Avian Flu Diary: Reassortment After Co-infection Of Chickens With H4N6 and H9N2 influenza Viruses

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Until 20 years ago, avian influenza viruses were thought to pose only a very limited threat to human health, and the few novel human infections that had been detected, had generally been mild.

That perception abruptly changed in 1997 when 18 people contracted H5N1 in Hong Kong, and six of them died.

After a remarkable poultry eradication program across all of Hong Kong, the H5N1 virus went silent - at least until 2003 - when 2 members of a family turned up in Hong Kong with the virus after visiting Fujian Province, China (see WHO H5N1 timeline).

A third family member had died on the Mainland, but no tests were taken.

By the end of 2003, H5N1 would turn up in South Korean Poultry, and infected chicken carcasses in Thailand would lead to the infection and subsequent deaths of rare captive leopards and tigers. In January of 2004, H5N1 would turn up in Vietnam, before beginning it's world tour.

H5N1 wasn't the only - or even the first - avian influenza virus to jump to humans, although due to its high morbidity and mortality in humans, it was viewed as being in a league of its own.

By 2003, avian H7 viruses had more than a 40-year documented history of infecting humans, albeit mostly causing minor respiratory illness and/or conjunctivitis (see 2009 EID Journal Article Past, Present, and Possible Future Human Infection with Influenza Virus A Subtype H7).

While lagging behind H5N1, in 2003 human infections with avian H7 viruses began to pick up their pace (see 2003-2007 chart below), and we were seeing warnings that H7 viruses might be adapting to human physiology (see H7's Coming Out Party)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727350/

Less than 4 years later (spring 2013), a highly lethal H7N9 virus would emerge in China - the product of a series of reassortments between H7 avian viruses and avian H9N2 - that would, in relatively sort order, knock avian H5N1 from the top perch of our pandemic threats list (see CDC's IRAT).

Suddenly, H7 viruses were viewed as potentially just as dangerous as H5 viruses, although only the Chinese H7N9 virus had shown such abilities (Note: 1 case of human infection with avian H7N4 was reported in 2018).

A few months later, 3 cases of H10N8 - two of which proved fatal - appeared in China. In June of 2014, Eurosurveillance carried a report that showed the H10N8 virus – like so many of the other emerging avian subtypes in China – also carried the internal genes of the H9N2 virus.

As we've discussed previously (see here, here, and here), the common denominator between nearly all of the avian viruses (including H5N1, H5N6, H10N8 and LPAI H7N9) that have emerged from China in recent years is that their internal genes can be traced back to the highly promiscuous LPAI H9N2 virus.

Which brings us to a new report - published in Veterinary Microbiology - that shows that H9N2 easily reassorts with avian H4N6 in co-infected chickens, and that some of these reassortants show signs of increased virulence.

Adding some gravitas - one of the authors is Chen Hualan - director of China's National Avian Influenza Reference Laboratory. First a look at the abstract (the full article is behind a pay wall), then I'll return with more on the H4N6 virus.

High frequency of reassortment after co-infection of chickens with the H4N6 and H9N2 influenza A viruses and the biological characteristics of the reassortants
Xuyong Li a, , , Baotao Liu c, Shujie Ma b, Pengfei Cui b, Wenqiang Liu a, Yubao Li a, Jing Guo a, , , Hualan Chen b,

https://doi.org/10.1016/j.vetmic.2018.06.011

Highlights

High frequency of reassortment was detected after co-infection of chickens with the H4N6 and H9N2 avian influenza A viruses.

Nine reassortants were purified successfully from the co-infection samples and their biological properties were evaluated. The H4N6 wt originated PA gene plays important role in the enhanced polymerase activity of RNP complex and the increased replication and virulence of the reassortants in vitro and vivo.

Reassortment during co-infection with different subtype influenza viruses in chickens is most likely a major feature and plays an important role in the evolution and mutation of the influenza A virus.

Abstract

H4 and H9 avian influenza viruses (AIVs) are two of the most prevalent influenza viruses worldwide. The co-existence of H4 and H9 viruses in multiple avian species provides an opportunity for the generation of novel reassortants and for viral evolution. The diversity of the biological characteristics of the reassortants enhances the potential threat to the poultry industry and to public health.

To evaluate the reassortment of these viruses and the potential public risk of the reassortants, we co-infected chickens with H4N6 and H9N2 viruses derived from poultry and tested the replication and virulence of the reassortant viruses in mice. A high frequency of reassortment was detected in chickens after co-infection with these two viruses and nine reassortants of six genotypes were purified from the chicken samples.

Two H9N2 reassortants containing the PA of the parent H4N6 virus showed higher virulence than the parent H9N2 virus, revealing the significant role of the H4N6 wt virus PA gene in viral reassortment.

Analysis of the polymerase activity of the ribonucleoprotein (RNP) complex in vitro suggested that the PA of H4N6 wt origin enhanced polymerase activity.

Our results indicate that co-infection of an avian individual with the H4N6 and H9N2 viruses leads to a high frequency of reassortment and generates some reassortants that have higher virulence than the wild-type viruses in mammals.

These results highlight the potential public risk of the avian influenza reassortants and the importance of surveillance of the co-existence of the H4N6 and H9N2 viruses in avian species and other animals.

(Continue . . . )

H4N6 hasn't been completely absent from our radar.

Two years after it was detected in Canadian swine in 1999, the USDA published: Pathogenesis of H4N6 Influenza A Virus Infection in Pigs and Turkeys: A Newly Emerging Disease Concern.

In addition to wild birds, poultry, and pigs - H4N6 has also been detected in marine mammals and raccoons. While surveillance around the world is sorely lacking, in recent years we've seen reports of avian H4 viruses detected in Chinese pigs (see Epidemic Status of Swine Influenza Virus in China).

Over the past couple of years, new research has come out suggesting that avian H4 viruses may pose a greater threat to human health than previously believed.

Less than a year ago, 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.

While last August, in Virology: Detection & Characterization Of Avian H4N6 In Midwestern Swine (2015) we looked at the first discovery (in 2015) of avian H4N6 in swine from America's Midwest.

None of this means that H4N6 is on the verge of becoming a pandemic threat, only that our list of novel flu subtypes that could pose a human health threat continues to evolve and expand.

From 1997 to 2012, it was all HPAI H5N1.

But over the past 6 years we've seen it joined by H7N9, H10N8 and H5N6 - along with several other `lesser' viruses (i.e. H6N1, H7N4, H7N2, H7N3, H10N7, etc.) - all demonstrating at least some degree of zoonotic ability.

A pattern that means we shouldn't be terribly surprised if the next big threat comes from out of left field, via a virus we've rarely, if ever, viewed as a serious pandemic risk.