Sunday, September 20, 2020

Viruses: PA From Recent H9N2 AIV Enhances H5N1 Infection In Mammalian Systems


In March of 2013 - just two weeks before we learned of the first H7N9 outbreak in humans in China - a study appearing in the EID Journal: Predicting Hotspots for Influenza Virus Reassortment, found  that both Eastern China and the Nile River Valley of Egypt were at the top of the list of areas likely to spawn the next novel flu virus.

EID Influenza Hotspots For Reassortment - 2013

China had spawned H5N1 in the mid-1990s, followed by a long  line of descendants, and within two years would launch not only H7N9, but also H10N8, H5N8, and H5N6.  Of those, only H5N8 has not infected humans.

Egypt, meanwhile, was becoming a hotbed of H5N1 activity and evolution, and starting in late 2014 - and lasting for about 6 months - became the scene of the large human outbreak of H5N1 on record (see EID Dispatch: Increased Number Of Human H5N1 Infection – Egypt, 2014-15). 

Both countries lie beneath major migratory flyways, both are major poultry producers (with many backyard flocks), and both have numerous LPAI and HPAI subtypes circulating in local birds. 

While China has gained better control of avian flu the past few years following their roll out of a new experimental H5+H7 poultry vaccine in 2017, Egypt continues to struggle with spotty poultry vaccination campaigns that often rely on older, mismatched vaccines (see Egypt: A Paltry Poultry Vaccine)which can potentially drive vaccine escape mutations.

As a result, multiple clades of HPAI H5 viruses (H5N1 and H5N8) - along with H9N2 - have all become endemic in Egyptian poultry.

As we've discussed often  (see The Lancet: H9N2’s Role In Evolution Of Novel Avian Influenzas), the avian H9N2 virus is a highly promiscuous virus, reassorting with many other subtypes easily.  In addition, we've seen the H9N2 continue to evolve towards a more `humanized' virus (see Virology: Receptor Binding Specificity Of H9N2 Avian Influenza Viruses).

While H9N2 has circulated in the Middle East for more than two decades, it was only first detected in Egyptian poultry in 2010 (cite), prompting concerns over how it might interact with HPAI H5N1.
The H5N1 virus already carries some internal genes from the H9N2 virus, and so the concern is another exchange - of more recent, potentially more `mammalian adapted'  H9N2 genes - could happen again with uncertain results. 

In early 2019, in J. Virology:Genetic Compatibility of Reassortants Between Avian H5N1 & H9N2 Influenza Viruses, we looked at the results of experimental reassortments of Egyptian H5N1 and H9N2 viruses in the laboratory, which warned:
In conclusion, our analyses indicated a substantial emergence potential of influenza virus reassortants derived from the H5N1 and H9N2 viruses currently cocirculating in Egypt, as well as the possibility of their high public health risk for humans relative to the parental H5N1 and H9N2 viruses. Cocirculation of the two influenza virus subtypes in birds may accelerate the emergence of novel viruses that may be a public health risk.
Last December, in EID Journal: Novel Reassortant HPAI A(H5N2) Virus in Broiler Chickens, Egyptwe saw a new proof of concept in the field, after an HPAI H5N8 virus reassorted with an LPAI H9N2 virus, and produced a new HPAI H5N2 virus in Egyptian poultry.

A reminder that while avian flu activity has been subdued globally for the past 3 years, the threat has not gone away.  

While a far less virulent virus than HPAI H5N1, LPAI H9N2 raises pandemic concerns because it continues to evolve towards a more mammalian-adapted virus. Add in its legendary ability to reassort with other influenza subtypes, and it becomes a virus to watch.

Which brings us to a new study that experimentally reassorts a recent LPAI H9N2 virus - carrying a mutated PA segment (Lysine 367) - with a recently circulating Egyptian H5N1 virus. 

Not only were these two viruses genetically compatible, their reassortant offspring showed enhanced replication in Madin-Darby canine kidney (MDCK-II) cells at 33 °C and 39 °C,  increasing their fitness to replicate in human hosts. 

While this reassortant combination has not been observed in the wild, with both viruses circulating in Egyptian poultry, there are legitimate concerns that they could someday conspire to spawn a more dangerous (to humans) H5Nx virus. 

I've only included the link, the Abstract, and an excerpt from the study's conclusion. Follow the link to read the paper in its entirety. 

by Ahmed Mostafa 1,2,*,,Sara H. Mahmoud 1,†,Mahmoud Shehata ,Christin Müller ,Ahmed Kandeil ,Rabeh El-Shesheny 1,3,Hanaa Z. Nooh 4,Ghazi Kayali 5,6,Mohamed A. Ali 1,* and Sephan Pleschka 2,*

Viruses 202012(9), 1046; (registering DOI)
Received: 26 August 2020 / Revised: 15 September 2020 / Accepted: 18 September 2020 / Published: 20 September 2020

Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet.
Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1EGY and H9N2EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2EGY, expressed individually and in combination in the genetic background of H5N1EGY, were genetically compatible with the other H5N1EGY segments.
At 37 °C the replication efficiencies of H5N1EGY reassortants expressing the H9N2EGY polymerase subunits PB2 and PA (H5N1PB2-H9N2EGY, H5N1PA-H9N2EGY) were higher than the wild-type H5N1EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1PB2-H9N2EGY, but reduced for H5N1PA-H9N2EGY. At 33 °C and 39 °C, H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY replicated to higher levels than the wild-type H5N1EGY in human Calu-3 and A549 cell lines.
Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1EGY. Genetic analysis of the polymerase-encoding segments revealed that the PAH9N2EGY and PB2H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1EGY enabled the mutant virus H5N1PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1PA-R367K caused higher mortality in BALB/c mice.
These findings demonstrate that H5N1 (Clade reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.


Taken together, our results demonstrate that the co-circulation of H5N1-subtype HPAIV and H9N2-subtype LPAIV in Egyptian poultry may result in co-infection and viral reassortment, thus H5N1 reassortants with an increased zoonotic potential may emerge and pose a public health risk. Specifically the lysine at position 367 of the PA seems to have a strong impact.
The data presented here question the role of genetic compatibility or viral replication fitness of H5N1/H9N2 reassortants to the low or rare detection of natural genetic reassortment events among HPAI H5N1 and LPAI H9N2 viruses in Egypt. Additionally, the ability of H9N2 strains to potentiate other AIVs may be attributed to the fact that H9N2 viruses accommodate for several human adaptation markers in their genome, which may enable the reassortant AIVs to cross the species barrier.

Even as we struggle to deal with a roughly 1% CFR (Case Fatality Rate) COVID-19 pandemic, there are other threats in the wild that could prove far more challenging.  Whether its avian influenza, a new swine-origin novel flu, or perhaps another exotic bat-borne virus, the specter of another pandemic is always on the horizon. 

We pretend it isn't at our own risk.