Credit ECDC
#18,063
Although we constantly hear that HPAI H5N1 still needs to acquire a number of specific genetic mutations before it could pose a human pandemic threat, no one really knows what those changes might be.
We do have a short list of known or suspected mammalian adaptations - amino acid changes that occur at specific locations in the genome - that are believed to enhance its ability to infect, replicate, or transmit among mammals.
But we certainly don't know all of them, or how they may `play together' in different combinations.
A dozen years ago, Virologist Ron Fochier controversially demonstrated, that an older clade of H5N1 could be made to transmit via the airborne route (see CIDRAP Fouchier study reveals changes enabling airborne spread of H5N1).
Our current clade (2.3.4.4b) is quite different from that older virus, and while we've seen a huge increase in mammalian infections - and mammal-to-mammal transmission - evidence of airborne spread remains scant.
Alarms were raised, however, in the fall of 2022 when H5N1 began spreading rapidly through a large mink farm in Spain (see Eurosurveillance: HPAI A(H5N1) Virus Infection in Farmed Minks, Spain, October 2022).
Mink are a member of the Mustelidae family of carnivorous mammals, which also includes otters, badgers, weasel, martens, ferrets, and wolverines. Many of these species are susceptible to flu viruses – most notably ferrets – which are often used in influenza research.
Spillovers into farmed animals are particularly worrisome, because they allow for serial transmission across a large number of hosts, which may result in host adaptation (a technique used by Fouchier in creating his transmissible H5N1).
This mink-derived H5N1 virus from Spain carried a rare mutation (PB-T271A), which is believed to `enhance the polymerase activity of influenza A viruses in mammalian host cells and mice', and which has subsequently been reported in outbreaks in fur farms in Finland.
Last summer, the CDC issued an IRAT Risk Assessment On Mink Variant of Avian H5N1, finding it's scores had risen in 6 of the 10 parameters used to evaluate their zoonotic potential (see chart below).
Curiously, transmission in both cases was delayed by several days, although there was no indication of in-host mutations causing this.
This is a lengthy, highly detailed, report with a lot to unpack. I've posted some excerpts below, but is really should be read in its entirety. I'll return after the break with more.
Risk assessment of a highly pathogenic H5N1 influenza virus from mink
Katherine H. Restori, Kayla M. Septer, Cassandra J. Field, Devanshi R. Patel, David VanInsberghe, Vedhika Raghunathan, Anice C. Lowen & Troy C. Sutton
Nature Communications volume 15, Article number: 4112 (2024)
Abstract
Outbreaks of highly pathogenic H5N1 clade 2.3.4.4b viruses in farmed mink and seals combined with isolated human infections suggest these viruses pose a pandemic threat. To assess this threat, using the ferret model, we show an H5N1 isolate derived from mink transmits by direct contact to 75% of exposed ferrets and, in airborne transmission studies, the virus transmits to 37.5% of contacts.Sequence analyses show no mutations were associated with transmission.The H5N1 virus also has a low infectious dose and remains virulent at low doses. This isolate carries the adaptive mutation, PB2 T271A, and reversing this mutation reduces mortality and airborne transmission.This is the first report of a H5N1 clade 2.3.4.4b virus exhibiting direct contact and airborne transmissibility in ferrets. These data indicate heightened pandemic potential of the panzootic H5N1 viruses and emphasize the need for continued efforts to control outbreaks and monitor viral evolution.
(SNIP)
To cause a pandemic, an influenza A virus must be able to replicate efficiently in humans and transmit via the airborne route from person-to-person. Owing to similarities to humans in their susceptibility, ferrets are a valuable model in which to evaluate influenza virus transmission and pathogenesis, and ferrets are routinely used to assess pandemic risk.
Ferrets possess a similar distribution of viral receptors (i.e.,α 2,6-linked sialic acids) to that observed in humans and, upon infection with human influenza viruses, ferrets develop clinical illness and shed high levels of infectious virus. Also consistent with influenza in humans, ferrets infected with human-adapted strains transmit the virus through the air to contact animals8.
To date, no subclade 2.3.4.4b highly pathogenic H5N1 virus has exhibited the ability to transmit by the airborne route, a feature thought to be critical in limiting their outbreak potential in humans. However, experimental studies have demonstrated the potential for an ancestral clade 2.1.3.2 H5N1 virus to become airborne transmissible in ferrets9.
(SNIP)
To assess the risk to humans, we evaluated the potential for an H5N1 isolate from this mink outbreak to infect, cause disease, and transmit in ferrets. As isolates from the mink outbreak could not be readily obtained, we generated recombinant influenza A/mink/Spain/3691-8_22VIR10586-10/2022 (H5N1) virus [A/mink (H5N1)] using reverse genetics.
(SNIP)
All viruses from this outbreak carried the mammalian-adaptive mutation T271A, and several additional mutations were identified throughout the genome; however, as previously reported, the function of these later mutations is unknown10.
Virus rescue or regeneration, and all subsequent experiments were performed following strict biosafety protocols in our biosafety level 3 enhanced facility, and all studies were conducted following all local, state, and federal rules and regulations.
(SNIP)
Discussion
Collectively, our studies show that the A/mink (H5N1) virus transmitted efficiently by direct contact, with 75% of contact animals infected, and inefficiently via the airborne route with 37.5% of respiratory contact animals developing an infection.
Sequence analyses of viruses shed in the nasal wash from infected donor and contact animals did not show evidence of positive selection acting during direct contact or airborne transmission. In dose de-escalation studies, the A/mink (H5N1) virus had a low infectious dose, and the virus was highly virulent across a wide range of doses as all infected animals developed severe disease.
Although we observed efficient direct contact transmission, it was delayed relative to that seen previously with pandemic influenza viruses. For pandemic influenza viruses, viral shedding in direct contacts often begins on day 1 p.c. 14,15. Here, the onset of shedding was on day 3 p.c. in one contact, and on days 7 and 9 p.c., respectively, in the others. While airborne transmission was observed, it occurred at lower efficiency than is typical for pandemic influenza viruses.
(SNIP)
The low median infectious dose observed for A/mink (H5N1) was comparable to the two most recent pandemic influenza viruses, indicating that the ability to infect and replicate in ferrets is not likely limiting transmission. Prior adaptation studies have shown that changes in the HA to reduce the pH of fusion and enhance binding to α2-6-linked sialic acids combined with mutations to enhance polymerase activity in mammalian cells are required for airborne transmission of a highly pathogenic clade 2.3.1.2 H5N1 virus 9,32.
The A/mink (H5N1) virus does not carry mutations known to reduce the pH of fusion or enhance binding to α2-6-linked sialic acids; however, the virus does carry the PB2 T271A mutation which has been shown to enhance polymerase activity and replication of avian viruses in mammalian cells. Consistent with this observation, we show that reversing the PB2 mutation (i.e., 271T) in A/mink (H5N1) reduced viral polymerase activity in mini-genome assays.
Moreover, introducing the PB2 A271T mutation reduced mortality and resulted in a reduction in the number of RC ferrets that shed virus in airborne transmission studies. This was associated with reductions in viral titers in the nasal wash which were not significant at lower inoculation doses used to assess virulence but were significant at high inoculation doses used for transmission studies. These findings indicate the PB2 T271A mutation is enhancing viral replication of the A/mink (H5N1) virus contributing to both virulence and transmission in ferrets.
As our studies assessed viral load in the nasal wash, future studies are warranted to assess the impact of the PB2 T271A mutation on viral replication in the lungs and systemic dissemination of the virus. Moreover, evaluating the role of the PB2 T271A mutation in the context of direct contact transmission will yield additional insight on the contribution of this mutation to the overall transmissibility of the virus.
An important consideration in interpreting our results with respect to the risk posed to humans is that the ferrets used in these studies have no pre-existing immunity to influenza, whereas the majority of humans have been exposed to H1N1 and H3N2 seasonal influenza viruses. While different influenza A virus subtypes are antigenically distinct, some degree of cross-protection against H5N1 may be conferred by prior exposure to these seasonal strains, especially against the N1 neuraminidase. Indeed, antibodies and T cells against seasonal influenza viruses have been shown to cross-react with H5N1 viruses33,34,35,36,37,38.
Future studies are warranted to determine if prior immunity reduces disease severity and/or transmission.
In conclusion, this is the first report of both direct contact and limited airborne transmission in a mammalian model of a subclade 2.3.4.4b H5N1 virus indicating these viruses pose a significant pandemic threat. Therefore, ongoing risk assessment and enhanced surveillance in wild and domestic animals is warranted to monitor the threat posed by these viruses as they continue to evolve and spillover into mammals, including humans.
While the headline may be that ferrets were found able to transmit this virus via both contact and airborne routes, the bigger news for me is that it was able to do so without first acquiring any of the `usual suspects' from the mammalian adaptation list.
The A/mink (H5N1) virus does not carry mutations known to reduce the pH of fusion or enhance binding to α2-6-linked sialic acids; however, the virus does carry the PB2 T271A mutation
Furthermore, by changing out that PB2 T271A mutation to PB2 A271T, the virus immediately became less effective. The authors stating:
These findings indicate the PB2 T271A mutation is enhancing viral replication of the A/mink (H5N1) virus contributing to both virulence and transmission in ferrets.
While T271A alone may not be enough to make H5N1 a pandemic contender, it is doing an impressive amount of heavy lifting. And, of course, if T271A can do this, there may be other evolutionary shortcuts we aren't aware of.
This dispatch also touches on potential (albeit, likely limited) pre-existing immunity to H5N1.
This is something we've looked at before, in EID Journal: A(H5N1) NA Inhibition Antibodies in Healthy Adults after Exposure to Influenza A(H1N1)pdm09 and in Frontiers Vet. Sci: Influenza Virus Immune Imprinting - Clinical Outcome In Ferrets Challenged with HPAI H5N1.
Whether it is occurring birds, in farmed mink, in dairy cattle, in peridomestic mammals, or in humans, H5N1 is engaged in a relentless series of GOF (Gain of Function) experiments.
With each passing day new hosts are infected, new genotypes are produced, and random amino acid changes are introduced. While the virus still lacks the `spark' to ignite a pandemic, it appears to be a lot closer today than at any time we've seen it in the past.
And if we get very lucky - and H5N1 doesn't have the `right stuff' - we can be pretty certain there are other novel flu viruses that do.
It's just a matter of time.
