PLoS Path: Genetics, Receptor Binding, and Transmissibility Of Avian H9N2

Photo Credit – FAO

 

# 9364

 

While the superstars of avian influenza tend to be those viruses that can infect, and sometimes kill, humans (H5N1, H7N9, H10N8) behind each of these deadly viruses is an obscure `parental’ virus called H9N2 that has lent a good deal of its backbone – it’s internal genes – to the creation of these emerging threats.

 

I’ve previously described H9N2 as the Professor Moriarty of avian flu viruses. 

 

Whenever something untoward happens with an avian flu strain – if you look deep enough – you often find clues that H9N2 was the viral `mastermind’ behind it all.

 

Last May, in EID Journal: H7N9 As A Work In Progress, we looked at a study that found the H7N9 avian virus continues to reassort with local H9N2 viruses, making the H7N9 viruses that circulated in wave 2 genetically distinct from those that were seen during the 1st wave.

 

Although categorized by their two surface proteins (HA & NA) Influenza A viruses have 8 gene segments (PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, NS2).

Shift, or reassortment, happens when two different influenza viruses co-infect the same host swap genetic material.  New hybrid viruses may be the result of multiple reassortments, with gene contributions coming from several parental viruses.

 

Of the three avian flu viruses we are currently watching with the most concern – H5N1, H7N9, and H10N8 – all  share several important features (see Study: Sequence & Phylogenetic Analysis Of Emerging H9N2 influenza Viruses In China):

 

• They all first appeared in  Mainland China
• They all  have come about through viral reassortment in poultry
• And most telling of all, while their HA and NA genes differ - they all carry the internal genes from the avian H9N2 virus

 

This ubiquitous, yet fairly benign H9N2 virus is apparently very promiscuous, as we keep finding bits and pieces of it turning up in new reassortant viruses.  Last June, in Eurosurveillance: Genetic Tuning Of Avian H7N9 During Interspecies Transmission, we saw evidence of even more influence of H9N2 on the ongoing evolution of H7N9.

 

Last January, The Lancet carried a report entitled Poultry carrying H9N2 act as incubators for novel human avian influenza viruses by Chinese researchers Di Liu a, Weifeng Shi b & George F Gao that warned:

 

Several subtypes of avian influenza viruses in poultry are capable of infecting human beings, and the next avian influenza virus that could cause mass infections is not known. Therefore, slaughter of poultry carrying H9N2—the incubators for wild-bird-origin influenza viruses—would be an effective strategy to prevent human beings from becoming infected with avian influenza.

We call for either a shutdown of live poultry markets or periodic thorough disinfections of these markets in China and any other regions with live poultry markets.

 

In the past, we’ve looked at the propensity of the H9N2 virus to reassort with other avian flu viruses (see PNAS: Reassortment Of H1N1 And H9N2 Avian viruses & PNAS: Reassortment Potential Of Avian H9N2) which have shown the H9N2 capable of producing `biologically fit’ and highly pathogenic reassortant viruses.

 

And in 2010 (see Study: The Continuing Evolution Of Avian H9N2) we looked at computer modeling (in silica) that warned the H9N2 virus has been slowly evolving towards becoming a `more humanized’ virus.

 

And while we have only seen a handful of human infections with this virus (see Hong Kong: Isolation & Treatment Of An H9N2 Patient), it is also true that in areas where this virus is most common, testing and surveillance for the virus is extremely limited.  Like so many other novel viruses, we can only guess at is true burden in the human population.

 

This week, we’ve a new study that finds a diverse set of H9N9 genotypes have been circulating in Chinese poultry between 2009-2013, with the majority sharing a remarkably stable “internal-gene-combination”.  This internal gene structure has been `lent’ to the emerging H7N9 and H10N8 viruses as well.

Perhaps most surprising, of 35 viruses tested, all bound preferentially to alpha 2,6 receptor cells -  the type commonly found in the human upper respiratory tract, rather than to alpha 2,3 receptor cells which are found in the gastrointestinal tract of birds.

This is viewed as one of the crucial steps in the adaptation of an avian influenza virus to a mammalian host (see Nature Comms: Host Adaptation Of Avian Influenza Viruses). 

 

Additionally, six of nine viruses tested in ferrets transmitted via respiratory droplets (two being highly transmissible) and inoculated ferrets readily developing spontaneous viral mutations conducive to greater virulence and better transmission in mammals. 

 
For more details, follow the link below to read:

 

Genetics, Receptor Binding Property, and Transmissibility in Mammals of Naturally Isolated H9N2 Avian Influenza Viruses

Xuyong Li equal contributor, Jianzhong Shi equal contributor, Jing Guo equal contributor, Guohua Deng, Qianyi Zhang, Jinliang Wang,  Xijun He, Kaicheng Wang,  Jiming Chen,  Yuanyuan Li,  Jun Fan,  Huiui Kong, Chunyang Gu,  [ ... ], Hualan Chen mail

Abstract

H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013.

Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China.

All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals.

Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans.

(Continue . . . )

PLoS Pathogens: Fitness Advantage From Permissive NA Mutations In Oseltamivir Resistant pH1N1

 

 

# 8429

 

Hopefully today’s blog won’t be as tedious as the title might first suggest.

 

Oseltamivir (aka Tamiflu ®) – an NAI (Neuraminidase Inhibiting) antiviral drug – is our primary pharmaceutical weapon against influenza.   While it doesn’t `cure’  the flu - when started early enough (preferentially within 48 hrs of onset of symptoms) - it can reduce both the severity and duration of infection (see Effectiveness Of NAI Antivirals In Reducing Mortality In Hospitalized H1N1pdm09 Cases).

Up until the middle of the last decade, we had another class of antiviral drugs - M2 ion channel blockers (e.g. Amantadine, Rimantadine) –  which were first developed in the late 1950s. But excessive use over the years (including in agricultural settings) eventually led to widespread resistance.

 

The replacement antivirals introduced during the last decade include Oseltamivir (Tamiflu), Zanamivir (Relenza), and Peramivir. Of these, Oseltamivir is by far the most widely used, and has been stockpiled by many governments for use in the event of a pandemic.

 

While occasional instances of Oseltamivir resistance was recorded prior to 2007, in nearly every case, it developed after a person was placed on the drug (`spontaneous mutations’).  While of concern to the patient being treated, it occurred in only about 1% of treated cases, and studies suggested that these resistant strains were `less biologically fit’, and were therefore believed to be unlikely to spread.

 

Which of course, is exactly what they did do.  Between 2007 and 2008, the incidence of resistant seasonal H1N1 viruses literally exploded around the globe. 

 

So much so, that by the end of 2008, nearly all of the H1N1 samples tested in the United States were resistant to oseltamivir and the CDC was forced to issue major new guidance for the use of antivirals (see CIDRAP article With H1N1 resistance, CDC changes advice on flu drugs).

This 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)). 

 

While this mutation had been seen before, obviously something had changed between 2006 and 2008 to allow the resistant form of the virus to spread so quickly.

 

In 2010  Bloom, Gong & Baltimore discussed these `enabling’ changes in the Journal Science in  a report called Permissive Secondary Mutations Enable the Evolution of Influenza Oseltamivir Resistance.

ABSTACT

The His²⁷⁴→Tyr²⁷⁴ (H274Y) mutation confers oseltamivir resistance on N1 influenza neuraminidase but had long been thought to compromise viral fitness. However, beginning in 2007–2008, viruses containing H274Y rapidly became predominant among human seasonal H1N1 isolates. We show that H274Y decreases the amount of neuraminidase that reaches the cell surface and that this defect can be counteracted by secondary mutations that also restore viral fitness.

Two such mutations occurred in seasonal H1N1 shortly before the widespread appearance of H274Y. The evolution of oseltamivir resistance was therefore enabled by “permissive” mutations that allowed the virus to tolerate subsequent occurrences of H274Y. An understanding of this process may provide a basis for predicting the evolution of oseltamivir resistance in other influenza strains.

 

In 2011 Abed,  Pizzorno,  Bouhy &  Boivin identified several `permissive’ neuraminidase mutations that occurred just prior to the spread of resistant H1N1 - that when combined with H275Y -  `enabled’ its efficient transmission (see PLoS Pathogens Role of Permissive Neuraminidase Mutations in Influenza A/Brisbane/59/2007-like (H1N1) Viruses).

 

This pervasive spread of resistant H1N1 would have been a much bigger deal had it not been for the arrival of the 2009 H1N1 pandemic virus, which effectively supplanted the old (resistant) H1N1, and replaced it with a new – but fortunately, still susceptible to NAIs – H1N1 virus.

 

Fast forward five years, and the (now seasonal, formerly pandemic) pH1N1 virus remains overwhelmingly susceptible to Oseltamivir and other NAI antiviral drugs, although we have seen a few signs of `creeping resistance’. 

 

Reassuringly, the latest FluView report (week 12) indicated that of 4524 viruses tested this flu season in the United States, only 54 (1.2%) showed signs of NA Inhibitor resistance.

 

But we have seen a few worrisome clusters of NAI resistant flu (see Eurosurveillance: Community Cluster Of Antiviral Resistant pH1N1 in Japan & NEJM: Oseltamivir Resistant H1N1 in Australia), which has raised concerns that we could see a repeat of the 2007-2008 rise in antiviral resistance in our current H1N1 strain.

 

All of which serves as prelude to a new study that appears in PloS Pathogens, that looks at the potential of pH1N1 following the same course as its predecessor.  Their assessment is not particularly rosy.

 

Estimating the Fitness Advantage Conferred by Permissive Neuraminidase Mutations in Recent Oseltamivir-Resistant A(H1N1)pdm09 Influenza Viruses

Jeff Butler, Kathryn A. Hooper, Stephen Petrie, Raphael Lee, Sebastian Maurer-Stroh, Lucia Reh, Teagan Guarnaccia, Chantal Baas, Lumin Xue, Sophie Vitesnik, Sook-Kwan Leang, Jodie McVernon, Anne Kelso, Ian G. Barr, James M. McCaw, Jesse D. Bloom, Aeron C. Hurt mail

Published: April 03, 2014  DOI: 10.1371/journal.ppat.1004065

Abstract

Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses.

Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses.

In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein.

These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.

 

Fair warning: the methods and materials section is lengthy, complex, and pretty tough sledding for those without a solid background in virology.  Those interested in the details (or with a masochistic bent) will want to read this report in its entirety.

 

The bottom line, however, is that since the 2009 H1N1 virus emerged five years ago, it has managed to pick up a series of `permissive’ mutations that are believed to increase its ability to replicate when it carries the H275Y resistance mutation.

 

Which in theory, should promote its spread.

 

Given that these mutations are already entrenched, it is a bit surprising we haven’t already seen an expansion in resistant pH1N1, beyond a couple of documented clusters in Australia and Japan.  The authors write:

One explanation is that a high level of circulating A(H1N1)pdm09 viruses may be required for a A(H1N1)pdm09 OR virus to become established and spread. The Australian HNE2011 virus cluster emerged [25], [26] during a season when A(H1N1)pdm09 viruses accounted for almost 40% of all influenza A and B viruses detected globally but, in 2012 and 2013, the proportion of A(H1N1)pdm09 viruses circulating has been considerably lower (9% and 25% respectively) [52].

In the most recent 2013/14 Northern Hemisphere influenza season, a cluster of A(H1N1)pdm09 H275Y OR viruses that contained both the V241I and N369K PPMs plus an additional N386K NA mutation, was detected in Sapporo, Japan [53], during a period of the season where A(H1N1)pdm09 viruses contributed approximately 50% of the circulating influenza strains [54].

We’ve just come through an H1N1 dominated flu season in North America, and the incidence of H274Y has remained low, so other factors may be involved. The authors suggest:

Apart from NA PPMs, it may be that other properties, such as antigenic novelty, are also necessary for an OR virus to spread widely. In 2007–2008, the H275Y NA mutation became fixed in a new seasonal A(H1N1) antigenic variant (A/Brisbane/59/2007-like), suggesting that the antigenic novelty of the OR virus assisted its prolific spread.

In this vein, the authors warn:

A(H1N1)pdm09 viruses have now been circulating in humans for over four years, but are yet to undergo a significant antigenic change (as evidenced by the continued inclusion of A/California/7/2009 in the human seasonal influenza vaccine since 2009).

As the H1 component of the vaccine has been updated, on average, every 2.8 years (range 1 to 8 years), and the H3 component every 1.8 years (range 1 to 4 years) since 1980, it is reasonable to anticipate that A(H1N1)pdm09 viruses will undergo antigenic change in the near future.

The significance being that an antigenic change in the virus might be the spark needed to spread the resistant mutation, and at the same time would reduce the effectiveness of the current vaccine (and evade herd immunity), and therefore increase our need for effective antiviral medications.

 

Viruses and bacteria evolve and adapt very quickly. The sobering truth is pharmacological victories over them tend to be fleeting. New classes of drugs are going to be needed, along with prudent stewardship of the drugs currently in our arsenal.

 

The authors of this study wrap up by saying:

 

Here we demonstrate that contemporary A(H1N1)pdm09 viruses have acquired NA mutations which permit the acquisition of NA H275Y without compromising viral fitness. These mutations, which are now present in virtually all circulating A(H1N1)pdm09 viruses, enhance the surface expression and enzymatic activity of the A(H1N1)pdm09 H275Y NA protein in vitro and result in enhanced viral fitness in vivo.

Hence, the risk that H275Y A(H1N1)pdm09 viruses will spread globally, in a similar manner to OR seasonal A(H1N1) viruses in 2007–2008, now appears greater than at any time since the A(H1N1)pdm09 lineage emerged in 2009.