identified that prevent this rearrangement — Arg292Lys, Asn294Ser, and His274Tyr — and the presence of any of them confers oseltamivir resistance in H1N1 influenza viruses.
H1N1 viruses containing the His274Tyr resistance mutation became widespread beginning with the 2007–2008 influenza season in the Northern Hemisphere, with a prevalence of about 10% in the United States and about 25% in Europe (except for an astonishing prevalence of about 70% in Norway). These resistant viruses then predominated during the Southern Hemisphere's 2008 influenza season. In the United States today, H1N1 is the dominant circulating strain and is virtually 100% oseltamivir-resistant. The urgency of the situation is tempered by the fact that this season's oseltamivir-resistant viruses are sensitive to zanamivir and by the tendency for the H1N1 strain of viruses to cause milder disease and fewer deaths than the H3N2 strain.
Oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y mutation in the neuraminidase emerged independently of drug use. By country, the proportion of ORVs ranged from 0% to 68%, with the highest proportion in Norway. The average weighted prevalence of ORVs across Europe increased gradually over time, from near 0 in week 40 of 2007 to 56% in week 19 of 2008 (mean 20%).
The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold).
Many mutations causing oseltamivir and peramivir resistance, including the common H275Y mutation in A(H1N1)pdm09 influenza, do not confer resistance to zanamivir and inhaled zanamivir may be used to treat these strains.
In H5N1 viruses containing the NA H275Y mutation, the antiviral activity of peramivir against the variant was lower than that against the wild-type. Evaluation of the in vivo antiviral activity showed that a single intravenous treatment of peramivir (10 mg/kg) prevented lethality in mice infected with wild-type H5N1 virus and also following infection with H5N1 virus with the H275Y mutation after a 5 day administration of peramivir (30 mg/kg). Furthermore, mice injected with peramivir showed low viral titers and low levels of proinflammatory cytokines in the lungs. These results suggest that peramivir has therapeutic activity against HPAI viruses even if the virus harbors the NA H275Y mutation.
Of the 22 S247N variants detected, nine were cultured and in an NA inhibition assay showed a mean six-fold reduction in oseltamivir sensitivity, a three-fold reduction in zanamivir sensitivity, and no significant reduction in peramivir sensitivity compared to the mean IC50 (concentration required to inhibit 50% of NA activity) of sensitive influenza A(H1N1)2009 viruses (Table).
There have now been several reports that oseltamivir-resistant influenza A (H5N1) viruses with the H274Y mutation have been isolated from humans with avian influenza infection who were treated with oseltamivir.
Frequency of viruses showing reduced inhibition by NA inhibitors remained low (0.5–0.6%) during 2018–2020. Frequency of viruses showing reduced baloxavir susceptibility was low (0.1–0.5%) but elevated (4.5%) in Japan in 2018–2019.
Viruses bearing the PA/I38T/F/M/N/S mutation selected in vitro or in clinical studies show reduced
susceptibility to baloxavir with changes in EC50 values ranging from 11 to 57-fold for influenza A
viruses and 2 to 8-fold for influenza B viruses.
In fact, baloxavir represented 40 percent of the market share of influenza drugs in Japan during the 2018-2019 flu season.
susceptibility to baloxavir with changes in EC50 values ranging from 11 to 57-fold for influenza A
viruses and 2 to 8-fold for influenza B viruses.
The studies have shown that peramivir resistance among influenza A/H1N1pdm09 viruses ranges from 1.3%–3.2% and is less than 1% for influenza A/H3N2 and B viruses. The most commonly occurring mutation leading to reduced effectiveness of peramivir is H274Y (H275Y N1 numbering), resulting in a 100–400-fold increase in IC50. (...) In vivo studies on mice showed that peramivir could be effective in the treatment of infections caused by oseltamivir-resistant A/H1N1/H274Y influenza virus. (...) In vitro studies demonstrated that passaging of the A/H3N2 virus in the presence of peramivir led to the emergence of strains with reduced susceptibility to peramivir, which was related to the K189E mutation in the HA protein. The R294K mutation in the N9 neuraminidase resulting in high-level resistance to peramivir was detected in clinical strains.
The studies revealed numerous mutations leading to the emergence of influenza strains resistant to laninamivir.
The E119G mutation in N9 neuraminidase confers a 150-fold resistance to the drug. Another example is a double H275Y + I436N mutation, which reduces influenza virus susceptibility to laninamivir. Two variants of the A(H1N1)pdm09 strain significantly increased laninamivir IC50. The Q136K A and Q136R variants conferred a 25.5- and a 131.8-fold increase, respectively, in comparison to the wild-type. Furthermore, an NA N142S amino acid substitution in the A/H3N2 strain resulted in a 53-fold IC50 increase.
The zanamivir-resistant influenza A/H3N2 variants with framework E119G or E119A mutations remain susceptible to peramivir and oseltamivir. Using reverse genetics, it was shown that these two mutations, as well as the E119D substitution, induce resistance to zanamivir but not to peramivir or oseltamivir in the H1N1pdm09 virus background. Surprisingly, laboratory generated H5N1 virus with the E119D mutation possessed high level of resistance to all NAIs. While the E119V mutation in the H3N2 context conferred resistance to oseltamivir only.
The E119G mutation in N9 neuraminidase confers a 150-fold resistance to the drug. Another example is a double H275Y + I436N mutation, which reduces influenza virus susceptibility to laninamivir. Two variants of the A(H1N1)pdm09 strain significantly increased laninamivir IC50. The Q136K A and Q136R variants conferred a 25.5- and a 131.8-fold increase, respectively, in comparison to the wild-type. Furthermore, an NA N142S amino acid substitution in the A/H3N2 strain resulted in a 53-fold IC50 increase.
Notably, three of ten (30%) A(H1N1)pdm09 strains collected in October, 2023, harboured both I223V and S247N mutations.
(...) None of the strains collected before July, 2023, harboured dual I223V/S247N mutations; however, since July, 2023, nine of 1023 strains (880 per 100 000 individuals) harboured dual I223V/S247N mutations, with the highest incidence in October, 2023 (five of 169 strains [2959 per 100 000 individuals]). Of the nine strains with dual I223V/S247N mutations, five were collected from Europe (two from the Netherlands and one each from Norway, Sweden, and France), and four were collected from Oman.
Previous studies have shown that mutations at neuraminidase amino acid residue 223 are associated with an 11–28-fold increase in the half-maximal inhibitory concentration (IC50) for oseltamivir, whereas mutations at residue 247 are linked to a six-fold increase in the oseltamivir IC50, compared with the reference median IC50 values.
However, investigations into such associations for dual I223V/S247N mutations have not been reported. Therefore, in this study, we assessed the susceptibility of a strain with dual I223V/S247N mutation (HKU-231217-085) to neuraminidase inhibitors using a chemiluminescent neuraminidase inhibition assay. The IC50 value for HKU-231217-085 was 10·63-fold higher (from 0·429 nM to 4·563 nM) against oseltamivir and 3·38-fold higher (from 0·924 nM to 3·120 nM) against zanamivir than the average IC50 value for the three A(H1N1) strains (HKU-231217-099/2023, HKU-231217-100/2023, and 415742/2009) without the dual mutation.
However, investigations into such associations for dual I223V/S247N mutations have not been reported. Therefore, in this study, we assessed the susceptibility of a strain with dual I223V/S247N mutation (HKU-231217-085) to neuraminidase inhibitors using a chemiluminescent neuraminidase inhibition assay. The IC50 value for HKU-231217-085 was 10·63-fold higher (from 0·429 nM to 4·563 nM) against oseltamivir and 3·38-fold higher (from 0·924 nM to 3·120 nM) against zanamivir than the average IC50 value for the three A(H1N1) strains (HKU-231217-099/2023, HKU-231217-100/2023, and 415742/2009) without the dual mutation.