Molecular epidemiology of H9N2 avian influenza viruses in Pakistan

Lead Research Organisation: Royal Veterinary College
Department Name: Graduate School


Avian influenza viruses (AIV) of the H9N2 subtype are causing devastating losses in poultry in many countries throughout Asia, the Middle East and North Africa including Pakistan. Recent evidence suggests that H9N2 viruses have a particularly high tendency for donating internal genes by reassortment to other co-circulating AIV, and these internal genes may contain molecular markers associated with mammalian transmissibility, thus posing a risk for pandemic emergence of novel human-transmissible genotypes. To reduce the impact of H9N2 in poultry, several countries employ vaccination, however the efficacy of these programs is compromised by continued rapid adaptive evolution of the virus, and the emergence of variants that are not covered by current vaccine formulations.

This project will investigate the epidemiology of H9N2 in Pakistan, a country that has experienced severe outbreaks of H9N2 avian influenza in poultry. The aims include (i) characterising diversity of H9 viruses among poultry productions systems at varying levels of industrialisation; (ii) examining the role of live bird markets in H9 transmission; and (iii) evaluating the extent of antigenic diversity and implications for vaccine effectiveness. The project will involve surveillance activities in Pakistan; field trials of novel diagnostics tools; and phylogenetic analysis of H9 sequences. The project will be conducted within the larger framework of a ZELS project to develop novel avian influenza vaccines, with the goal of understanding how vaccine intervention strategies impact viral diversity and industrialisation of poultry production. There will be opportunities for comparative analysis of AIV and H9 epidemiology across different countries.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/N503563/1 01/10/2015 30/09/2019
1811252 Studentship BB/N503563/1 05/10/2015 04/04/2019 Joshua Sealy
Description Through characterizing the antigenic diversity of H9N2 avian influenza viruses form Pakistan we have identified a subset of circulating viruses with antigenic diversity compared to previous H9N2 strains from Pakistan. The immediate implication is the potential for vaccine failure if current vaccines are poorly matched to the diverse H9N2 strains infecting poultry in Pakistan. Further, the causative mutation for this antigenic divergence is in the haeamagglutinin glycoprotein with the added effect of facilitating receptor binding to mammalian cellular receptors. The implications are therefore two-fold; there are circulating H9N2 avian influenza viruses in Pakistan which can escape vaccine-induced immunity and they also have enhanced zoonotic potential. This information can be used in future vaccine design and can provide impetus to active surveillance efforts. This work has now been published in the journal Emerging Infectious Diseases.

A field study in Vietnamese live bird markets has also been completed and a manuscript submitted for review in a peer reviewed journal. In this study, surveillance of avian influenza viruses was conducted in poultry in live bird markets and viruses were isolated and sequenced. In this study we determine the prevalence of avian influenza infection in chickens and use the prevalence data to identify risk factors in poultry trading practices of poultry traders.
Exploitation Route Regarding the antigenic diversity of circulating strains of H9N2 in Pakistan, vaccines that are cross-protective can be developed.

A targeted cost-effective surveillance of avian influenza viruses can be conducted in the future in endemic regions.
Sectors Agriculture, Food and Drink,Communities and Social Services/Policy,Healthcare,Pharmaceuticals and Medical Biotechnology

Description PhD training funds
Amount £500 (GBP)
Organisation The Pirbright Institute 
Sector Academic/University
Country United Kingdom
Start 05/2018 
End 05/2018
Description Society Conference Grant
Amount £300 (GBP)
Organisation Microbiology Society 
Sector Learned Society
Country Unknown
Start 04/2017 
End 04/2017
Title Sequencing data generated for public access 
Description H9N2 avian influenza viruses were isolated from poultry in Pakistan, and H9N2 and H5N6 avian influenza viruses were isolated from poultry in Vietnam. Viruses were sequenced and sequencing data submitted to a public access database (NCBI). 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Analysis of sequencing data was followed by publication in a peer-reviewed journal. 
Description Collaboration with University of Veterinary and Animals Sciences, Lahore, Pakistan for poultry surveillance against H9N2 aviain influenza. 
Organisation University of Veterinary & Animal Sciences
Country Pakistan 
Sector Academic/University 
PI Contribution Provide reagents for the PCR amplification of viral RNA extracted from isolated H9N2 avian influenza viruses from poultry farms in Pakistan. This PCR work was done by myself during a trip to UVAS, Pakistan. The samples being amplified were generated from surveillance activities carried out by staff at UVAS between 2014 and 2016.
Collaborator Contribution Surveillance of poultry farms in Pakistan was carried out against avian influenza viruses infecting poultry. This surveillance was carried out by staff and students enrolled at UVAS supervised by one of the co-supervisors of this project. This work was conducted between 2014 and 2016 and involved swabbing poultry, screening samples and isolating viruses for subsequent PCR amplification by myself. Further characterization of Pakistan H9N2 viruses was conducted at Pirbright by myself.
Impact A manuscript has been written that details the surveillance activity, sequencing and characterization of viruses. This manuscript is in review by the authors prior to submission
Start Year 2014
Description Field research in northern Vietnam, partnership with Oxford University Clinical Research Unit, Hanoi and National Centre for Veterinary Diagnosis, Hanoi 
Organisation University of Oxford
Department Oxford University Clinical Research Unit Vietnam (OUCRU)
Country Viet Nam 
Sector Academic/University 
PI Contribution I conducted field studies on avian influenza in live bird markets in northern Vietnam for six months. During this time I generated data on prevalence of influenza among chickens in northern Vietnam, and will aim to generate risk assessment of poultry trade behaviors for avian influenza infection in chickens. This contributes to diseases estimates in northern Vietnam and will contribute to understanding poultry trade behaviors which may enhance infection rate of chickens. Samples containing influenza viruses are now stored at NCVD for use in further virus characterization if required.
Collaborator Contribution The two partnering institutes hosted me in their laboratories and offices, supplying me with space to work and facilities to receive research consumables which were paid for by our common budget. They helped with getting the government-level approval and permissions to conduct the studies and provided supervision when necessary.
Impact Although the influenza prevalence data has been generated, further analysis of questionnaires linked to trade behaviors and influenza infection needs to be carried out. Sequencing data for virus isolates needs to be generated. Once the analysis is complete and written it will be communicated back to the partnering institutes. The potential impact for this work is economic and policy & public services by facilitating more targeted influenza surveillance activities in the future and understanding trade behaviors that enhance risk of chicken infection.
Start Year 2017
Description Next generation sequencing at the Crick Worldwide Influenza Centre 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provided PCR products to Crick-WIC for next generation sequencing
Collaborator Contribution Crick-WIC conducted NGS on influenza PCR products which were generated from surveillance activities in poultry farms in Pakistan. The raw data was assembled and analysed at Pirbright by myself.
Impact A manuscript has been written that details the surveillance, sequencing and characterization of H9N2 avian influenza viruses from Pakistan and is in review by the authors prior to submission.
Start Year 2015
Description Flu fighters 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Greater than 200 people attended the Guildford Live facility that hosted an exhibition on a wide variety of companies, technologies and entertainment. The Pirbright institute maintained a stall at this event and I represented the institute. This involved public engagement on research conducted at the institute, including influenza research and research into other animal viruses and vaccines.
Year(s) Of Engagement Activity 2016,2017
Description Participation in the Young Entrepreneur Scheme 2018 competition 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Participation in this event was not linked to my PhD research work.

In this competition,myself and three other postgraduate students/employees competed against other postgraduate students/employees from UK academic institutions for the chance to win a cash prize. This competition was the Young Entrepreneur Scheme (YES) 2018 competition where scientists with no business background are tasked with formulating a business plan that relies on a biotechnological achievement for the purpose of commercialization.
Year(s) Of Engagement Activity 2018