Wednesday, April 02, 2014

Study: Sequence & Phylogenetic Analysis Of Emerging H9N2 influenza Viruses In China


Poultry Vaccination - Photo Credit OIE


# 8425


Among the avian flu viruses we watch with concern, the H5N1 and H7N9 viruses have gained the most notoriety.  Yet, behind each of these rising stars is an unindicted co-conspirator – the ubiquitous, but little noticed H9N2 avian virus – which has lent crucial internal genes to both of these emerging threats.

We looked at these contributions six weeks ago in The Lancet: H9N2’s Role In Evolution Of Novel Avian Influenzas, but briefly - H5N1, H7N9, and the recently observed H10N8 – all share several important features:

    • They all first appear to emanate from Mainland China
    • They all appear to 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

Since H9N2 rarely causes serious illness in humans, and as it is already pretty much widespread in Asian poultry, it tends to fly below the radar.  Unlike H5 and H7 avian viruses, it isn’t considered enough of a threat by the OIE to be reportable, and vaccination – not culling – is the standard practice to prevent its spread.

Yet, some researchers view it with concern.


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.


All of which brings us to a new study, which appears in the journal Virus Genes, that takes a look at H9N2 viruses collected from two regions in China (Zhejiang & Guangdong Provinces) between October & December 2011,  where poultry vaccination is practiced (Note: an inactivated H9N2 vaccine was introduced in China in 1998).


What they found was an array of viruses that have evolved antigenically away from the vaccine strain, with a majority of isolates (14 out of 18) showing an amino acid change in the receptor binding site suggestive of an enhanced ability to bind to human receptor cells.


H9N2 influenza viruses isolated from poultry in two geographical regions of China

Yu Xue, Jing-Lan Wang, Zhuan-Qiang Yan, Guang-Wei Li, Shun-Yan Chen, Xiang-Bin Zhang, Jian-Ping Qin, Hai-Yan Li, Shuang Chang, Feng Chen, Ying-Zuo Bee, Qing-Mei Xie


Subtype H9N2 avian influenza viruses (AIVs) circulating in China have aroused increasing concerns for their impact on poultry and risk to public health. The present study was an attempt to elucidate the phylogenetic relationship of H9N2 AIVs in two geographically distinct regions of China where vaccination is routinely practiced.

A total of 18 emerging H9N2 isolates were identified and genetically characterized. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that the isolates belonged to the Y280 lineage. Based on the HA genes, the isolates were subdivided into two subgroups. The viruses from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II.

Antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the receptor-binding site, which indicated that the virus had potential affinity of binding to human like receptor.

These results suggest that the emerging viruses have potential risk to public health than previously thought. Therefore, continuous surveillance studies of H9N2 influenza virus are very important to the prognosis and control of future influenza pandemics.

Yu Xue, Jing-Lan Wang, and Zhuan-Qiang Yan have contributed equally to this study.

One of the concerns with long-term vaccination schemes, such as China has employed over the past 16 years, is that the widespread use of vaccines can sometimes drive the development of new viral strains. And a failure to continually update the vaccines being used to match these new strains can further exacerbate the problem, as a poorly matched vaccine may mask symptoms without actually preventing infection.


Last year, in the Journal Clinical and Experimental Vaccine Research, Dong-Hun Lee and Chang-Seon Song penned a study called H9N2 avian influenza virus in Korea: evolution and vaccination, where they wrote:


Compared to human influenza virus, the antigenicity of AIV is relatively stable, which may be due to the lack of immune pressure. However, as large-scale and long-term vaccinations against AIV have been performed in several countries, AIVs have also undergone antigenic drift due to the presence of immune pressure [48].


Concluding Remarks

Wide use of AIV vaccine in animal population could enhance the immune pressure and drive the mutation resulting in rapid antigenic drift at the antigenic sites [52]. Therefore, improved vaccination strategies and periodic updates of vaccine seed strains are required to increase immunogenicity and cross protective efficacy in chicken farms. These strategies could include: the selection of highly immunogenic vaccine seed strains, the use of effective adjuvants for chickens, and the use of new technology vaccines. Several studies reported that the recent Korean LPAI H9N2 virus underwent antigenic drift and could escape from vaccine protection.

Thus, continued active surveillance of poultry farms and LBMs to reveal new variant LPAI H9N2 viruses in Korea and analyzing appropriate vaccine seed viruses should be considered to prevent new outbreaks.


The use of vaccines to control avian viruses in poultry is not without controversy.  The OIE has repeatedly warned that vaccines are a short-term solution to avian flu problem, and that countries should have an `exit strategy’ away from vaccines.  This from the H7N9 FAQ issued last year:


Does OIE recommend vaccination of animals to control the disease?

When appropriate vaccines are available, vaccination aims to protect the susceptible bird populations from potential infection. Vaccination reduces viral excretions by animals and the virus’ capacity to spread. Vaccination strategies can effectively be used as an emergency effort in the face of an outbreak or as a routine measure in an endemic area. Any decision to use vaccination must include an exit strategy, i.e. conditions to be met to stop vaccination.

Careful consideration must be given prior to implementing a vaccination policy and requires that the recommendations from the World Organisation for Animal Health (OIE) on vaccination and vaccines are closely followed ( INFLUENZA/Guidelines on AI vaccination.pdf).

In short, vaccination should be implemented when culling policies cannot be applied either because the disease is endemic and therefore widely present, or the infection in affected animals is too difficult to detect.


Unfortunately, after 16 years of use, there are no signs that an exit strategy is likely to be proffered by the Chinese anytime soon.


A legitimate concern due to growing evidence that the use of (particularly mismatched)  vaccines can drive potentially dangerous evolutionary changes in the very viruses they are designed to prevent.


All of which means that while we watch H7N9, H5N1, or H10N8 for signs of pandemic potential, we can’t afford to ignore the evolving threat posed by the H9N2 virus – which either directly or indirectly – could play a substantial role in generating the next pandemic virus.