Wednesday, May 15, 2019

J. Gen. Virology: Mutations in PB2 and HA Enhanced Pathogenicity of H4N6 Avian Influenza Virus in Mice


Influenza A viruses, being negative-sense single-stranded segmented RNA viruses, are prolific but notoriously sloppy replicators. They make millions of copies of themselves while they infect a host, but in the process, often make small transcription errors.
Most of these `errors' do little to help the virus, and many can be detrimental to its survival.
Those that accidentally favor replication in the host, however, are able to go on to produce more progeny, and if biologically`fit' enough, they can drown 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 graphic above).
You basically inoculate a host (mouse, ferret, guinea pig, etc.) with a `wild type’ strain of a virus, let it replicate for 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), often increasing replication, virulence, and/or transmissibility.

This is one of the big reasons why - when dealing with a spillover of a novel virus into humans - we look carefully for any signs of clusters. The more times a novel virus successfully jumps from one human host to the next, the more likely it is to accrue host adaptations.

Among the novel avian flu viruses, we watch the H5 and H7 viruses with the greatest interest because H5N1, H7N9, and H5N6 have demonstrated their ability to cause significant morbidity and mortality in humans.  
What they lack is the ability to transmit efficiently from human-to-human. 
But avian viruses continue to surprise us. Until the spring of 2013, H7 viruses were considered relatively minor threats to human health, a notion that was quickly dispelled with the emergence of a LPAI H7N9 virus in China.

H9N2 has a 20 year history of infection humans (see FluTrackers List), albeit generally producing mild illness, but over the past few years has shown signs of increasing mammalian adaptation (see Virology: Receptor Binding Specificity Of H9N2 Avian Influenza Viruses).

We've seen H10 viruses infect humans, both mildly and severely, while H6 avian viruses, which are ubiquitous in Asian poultry, and have recently:
Another virus increasingly on our `suspect list' is LPAI H4N6 - which while common in wild and migratory birds - has also been found in Chinese, Canadian (1999) and American swine (see Virology: Detection & Characterization Of Avian H4N6 In Midwestern Swine (2015)).

In August of 2017, in Cell: Avian-to-Human Receptor-Binding Adaptation by Influenza A Virus Hemagglutinin H4, researchers presented evidence that avian H4N6 viruses can adapt to human receptor cells while in a swine host (using the 1999 Canadian isolate), warning:
These results clearly implicate the potential threat posed by H4 viruses to public health. Therefore, early-warning study of H4 subtype human receptor-binding property is highly appreciated.
H4N6 has also been detected in other mammals, including seals.  In 2017's PLoS One study Isolation and characterization of H4N6 avian influenza viruses from mallard ducks in Beijing, China, the authors warned: 
All of our isolates belonged to a novel genotype that was different from other H4N6 viruses isolated in China. We further evaluated the virulence and transmission of two representative H4N6 strains in mammalian models. We found that both of these H4N6 viruses replicated efficiently in mice without adaptation.
Additionally, these two strains had a 100% transmission rate in guinea pigs via direct contact, but they had not acquired respiratory droplet transmissibility.
These results reveal the potential threat to human health of H4N6 viruses in migratory birds and the need for enhanced surveillance of AIVs in wild birds.
All of which serves as prelude to a new paper, just published in the Journal of General Virology, which takes the H4N6 virus and runs it through the classic serial passage study described above.
After just 12 passages a virulent mouse-adapted virus emerged, with four significant mutations appearing in PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering).
The E627K substitution in the PB2 protein (swapping out Glutamic acid (E) for Lysine (K)) makes avian influenza viruses better able to replicate at the lower temperatures (roughly 33C) found in the upper respiratory tract of mammals.
Although the two HA mutations did not individually raise the pathogenicity of the virus, they did so in concert . . . in mice.
While mice are not a perfect analog for humans, and the initial transmission of the wild-type virus was artificially propelled by researchers, this study - when combined with the earlier work we've seen on H4N6 - suggest that H4 viruses are not off the table as a human health threat.

While the full study is behind a paywall, you will find a link to a preview page with additional text, in the abstract.
Mutations in PB2 and HA enhanced pathogenicity of H4N6 avian influenza virus in mice

Authors: Guanlong Xu1​,† , Fang Wang1​,† , Qiuchen Li1​,2​,† , Guoxia Bing3​ , Shijie Xie1​ , Shijing Sun1​ , Zengjie Bian1​ , HaoJie Sun1​ , Yu Feng1​ , Xiaowei Peng1​ , Hui Jiang1​ , Liangquan Zhu1​ , Xuezheng Fan1​ , Yuming Qin1​,* , Jiabo Ding1​,*

First Published Online: 13 May 2019, Journal of General Virology doi: 10.1099/jgv.0.001192

The H4 subtype avian influenza virus (AIV) continues to circulate in both wild birds and poultry, and occasionally infects mammals (e.g. pigs). H4-specific antibodies have also been detected in poultry farm workers, which suggests that H4 AIV poses a potential threat to public health.
However, the molecular mechanism by which H4 AIVs could gain adaptation to mammals and whether this has occurred remain largely unknown. To better understand this mechanism, an avirulent H4N6 strain (A/mallard/Beijing/21/2011, BJ21) was serially passaged in mice and mutations were characterized after passaging.
A virulent mouse-adapted strain was generated after 12 passages, which was tentatively designated BJ21-MA. The BJ21-MA strain replicated more efficiently than the parental BJ21, both in vivo and in vitro. Molecular analysis of BJ21-MA identified four mutations, located in proteins PB2 (E158K and E627K) and HA (L331I and G453R, H3 numbering). Further studies showed that the introduction of E158K and/or E627K substitutions into PB2 significantly increased polymerase activity, which led to the enhanced replication and virulence of BJ21-MA.
Although individual L331I or G453R substitutions in HA did not change the pathogenicity of BJ21 in mice, both mutations significantly enhanced virulence. In conclusion, our data presented in this study demonstrate that avian H4 virus can adapt to mammals by point mutations in PB2 or HA, which consequently poses a potential threat to public health.
Due to their present low pathogenicity, H4 viruses would seem a pretty poor pandemic candidate. But then, the same could have been said about H7N9 seven years ago.   

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