This is the threat that’s going to keep knocking at our door until it will indeed, I assume, cause a pandemic. Because there is no way back.
Risk Accumulation
Most infections of mammals are short-term risks. Usually the mammal dies and any adaptive mutations the virus may have gained disappear with the host. But sometimes those variants better adapted to mammals could find their way back into the bird population and replicate for a long time, for example due to scavenging birds. This would increase the pandemic risk for a long time. It could also increase the ability of birds to infect mammals.
In a very simplified model, 4 mutations are needed for aerosol-based mammal-to-mammal transmission as shown in the Fouchier et al gain-of-function experiment. One of those mutations circulating in the bird population would reduce the number of mutations to be gained in a mammal (population) from 4 to 3. Of course reality is much more complex, but some adaptations clearly increase the pandemic risk.
However the prevalence of mutations in the various variants of H5N1 is quite diverse. While the PB2-E627K prevalence in clade 2.2 is 92.1%, it is only 1.1% in clade 2.3 and 1.0% in clade 2.3.4.4b, which has caused the current panzootic. So far this seems like a positive development. But the spread of the virus and the absolute number of cases have increased. Some concerning mutations associated with mammals don't seem to be decrease the transmissibility in bird populations. Others are only a minor evolutionary disadvantage and may become more prevalent at any time.
And there are signs of these adaptations circulating in wild birds.
The most worrying H5N1 variant detected so far, although not yet peer-reviewed, was found in a bird with a preference for scavenging, the Red Tailed Hawk. This variant is capable of ferret-to-ferret transmission by direct contact.
A PB2-T271A mutation has been found in all infected animals from the mixed farm in Brescia, Italy. This includes five dogs, one cat, and poultry. This implies that the wild bird vector already carried the mutation.
Another concerning development is that the virus managed to infect approximately 30 fur farms in Finland in a short amount of time. Before there has been only a single infected fur farm in Galicia, Spain in 2022. This may indicate an improved ability to infect mammals and widespread circulation within the bird population, with the potential to spread to many more fur farms.
Just as in Spain there is a high probability of mammal-to-mammal transmission on the fur farms in Finland, which makes this very concerning.
It is important to not jump to conclusions: There was possible evidence of mammal-to-mammal transmission or an avian reservoir in South America in form of a cluster of rare PB2-D701N mutations including a bird, two sea lions, and a human. However phylogenetic analysis showed that these rare mutations developed independently from each other. In another case in Argentina however, the sea lions carried the same rare mutations as a tern from the same area, PB2-Q591K and PB2-D701N, suggesting circulation in local birds and transmission of mammalian adaptations from mammals to bird.
PB2-E627K and HA-T156A or functionally similar mutations are found fairly often in H5N1 viruses. Variants with the mutations PB1-H99Y, HA-H103Y, and HA-Q222L have been found to be transmissible between birds in laboratory experiments. Variants with HA-G224S were not transmissible between birds. All mutations combined reduced the virulence significantly, an evolutionary disadvantage. While in reality adaptations specifically to the human immune system have to be considered, the basic idea that most mutations required for mammal-to-mammal transmission could at least locally accumulate in avian hosts seems to be realistic.
A very concerning recent discovery is the HA-N193D mutation, which enables dual receptor binding specificity and other dangerous properties with just a single mutation.
The confirmed spillover of the reassorted virus from infected cows into multiple poultry farms, cats, a racoon, and at least three wild birds elevates the risk.
The sequence data indicate that, at least originally, transmission likely occurred from birds to the fur animals, most probably through contacts in the shade houses. Birds have easy access to the interior of the shade houses and gulls have frequently been observed in the vicinity of the farms. Mass deaths of gulls have also occurred in the same general region.
One isolate, A/Red Tailed Hawk/ON/FAV-0473-4/2022, efficiently transmitted by direct contact between ferrets, resulting in lethal outcomes.
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.
At this time, multiple transmission scenarios involving wild birds, marine mammals, and humans could potentially explain the clusters of viruses with Q591K and D701N mutations in Chile/Peru. Additional sequence data is needed from avian and mammalian hosts in Chile to fill in gaps in the tree and distinguish which scenario best fits the data.
Both of these viruses have the PB2 D701N mutation that is associated with enhanced mammalian transmission in mammals (see below), however, these Arequipa viruses were collected in sea lions a month apart (February 7, 2023 vs. March 6, 2023) and they do not cluster together on the same phylogenetic trees. One of the Arequipa viruses (A/South American sea lion/PeruAQP-SER00R/2023) with the D701N mutation clusters on the PB2 tree with the H5N1 virus isolated from a human in Chile22 a few weeks later (March 24, 2023) that also has the D701N mutation, but this pattern does not hold for trees inferred using other genome segments.
Taken together, these data provide evidence that the D701N mutation is emerging repeatedly in H5N1 viruses that infect mammalian hosts along South America’s Pacific coast, however, these data cannot yet ascertain whether viruses with this mutation are transmitting within mammals.
These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.
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.
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.
At 24 hpi, all mutant viruses were detected in the vast majority of collected tissues, demonstrating that they were all able to spread systemically in chickens, even the viruses carrying the receptor binding substitutions HA-Q222L and/or HA-G224S.
Chickens inoculated with INDOWT succumbed to the infection at approximatively 36 hpi, which corresponded to 5 h to 10 h after the onset of symptoms (lethargy, ruffled feathers). In contrast, the mean time to death of the chickens inoculated with INDOAT6 was >6.25 days .
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.
Furthermore, although the genetic changes that have been described to promote airborne transmission of avian A/H5 viruses are the result of adaptation to mammalian hosts, some of these changes have also been detected in avian isolates. PB2-E627K has been found in 11% of the avian A/H5 viruses, as compared to 38% of the human A/H5 viruses. Interestingly, HA-H103Y has been found in only 5 avian A/H5 strains but not in A/H5 human strains. HA-T156A is present in 69% and 47% of the avian and human A/H5 strains respectively. Moreover, HA-T156A and HA-H103Y have been found in combination in 5 avian A/H5 viruses. This suggests that the impact of -at least some of- the mammalian adaptive changes on viral fitness in avian hosts may be small, but our knowledge on this matter is virtually non-existent.
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.
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.
Possible spill-back of viruses from carnivores to wild birds has also been suggested, for example after predation of infected carcasses by birds of prey. Introductions of viruses with amino acid changes that facilitate replication in mammals into wild birds would greatly increase chances of further spread.
SNU-01 has shown multiple mammalian adaptations in several gene segments that enhance the polymerase activity of AIVs in mammalian hosts. Interestingly, except for D701N, which significantly contributes to mammalian adaptation, the remaining mammalian adaptive mutations were not limited to SNU-01; they were also observed in avian isolates. These mutations were prevalent among most avian isolates currently circulating in Eurasia, and additional mutations were identified in bird isolates that share a common ancestry with SNU-01 within phylogenetic trees. The D701N mutation has not been detected in avian isolates; however, its presence in wild birds cannot be ruled out, given recent cases in Chile. Meanwhile, the function of the unique mutations in PA and NA gene segments, found exclusively in SNU-01 requires further study to understand their implications.
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.
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.
Results showed that the rCT/W811-HA193N and rCT/W811-HA193D retained their infectivity even after 240 mins of incubation at 50°C (...). Taken together, these results demonstrate that the fine balance between affecting HA functions (dual receptor binding affinity for α2,6-SAs and α2,3-SAs and heat stability) may be crucial for the transmissibility of the rCT/W811-HA193D virus which was also observed in human infectious HPAI H5N1 viruses.
Specifically, we found Q591K and D701N mutations in polymerase basic 2 associated with increased pathogenicity to mammals. The virus we detected in the tern from South America 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.
“This confirms that while the virus may have adapted to marine mammals, it still has the ability to infect birds,” said lead researcher Agustina Rimondi, a virologist from the National Institute of Agricultural Technology in Argentina.
As of April 24, 2024, USDA has confirmed H5N1 virus
detections on 33 dairy cattle premises in 8 states
(Kansas, Idaho, Michigan, New Mexico, North Carolina,
Ohio, South Dakota, Texas). USDA has also confirmed -
based on specific phylogenetic evidence and
epidemiological information - that 8 poultry premises in
5 states (Kansas, Michigan, Minnesota, New Mexico and
Texas) have also been infected with the same distinct
H5N1 virus genotype detected in dairy cattle.
Phylogenetic analysis using genome sequencing suggests that there was a reassortment event in late 2023 between the current highly pathogenic 2.3.4.4b clade in wild birds and a low-pathogenic wild bird strain, which produced the B3.13 genotype now circulating in dairy cows.
The NP gene acquired during reassortment may have played a role in the emergence in cattle, they wrote, noting that the NP gene seems to allow influenza viruses to spread more easily in pigs.
Their analysis suggests there were as many as five B3.13 introductions from cattle to poultry, one to a raccoon, two to domestic cats, and three to wild birds. Though the findings track with epidemiological findings of spread through movement of herds to other states, they emphasized that there are still gaps. "We cannot exclude the possibility that this genotype is circulating in unsampled locations and hosts as the existing analysis suggests that data are missing and under surveillance may obscure transmission inferred using phylogenetic methods," they wrote.
H3N8
With H3N8 another avian influenza virus is proving that acquisition of most of the mutations discussed above is possible, although the specimen in question is from a human infection.
Here, we demonstrate that H3N8 viruses were able to infect and replicate efficiently in organotypic normal human bronchial epithelial (NHBE) cells and lung epithelial (Calu-3) cells. Human isolates of H3N8 virus were more virulent and caused severe pathology in mice and ferrets, relative to chicken isolates. Importantly, H3N8 virus isolated from a patient with severe pneumonia was transmissible between ferrets through respiratory droplets; it had acquired human-receptor-binding preference and amino acid substitution PB2-E627K necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naive to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.
Prevailing H3N8 viruses have evolved through a triple reassortment event with the Eurasian avian H3 gene, the North American avian N8 gene, and H9N2 internal genes. In 2022, H3N8 viruses were frequently detected in chicken farms and live poultry markets (LPMs) in China and animal studies indicated that the H3N8 virus was well adapted in chickens.
“We demonstrate that an avian H3N8 virus isolated from a patient with severe pneumonia replicated efficiently in human bronchial and lung epithelial cells, was extremely harmful in its effects in laboratory mammalian hosts and could be passed on through respiratory droplets,” says Professor Kin-Chow Chang, at the University of Nottingham.
“We demonstrate that an avian H3N8 virus isolated from a patient with severe pneumonia replicated efficiently in human bronchial and lung epithelial cells, was extremely harmful in its effects in laboratory mammalian hosts and could be passed on through respiratory droplets,” says Professor Kin-Chow Chang, at the University of Nottingham.
Infection and transmission studies in ferrets showed that the H3N8 virus transmitted efficiently between ferrets through direct contact but inefficiently through airborne exposure.
"Acid resistance of influenza virus is also an important barrier for avian influenza virus to overcome to acquire the adaptability and transmissibility in new mammals or humans. The current novel H3N8 virus has not acquired the acid resistance yet. So, we should pay attention to the change on acid resistance of the novel H3N8 virus,” says Professor Jinhua Liu at the China Agricultural University in Beijing.
Importantly, we discovered that the virus had acquired human receptor binding preference and amino acid substitution PB2-E627K, which are necessary for airborne transmission. Human populations, even when vaccinated against human H3N2 virus, appear immunologically naïve to emerging mammalian-adapted H3N8 AIVs and could be vulnerable to infection at epidemic or pandemic proportion.
H5N1 Spread
Another concerning development is the continuous spread of H5N1. It seems likely that H5N1 will sooner or later arrive on the remaining uninfected continent, Australia. This would maximize the number of available hosts, with H5N1 circulating almost everywhere. With hardly any geographical boundaries and a broad host range, H5N1 will probably be a persistent threat for decades. And it may continue to adapt to mammals. The unprecedented H5N1 infections of cows seemingly related to milking demonstrate how a large number of infected birds can lead to unexpected widespread infections, leading to spillovers into other species from the cows, including H5N1 in cats and a racoon.
The currently circulating HPAI H5N1 is derived from viruses of the goose/Guangdong/96- (gsGD)-
lineage that were first detected in commercially-farmed geese in China in 1996 and have circulated
and evolved in poultry. Multiple strains of virus within the gsGD lineage spread across east and
southeast Asia in 2003-04, some of which was the result of spread by wild birds (e.g. to Japan and
Republic of Korea). In 2005 the first intercontinental wave of transmission of gsGD viruses occurred
across Eurasia and into Africa and has been followed by multiple waves of intercontinental spread.
Viruses in the gsGD-lineage viruses have evolved into 5th order clades and have formed multiple
genotypes. The A(H5N1) virus that emerged in 2020 belongs to clade
2.3.4.4b and has caused numerous outbreaks in wild birds in Asia and Europe, typically during
autumn and winter, as well as in Africa, but has persisted year-round in wild birds in Europe since
2021. That same year, HPAI H5 spread across the Atlantic Ocean to North America, where it spread rapidly across the continent in 2022 and southwards to Central and South
America
On March 13, 2023, a 53-year-old male from the Region of Antofagasta in northern Chile began experiencing symptoms, including cough, sore throat, hoarseness, and sought medical treatment at a local hospital on March 21 after symptoms progressed. (...)As of June 16, 2023, the patient remains in respiratory isolation and
requires mechanical ventilation due to pneumonia.(...)Initial findings from the epidemiological investigation suggest that the most probable mode of transmission for this human case was through environmental exposure, considering the significant presence of deceased sea mammals and wild birds near the patient's residence, which is within 150 meters of the beach.
Here, we performed molecular and serological screening of over 500 dead wild carnivores and sequencing of RNA positive materials. We show virological evidence for HPAI H5 virus infection in 0.8%, 1.4%, and 9.9% of animals tested in 2020, 2021, and 2022 respectively, with the highest proportion of positives in foxes, polecats and stone martens. (...) Serological evidence for infection was detected in 20% of the study population.
Of the 701 stray cats examined, 83 were found to have antibodies to the bird flu virus. Some of the stray cats examined had mild symptoms of illness, but not specific to bird flu. Eating contaminated dead birds is a plausible route of infection for these stray cats. An analysis into different risk factors showed that stray cats originating from nature reserves had, on average, more frequent antibodies against the bird flu virus stray cats from other habitats, such as a livestock farm, holiday park or industrial area.
We detected viral RNA with cycle threshold values of 13.75–36.35 in the brains of 5 red foxes (4.5%), which were submitted with differing preliminary reports partly involving signs of disease specific for the central nervous system.
A strain of highly pathogenic avian influenza has been silently spreading in US cattle for months, according to preliminary analysis of genomic data. The outbreak is likely to have begun when the virus jumped from an infected bird into a cow, probably around late December or early January. This implies a protracted, undetected spread of the virus — suggesting that more cattle across the United States, and even in neighbouring regions, could have been infected with avian influenza than currently reported. (...)
Analysis of the genomes suggests that the cattle outbreak probably began with a single introduction from wild birds in December or early January. (...) The data also show occasional jumps back from infected cows to birds and cats. “This is a multi-host outbreak,” says Nelson.
According to the USDA’s Animal and Plant Health Inspection Service (APHIS), H5N1 has been identified in 33 herds in eight states. On Thursday, a senior FDA official said 1 in 5 milk samples have tested positive for H5N1.
As of April 24, 2024, USDA has confirmed H5N1 virus
detections on 33 dairy cattle premises in 8 states
(Kansas, Idaho, Michigan, New Mexico, North Carolina,
Ohio, South Dakota, Texas). USDA has also confirmed -
based on specific phylogenetic evidence and
epidemiological information - that 8 poultry premises in
5 states (Kansas, Michigan, Minnesota, New Mexico and
Texas) have also been infected with the same distinct
H5N1 virus genotype detected in dairy cattle.
Phylogenetic analysis using genome sequencing suggests that there was a reassortment event in late 2023 between the current highly pathogenic 2.3.4.4b clade in wild birds and a low-pathogenic wild bird strain, which produced the B3.13 genotype now circulating in dairy cows.
The NP gene acquired during reassortment may have played a role in the emergence in cattle, they wrote, noting that the NP gene seems to allow influenza viruses to spread more easily in pigs.
Their analysis suggests there were as many as five B3.13 introductions from cattle to poultry, one to a raccoon, two to domestic cats, and three to wild birds. Though the findings track with epidemiological findings of spread through movement of herds to other states, they emphasized that there are still gaps. "We cannot exclude the possibility that this genotype is circulating in unsampled locations and hosts as the existing analysis suggests that data are missing and under surveillance may obscure transmission inferred using phylogenetic methods," they wrote.