The development of neuraminidase inhibitor resistance in avian influenza: genetic determinants, fitness cost and zoonotic transmission.

Influenza A viruses bind sialic acid on the cell surface allowing entry. After genome replication, new virions exit through host cell membranes. Neuraminidase (NA) is present as an envelope surface glycoprotein and facilitates the final release and spread of infectious particles by cleaving sialic acid. There are 12 known NA subtypes. Neuraminidase inhibitors (NAI), such as Tamiflu are important antivirals, where the NAIs compete for binding with sialic acid in the active site of NA preventing viral replication. These drugs are utilised for humans prophylactically and for treatment upon influenza infection, both during seasonal epidemics and in pandemic scenarios.
The exact amino acid mutations that confer resistance to NAIs differ between NA subtypes and the drug to which the resistance is directed. Most of the functional resistance mapping has been undertaken in NAs of human influenza strains, the N1 and N2 subtypes, whilst only a small amount of information has been reported about the requirements in avian NA subtypes. Since the three major human influenza pandemics of the 20th Century were caused by influenza strains where the NA gene originated from an avian source and there continues to be frequent zoonotic infections from avian influenza viruses of many different subtypes, it seems critical to understand whether known NAI associated motifs confer resistance to avian NA subtypes and whether other unknown motifs for resistance exist that should be part of avian surveillance programmes. We will therefore introduce specific mutations in the NA of avian influenza viruses that threaten human health and address whether they confer a resistant profile to the current NAI drugs and those in development. We will also attempt to identify if any novel mutations in NA can cause resistance in avian subtypes.
In the mid-1990s the unregulated use of the influenza anti-viral Amantadine, which acts on the M2 protein of influenza, in poultry resulted in widespread resistance in avian influenza globally. This reduced the arsenal available to treat zoonotic influenza infection in humans to only the NAIs. Under continued threat from circulating avian influenza viruses the same unregulated use in poultry is likely with NAI drugs unless the regulation is more strongly enforced this time around. What virologists are unclear about is whether avian influenza viruses that are resistant to NAI drugs would be competitive in the poultry host and be stably maintained upon transmission. These are question we will endeavour to answer in this proposal by introducing the most prevalent NAI resistance mutations in to important avian influenza NA subtypes and measuring in chickens their ability to infect and transmit the viruses.
Of major concern is the potential for the next human influenza pandemic to harbour or rapidly acquire resistance to NAIs. The scenarios by which this could manifest are firstly by the direct introduction to the human population of an avian strain that already carries NAI resistance. Secondly, the cross-species introduction of an avian strain in to a new host environment (humans or swine from poultry) may result in the unbalancing of sialic acid binding and cleavage activity of the virus surface proteins due to a change in the target receptors and mucus environment. An unbalanced activity in the face of enthusiastic NAI use for treatment and prophylaxis, may result in NAI resistance motifs being better supported and more likely to develop. Therefore in this programme of work we will infect biological tissue substrates that mimic avian, swine and human hosts with avian influenza strains and apply the NAI drug Oseltamivir. Using next generation sequencing we can determine the rate of NAI resistance development in the NAs from important avian influenza strains such as H9N2 and H7N9 and the effect that the different host environments have on the likelihood that resistance will develop.

Frequently we are observing the establishment of novel avian influenza infection in poultry which threatens the poultry industry globally and is of significant concern with regards to human health. This current situation demands that influenza virologist's answer fundamental questions of influenza biology in the context of avian isolates which until now have been under-studied in contrast to human isolates. This proposal will assess the genetic determinants of NAI drug resistance in avian NA subtypes and identify novel signatures conferring resistance. Using reverse genetics we will generate recombinant viruses that carry matched avian HA and NA genes of various subtypes on a vaccine strain backbone. By mutating known residues in the NA gene we will assess whether resistance to NAI drugs is conferred by measuring the NA activity inhibition concentration. We will also assess if avian NAs can escape inhibition by NAIs in novel ways by passaging the recombinant viruses in the presence of NAI drug and sequencing the NA and HA viral segments of the resultant virus pools. Our second objective will be to determine any fitness advantage of recombinant influenza viruses, of H7N9 and H9N2 subtypes, bearing resistance mutations in chicken hosts. Viral shedding and clinical symptoms will be assessed alongside transmission to naïve birds. Mathematical modelling on the in vivo data will determine if there is a fitness cost/advantage to carrying NAI resistance within host or during transmission. Finally we will use ex vivo tissue substrates from chickens, swine and humans to passage chicken adapted H7N9 and H9N2 viruses in the presence of Oseltamivir and analyse the viral pools by next generation sequencing. Bioinformatics interrogation of the sequence data will allow the rates of genetic change in the different hosts to be measured and give us an understanding of whether the process of cross species transmission is actually a risk factor in the development of NAI resistance.

The global poultry industry has under gone a dramatic expansion in the last two decades and continues to grow steadily as the demand for cheap and healthy protein sources to sustain the global population also increases. The FAO suggests that poultry accounts for 88% of the world meat production. Despite significant research regarding the development of resistance to antivirals in human influenza isolates little work has been undertaken to characterise functionality and fitness cost of these motifs in avian subtypes, particularly in avian hosts. Frequent outbreaks in farmed poultry of novel avian influenza strains causes' economic burden to farmers and increases the exposure risk of human to possible zoonotic strains. This programme of work will contribute to our knowledge of influenza infection by assessing the likelihood of antiviral resistance development in different host species and by identifying resistance motifs that may alter the fitness of avian influenza viruses. The outcomes from this programme of work will be of great interest to academics in many different disciplines associated with infectious disease. However these outcomes could also benefit the poultry industry at large by providing risk assessment data of viruses circulating in local populations and the risk of increased virulence being developed. It will also provide scientific data to strengthen arguments about guarding against the use of human anti-viral drug in farmed animal populations. This project will identify which avian virus strains are most at risk of developing NAI resistance without an attenuation of viral fitness and thus the data produced will feed into the 'One medicine agenda' linking animal disease and human zoonotic disease. Information generated on the functionality of know NAI resistance motifs in avian subtypes and novel mutations that confer reduced sensitivity will be important for the multiple influenza surveillance networks such as those run by the OIE/FAO (OFFLU) and WHO (Global Influenza Surveillance and Response System (GISRS) and European Influenza Surveillance Network (EISN)) as well as national animal and human health programmes based at the Animal Health and Veterinary Laboratories Agency (AHVLA) and Public Health England (PHE). Data from this programme of work about the risk of different avian influenza subtypes to develop functional resistance to NAIs can be made available for those involved in determining national and international health policy.