Posted: 28 April, 2017 Experiments by researchers at The Pirbright Institute have identified the molecular mechanisms that enable H9N2 viruses - the most common type of avian influenza (bird flu) virus - to infect humans, helping improve risk assessments for its potential to cause pandemic. Although H9N2 viruses are considered less pathogenic than some types of avian influenza virus, they still cause significant losses for the poultry industry in many countries throughout Asia, the Middle East and North Africa - sometimes with death rates as high as 60%. Over the past 20 years there have also been a growing number of (generally mild) human infections of H9N2 in Hong Kong, mainland China, Bangladesh and Egypt, especially amongst poultry workers. Concerns increased however when other experiments demonstrated the potential for human-to-human airborne transmission; a property normally associated with the potential to cause pandemic. Despite the widespread global distribution and diversity of H9N2 viruses and their potential threat to human health, they have not been extensively characterised until now. In new research undertaken by Pirbright scientists and published in the science journal Nature's Emerging Microbes & Infections, researchers analysed the specific properties that enable some lineages of H9N2 to adapt for successful human infection. Led by Dr Munir Iqbal, head of the avian influenza virus group at Pirbright, researchers specifically focussed on the surface protein haemagglutinin, which enables the virus to bind and fuse with host cells for entry and infection. In particular scientists investigated two properties that facilitate adaption for human infection: its preference for binding to different host receptors (molecules on the surface of a cell responsible for communication) that allow cell entry and the pH level at which the virus can fuse with the host cells (pH of fusion) and therefore begin infection. Dr Iqbal's team aimed to explore the potential relevance of these biomarkers for zoonotic (disease capable of spreading from animals to humans) risk assessments. They characterised which host cell receptors the H9N2 haemagglutinin prefers to bind to, which is important as human and bird receptors are slightly different, which usually means that bird flu strains bind to 'bird-like' receptors and human strains bind to 'human-like' receptors. Bird flu strains can infect humans when a mutation occurs that enables a preference for binding receptors that are 'human-like'. This has been partially or entirely attributed to a single amino-acid change in the haemagglutinin. The team also assessed how the stability of H9N2 haemagglutinins affects the pH of fusion. In order to infect humans, the haemagglutinin must be stable enough to survive in respiratory droplets for airborne transmission and in the mammalian nasal tract, which is mildly acidic (less than a pH of 7). Haemaggluttinins of human flu strains have adapted to be stable and fuse at an acidic pH that is typically less than 5.5, whereas avian flu strain haemagglutinins are generally more stable above this level and are therefore unable to fuse in the acidic conditions of the human nasal tract. The H9N2 strains being investigated were found to possess haemagglutinins which were stable at lower pH levels, something which was mirrored in other bird flu strains which have adapted to infect humans in the past. It was established that the stability at a lower pH was a more important factor for virus fusion than the preference for binding to different receptors. Dr Iqbal said: "Based on the two properties we tested, our results indicate that the lineages with the highest zoonotic potential may be those currently circulating in southern China and Vietnam (G1 'Eastern' sub-lineage). However, evaluations in this study of the lineages prevalent in China and Vietnam (BJ94) and from Bangladesh to Morocco ('Western' G1 sub-lineage), suggest these viruses could also adapt to humans with relatively few additional mutations and merit further research. "This study has provided us with some important new insights which are helping us develop our understanding of these influenza viruses from molecular, biophysical and virological perspectives. We hope this will inform risk assessments of their zoonotic and pandemic potential and help improve global vaccine strategies". This research was funded by The Pirbright Institute studentship grant, Biotechnology and Biological Sciences Research Council (BBSRC) ZELS grant and the BBSRC Avian Disease Programme grant. Work at the World Health Organization (WHO) Collaborating Centre in London was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK, the Medical Research Council and the Wellcome Trust.

A new study by a group including Dr Munir Iqbal, from The Pirbright Institute, has identified a previously unknown factor which works to limit the host range of influenza viruses.

The research, recently published in Nature, has revealed that the avian protein ANP32A works as a species barrier. During replication, the avian influenza strains use an enzyme called RNA polymerase, which is supported by ANP32A. However, because mammals do not possess ANP32A, avian influenza viruses are not able to replicate as efficiently, and therefore their virulence is diminished in mammals.

The study found that the mutation E627K in PB2 - one of the RNA polymerase proteins, enables mammalian proteins similar to ANP32A to support the avian virus RNA polymerase, thereby increasing the virulence of avian viruses in mammals.

The incidence of influenza pandemics can be unpredictable when zoonotic influenza viruses with new mutations acquire the ability to transmit amongst humans. Host range is limited by incompatibilities between elements of the avian virus and the human host. These barriers include receptor preference, virion stability and poor activity of the avian virus RNA polymerase in human cells.

Dr Iqbal, Head of the Institute’s Avian Influenza Virus group in the Avian Viral Diseases Programme, said: “In this study we have shown that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells.

“In mammalian cells, avian ANP32A restored the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A prevented this activity, but its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function.

“We can therefore conclude that ANP32A represents an essential host partner, elected to enable influenza virus replication and is a candidate host target for novel antivirals”, he said.

 A new study of a strain of avian influenza virus, which can pose a risk to both poultry and human health, will help guide surveillance efforts and ensure vaccines are more effective; helping prevent serious outbreaks.

The H9N2 virus leads to significant losses in poultry production across Asia, but also has the potential for creating new viruses which can infect humans; with infections reported across China, Bangladesh and Egypt. It can also act as a donor of genes to other zoonotic avian influenza viruses such as the 1997 Hong Kong H5N1 outbreak, and the recent Chinese H7N9 and H10N8 outbreaks.

The use of vaccines is widespread, but because influenza viruses are constantly changing, the effectiveness of vaccines is often compromised.

The research was led by Dr Munir Iqbal, Head of the Institute’s Avian Influenza Virus group in the Avian Viral Diseases Programme, and was recently published in Scientific Reports. His team sought to gain a better understanding of the antigenic sites on the influenza virus major antigen: haemagglutinin.

Haemagglutinin is a glycoprotein on the surface of all influenza viruses which enables the virus to enter host cells. There are at least 18 different types of haemagglutinin (H1-H18) that influenza viruses may possess.

Dr Iqbal said, “During circulation in birds, the virus acquires genetic changes in the haemagglutinin gene that greatly influence its antigenic properties; resulting in viruses with the ability to escape natural or vaccine-induced immunity. This means that vaccines can fail in the field and lead to viruses circulating and spreading unhindered in vaccinated animals.

“To increase the effectiveness of vaccines we needed to understand more about the molecular factors that allow these viruses to escape from vaccine-induced immunity, as well as a better awareness of how a more potent, cross-protective immune response may be induced.”

The analysis and structural mapping revealed two novel, antigenic sites “H9-A” and “H9-B”. Additionally, a second subset of escape mutants contained amino acid deletions within the haemagglutinin receptor binding site. This constitutes a novel method of escape for this group of haemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity.

Dr Iqbal added: “Recently it has been suggested that the most effective method of preventing new zoonotic avian influenza subtypes from entering the human population would be better control of these viruses in poultry.

“This research gives us a better understanding of the basis of antigenicity of these viruses, and will enable more accurate vaccine matching with circulating field strains, with veterinary or human pandemic potential. It will also help virus surveillance efforts determine that antigenic variants are emerging or prevalent in a population”, he said.