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.

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Nature Comms: Host Adaptation Of Avian Influenza Viruses

Flu Virus binding to Receptor Cells – Credit CDC

 

# 9360

 

More than a decade after it re-emerged in Vietnam, H5N1 continues to circulate widely in Asian and Middle Eastern poultry providing numerous opportunities to infect humans, and yet only a few more than 600 human infections have been identified.  

 

Similarly, the H7N9 virus which appeared 2 years ago in China appears well distributed in Asia’s domesticated poultry population, but only about 450 human infections have been reported.

 

While both viruses are capable of infecting and causing severe illness in humans, neither has taken off as a human-adapted pathogen.  Transmission has almost always been from bird to human, with secondary human-to-human transmission a rarity. 

 

Despite our concerns over the future of these influenza subtypes, both viruses remain primarily adapted to avian hosts.  The concern of course, is that over time, that may change.

 

Why some influenza viruses – like seasonal H1N1, H3N2, H2N2, and others through the years – have successfully adapted to humans, while others like H5N1, H7N9, H5N6, H10N8 haven’t remains a mystery, although researchers are making progress in figuring it out.  

 

Avian flu viruses are preferentially adapted to birds, where it is primarily a gastrointestinal infection. What scientists look for are `mammalian adaptations’;  those that favor the infection and respiratory transmission among mammals – including humans. 

Unlike solving a Rubik’s cube, there is probably more than just one `winning’ combination.  And a change in one part of the virus that favors adaptation may either be enhanced, or blocked, by a change somewhere else along the14,000 nucleotide chain of the influenza A virion.

 

Avian adapted flu viruses bind preferentially to the alpha 2,3 receptor cells found in the gastrointestinal tract of birds. So the first barrier appears to be switching the RBS, or Receptor Binding Site (the area of its genetic sequence that allows it to attach to, and infect, host cells) to `fit’ the receptor cells commonly found in the human upper respiratory tract; the alpha 2,6 receptor cell (see Study: Dual Receptor Binding H5N1 Viruses In China)

But that, we are learning, isn’t enough on its own.

 

Birds run `hotter’ than mammals, with a normal body temperature  several degrees higher (and avian viruses replicate in the gut, which is warmer than the upper airway of humans).  Which means mammalian adapted viruses must be adapted to replicate at a lower temperature.

 

Researchers have determined the (E627K) substitution in the (PB2) protein - the swapping out of the amino acid Glutamic acid (E) at position 627 for Lysine (K) - makes the an influenza virus better able to replicate at the lower temperatures (roughly 33C) normally found in the upper human respiratory tract (see Eurosurveillance: Genetic Analysis Of Novel H7N9 Virus).

 

These are just two examples of species barriers that must be overcome before an avian virus can successfully adapt to human (or mammalian) physiology.  There are more, some we know about, some we probably don’t. 

 

Complicating matters – influenza viruses constantly develop multiple amino acid changes – and their combined effects on the virulence, transmission, antiviral resistance, `fitness’, and host range of the virus are far from fully understood.

 

All of which serves as prelude to a study recently published in Nature Communications, that finds another PB2 amino acid substitution (K526R) enhances the effects of the E627K mutation mentioned above.

 

First the link and abstract with the daunting title of:

 

The K526R substitution in viral protein ​PB2 enhances the effects of E627K on influenza virus replication

Wenjun Song, Pui Wang, Bobo Wing-Yee Mok, Siu-Ying Lau, Xiaofeng Huang, Wai-Lan Wu, Min Zheng, Xi Wen, Shigui Yang, Yu Chen, Lanjuan Li, Kwok-Yung Yuen & Honglin Chen

Host-adaptive strategies, such as the E627K substitution in the ​PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation ​PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970.

Polymerase complexes containing ​PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. ​PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. ​PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.

(Continue . . .)

Simply put, an influenza virus carrying both the E637K and K526R mutation in it’s PB2 protein replicates more efficiently in mammalian hosts. 

 

Interestingly, the H3N2 seasonal flu virus – which traditionally produces more severe flu seasons than does seasonal H1N1 – has also carried this dynamic duo of amino acid substitutions since the early 1970s.

 

HKU (Hong Kong University) – which did this study – published a press release (excerpts below) with a summary of their findings:

 

HKU medical research team finds host adaptation strategies of avian influenza A viruses for better replication in human

20 Nov 2014

(EXCERPT)

Research findings
In a recent study reported in the Nature Communications, a research team led by Dr. Honglin Chen, Associate Professor, and Professor Kwok-yung Yuen, Henry Fok Professor in Infectious Diseases, Chair Professor of Infectious Diseases from the Department of Microbiology, Li Ka Shing Faculty of Medicine, and State Key Laboratory for Emerging Infectious Diseases, the University of Hong Kong, found that avian influenza A H5N1 and H7N9, and seasonal H3N2 viruses may gain the ability to replicate in mammal and human cells through various adaptation changes in the viral replication enzyme complex called the PB2 subunit. 

They found that H7N9 avian influenza A virus is able to utilize multiple adaptive strategies to replicate in human cells, which may explain why H7N9 is distinct in causing human infections; This study identified a novel adaption marker, PB2-526R among some H7N9 viruses and almost exclusively among all H5N1 human cases from Indonesia.  It has been a puzzle why there is no known PB2 adaptation marker in the H5N1 virus from Indonesia human cases and the finding from HKU nicely explained how this Indonesian subclade of avian H5N1 virus may have adapted for human infections. 

This study also found PB2-526R is able to enhance replication and pathogenicity of other types of PB2 adaptations, such as previously known PB2-627K, in H7N9 and H3N2 viruses.  Since the human pandemic H3N2 virus emerged in 1968, it has gained an additional PB2-526R adaptation marker since 1970s and the PB2-526R-627K virus replicates better than the solely PB2-627K virus.  It is likely that the impression of more severe disease burden caused by H3N2  than that of H1N1 may be partly attributed to the better replication ability of PB2-526R-627K virus. 

These findings by HKU provided new insight for the understanding of cross species transmission and replication in human cells by avian influenza viruses.  The study provides a new genetic marker for the surveillance of avian influenza A virus with potential for human infection.

While this study provides us with a new genetic marker by which to track the evolution of avian flu viruses, it alone is obviously not the only barrier to seeing H7N9, H5N1, or any other novel flu virus become a pandemic. 

But given its sloppy replication habits, and promiscuous `mating habits (reassortment)’, influenza viruses get billions of throws of the genetic dice each and every day.

 

So the odds (and history) suggest that given enough time the `right’ combination will come up, and another novel virus will strike mammalian host gold.  We’ve seen that happen four times in the past century (2009, 1968, 1957, and 1918), and there is no reason to doubt it will happen again.

Serial Passage Of H5N2 In Mice

 

# 8854

 

Influenza viruses are prolific, but sloppy, replicators.  They make millions of copies of themselves inside every host, but in the process, often make small transcription errors – amino acid substitutions – that can change the way the replicated virus acts.  Often, these changes are of little or no effect, or are even detrimental to the survival of the virus.

Those that favor replication in the host, however, tend to carry on to produce more progeny, advancing their newfound lineage forward, often drowning out the earlier `wild type’ virus in the host.

 

This process is called host adaptation, and while it can (and does) happen in the wild, it can be easily simulated in the laboratory as well via a classic serial passage study (see chart above).  You simply inoculate a host with a `wild type’ strain of a virus, let it replicate awhile, then take the virus from the first host and inoculate a second, and then repeat the process five, ten, fifteen times or more.

 

Over time, the virus tends to adapt to the new host (assuming there are no species barriers to prevent it). 

 
This tendency to adapt over long chains of infection is one of the reasons we watch for clusters of novel flu infections in humans, as it could either indicate adaptation, or provide more opportunities for the virus to do so.

 

Although regrettably most of this article is behind a pay wall, the journal Veterinary Microbiology has published a study wherein researcher performed this serial passage experiment in mice using a naturally H5N2 reassortant virus.

 

One should note that the H5N2 virus (A/chicken/Hebei/1102/2010 (HB10)) used was a reassortant of the H5N1 virus and H9N2 avian flu.

 

Unlike it’s highly pathogenic, and infamous, sibling – H5N1 – there is only limited evidence suggesting that H5N2 viruses can infect mammals.

 

• In 2012,  in Taiwan: Three Poultry Workers Show H5N2 Antibodies, we looked at a report that three poultry workers and officials working in animal quarantine have tested positive for antibodies for the H5N2, but all remained healthy and asymptomatic (note: refer to article for other possible causes of seropositivity).
• Some earlier H5N2 studies have been  suggestive of prior H5N2 human infection – particularly among poultry workers – including: Human H5N2 avian influenza infection in Japan and the factors associated with high H5N2-neutralizing antibody titer  & Serological survey of avian H5N2-subtype influenza virus infections in human populations.
• And in 2009, two H5N2 viruses were isolated in South Korean pigs (see Isolation and genetic characterization of H5N2 influenza viruses from pigs in Korea).

 

Still, we haven’t seen any solid evidence that H5N2 has produced significant or serious human illness, although the possibility was explored in the J. Chinese Medical Association in 2012 in  The threat of highly pathogenic avian influenza H5: will H5N2 infections occur in humans?.

 

First the abstract from this latest study, which produced a virus with increased virulence and replication efficiency after just 15 serial passages,  then I’ll return with a bit more.

 

Adaptation of a natural reassortant H5N2 avian influenza virus in mice.

Li Q¹, Wang X¹, Zhong L¹, Wang X², Sun Z¹, Gao Z¹, Cui Z¹, Zhu J¹, Gu M², Liu X², Liu X³.

 

Abstract

It is reported that the H5N2 highly pathogenic avian influenza virus A/chicken/Hebei/1102/2010 (HB10) is a natural reassortant between circulating H5N1 and endemic H9N2 influenza viruses. To evaluate the potential of its interspecies transmission, the wild-type HB10 was adapted in mice through serial lung passages.

Increased virulence was detectable in 5 sequential lung passages in mice and a highly virulent mouse-adapted strain (HB10-MA) with a 50% mouse lethal dose of 10^2.5 50% egg infectious dose was obtained in 15 passages. The virulence and the replication efficiency of HB10-MA in mice were significantly higher than those of HB10 while HB10-MA grew faster and to significantly higher titers than HB10 in MDCK and A549 cells.

Only five amino acid mutations in four viral proteins (HA-S227N, PB2-Q591K, PB2-D701N, PA-I554V and NP-R351K) of HB10-MA virus were found when compared with those of HB10, indicating that they may be responsible for the adaptation of the novel reassortant H5N2 avian influenza virus in mice with increased virulence and replication efficiency.

The results in this study provide helpful insights into the pathogenic potential of novel reassortant H5N2 viruses to mammals that deserves further attentions.

 

Although I know better than to anthropomorphize viruses (they hate when you do that), as time goes on I’ve begun to think of H9N2 as the Professor Moriarty of avian flu viruses.  Whenever something worrisome happens with an avian flu strain – if you look deep enough – you often find clues that H9N2 was 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.

 

As we’ve discussed before, the genetic contributions from the avian H9N2 virus appear to be significant.

 

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 promiscuous, as we keep finding bits and pieces of it turning up in new reassortant viruses.  Most recently, 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.

 

For now, H5N2 is generally regarded as posing a low level of threat to human health. 

 

Of course, the same thing could have been said about all of the H7 family of avian flu viruses 18 months ago, before H9N2 lent its internal genes to H7N9 in China, sparking a serious threat to public health.

If there is one constant with influenza viruses, it is that they continually change.