IAH studentship:The role of AKT activation during Avian Infectious Bronchitis Virus infections

Lead Research Organisation: The Pirbright Institute
Department Name: UNLISTED

Abstract

Studentship: Research into many viruses such as Influenza virus, Hepatitis C virus and Papillomavirus has revealed that the PI3K/AKT pathway plays a crucial role in infection. The PI3K/AKT pathway is responsible for the control of many cellular processes including apoptosis, cell growth and proliferation. AKT (also known as PKB) is a protein kinase, ubiquitously expressed in most cells. It acts as an upstream signaling protein for many pathways that are vital for cell survival and replication. Studies of the Influenza virus have found that the viral protein NS1 is directly involved in activation of the PI3K/AKT pathway. Activation leads to negative regulation of apoptosis amongst other changes, which creates a more effective environment for virus entry and growth.
Although the AKT locus has been identified in chickens, few studies have characterised the involvement of this viral evasion mechanism in this important livestock species. In this project we will characterize the role of AKT within chicken cells when infected by a virus, using both proteomic and molecular techniques. The virus to be used in this study is avian gammacoronavirus, infectious bronchitis virus (IBV) that causes infectious bronchitis (IB) in poultry. IB is an economically important disease that causes severe respiratory pathology in chickens of all ages. It is extremely detrimental to the poultry industry due to reduced egg production and quality of both egg and meat. Although vaccines exist against the virus, the high level of mutation that is associated with virus replication and numerous strains of the virus with poor cross-protectively means that the disease is difficult to control.
 
Description The ability of some coronaviruses to transmit between animals and humans has been highlighted in recent years by the emergence of novel human coronaviruses, SARS-CoV and more recently MERS-CoV, both of which are thought to have originated in bats. The gammacoronavirus infectious bronchitis virus (IBV) is endemic in most countries around the world and causes infectious bronchitis in poultry. Infectious bronchitis is a highly contagious respiratory disease that results in reduced egg production and can be fatal in young birds. All viruses modulate cellular processes to establish themselves within the cell. The AKT signalling pathway is a host pathway often modified by viruses and plays a crucial role in the regulation of many cellular processes. We have studied the activation of the AKT signalling pathway and downstream processes such as apoptosis, autophagy and macropinocytosis during IBV infection. Here we show that IBV infection induces activation of AKT in a PI3K dependent manner. In avian cells this activation occurs in a biphasic manner at both an early and late time point during infection. We have shown that IBV requires an active PI3K/AKT pathway for efficient replication and that over expression of several viral proteins has been found to cause activation of AKT. Furthermore we have investigated how this activation allows modulation of downstream pathways such as apoptosis, autophagy and macropinocytosis.
Exploitation Route This study highlights the importance of the PI3K/AKT signalling pathway during IBV infection and may be applied to other human and livestock coronaviruses for development of therapeutics or novel vaccines. This was written up as Ambi Batra's PhD thesis.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Increased understanding of the impact of AKT signalling in disease pathogenesis
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Healthcare
Impact Types Societal
 
Description Contributed to Chief Medical Officers report- Genomics section
Geographic Reach Europe 
Policy Influence Type Implementation circular/rapid advice/letter to e.g. Ministry of Health
URL https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/6310...
 
Description Commercial Partner
Amount £30,000 (GBP)
Organisation HyLine 
Sector Private
Country United States
Start 08/2014 
End 06/2015
 
Title A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR 
Description Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes can be used to control random technical variation between samples. It is necessary to select reference genes with the lowest variation in representative samples. The candidate reference gene assays must be reliable. In particular, they should have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios, a tissue collection, and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where the use of this gene might provide misleading results. Widely used methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. 
 
Title SAL Locus 
Description SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2013 
Provided To Others? Yes  
Impact SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute. 
 
Description Roslin 
Organisation University of Edinburgh
Department The Roslin Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint Grant: 'Role of AKT1 & SIVA1 in resistance to avian salmonellosis' Stevens, Kaiser & Fife
Collaborator Contribution Poultry are a key reservoir of human Salmonella infections owing to the ability of some strains to colonise the avian intestines and reproductive tract. Birds often carry the bacteria in the absence of overt symptoms; however some types of Salmonella cause severe typhoid-like diseases in poultry that exert substantial welfare and economic costs. Global population growth and rising affluence are fuelling demand for poultry meat and eggs, and a need exists to enhance the supply and safety of such. Though vaccines are used in layers in some countries, most of the 55 billion chickens reared annually worldwide lack protection against Salmonella infection. We and others have discovered that some chickens exhibit heritable differences in resistance to Salmonella. It may be feasible to selectively breed for birds with improved resilience to Salmonella infection; however this requires the identification of resistance-associated factors and knowledge of how they act. By analysing the genetic material of birds that differ in resistance, we have located a region of the chicken chromosome that confers protection against typhoidal salmonellosis, both in laboratory studies and commercial poultry populations. Recent studies have now resolved the region associated with resistance to just a handful of genes. It is highly plausible that variation affecting two genes in this region (AKT1 and SIVA1) explains why birds react to Salmonella in different ways, as the encoded proteins control host processes that impact on the fate of bacteria. For example, AKT1 and SIVA1 control the death of infected cells and the induction of immune responses, but have opposing activities. It is not possible for us to predict how the genetic changes affecting these genes will alter their expression or activity. Moreover, it is unclear how such factors may control the growth and spread of Salmonella in birds. We therefore propose to: 1. Examine if birds normally respond to Salmonella infection by activating the expression or function of AKT1 and SIVA1. We will examine this in cells cultured from chickens, as well as in intact birds, and associate any differences with host responses and the fate of the bacteria. 2. Examine if lines of chicken known to differ in resistance to Salmonella infection vary in the levels or activation of AKT1 and SIVA1. 3. Use specific inhibitors and bacterial strains to establish that AKT1 activation is necessary for Salmonella to grow and spread in birds. 4. Define the nature, frequency and consequences of genetic changes affecting AKT1 and SIVA1 in commercial poultry populations. This will aid the selective breeding of chickens that show improved resilience to Salmonella infection. We are fortunate to have the support of one of the world's largest poultry breeding companies (Erich Wesjohann Group), who will provide birds, genome sequences, expertise and 10% of total project costs. This reflects the value of the proposed studies to the industry. The consortium has productively collaborated and the proposed studies are a timely, logical and feasible extension of our recent joint research.
Impact SAL1 Patent for Salmonella resistance in chickens"-. PCT/GB2010/000850. The Pirbright Institute.
Start Year 2012
 
Title Avian cells for improved virus production 
Description Type I interferons protect cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). Among these interferon stimulated proteins, are the IFITM (interferon-inducible transmembrane) genes which have been shown to restrict the replication of several highly pathogenic human viruses, including severe acute respiratory syndrome (SARS) coronavirus, filoviruses (Marburg virus and Ebola virus), influenza A viruses (IAVs), and flaviviruses (dengue virus). The Genetics and Genomics group have identified these antiviral proteins in the chicken (chIFITM) and have shown that a reduction in chIFITM expression results in an increase in the virus titre in CEFs infected with avian influenza A virus (AIV) H9N2, suggesting that chIFITMs have a functional role in the control of viral infections. The observation may have useful implications in terms of vaccine production. To this end, a patent was filed relating to the modification and testing of avian IFITMs, and has now been granted in multiple countries. Many vaccines have been produced in embryonated hen's eggs or continuous avian cell lines for more than 30 years. However, it is well established that the rate determining step in the manufacture of numerous vaccines is the induction of antiviral immune responses that prevents the replication of vaccine viruses. To generate chIFITM knock-down, we will use cutting edge genetic approaches such as siRNA and a CRISPR/Cas9 system which will directly target and knock-out chIFITM expression. We believe that this approach will overcome the rate limiting step in vaccine production, directly resulting in increased vaccine yields and improve the speed at which vaccines can be manufactured. We are currently in talks with major vaccine producers keen to adopt this internationally patented technology, to advance the field of both animal and human vaccine production. 
IP Reference WO2014195692 
Protection Patent application published
Year Protection Granted 2016
Licensed No
Impact This work has arisen as a result of an Animal Health Research Club grant. Involvement of the commercial sector has been encouraged and applauded by the ARC Steering committee. The IMPACT of this work is potentially very significant, and could provide considerable income to The Pirbright Institute.
 
Description AB International Nidovirus Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Batra, A., Maier, H.J., Britton P., Hiscox, J.A., Fife, M.S., 2014. AKT activation during infectious bronchitis virus infection. XIIIth International Nidovirus Symposium, Salamanca, Spain (Poster presentation)
Year(s) Of Engagement Activity 2014
 
Description AB Society of General Microbiology Annual conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Poster presentation. Batra, A., Maier, H.J., Britton, P., Hiscox, J.A., Fife, M.S. AKT activation during infectious bronchitis virus infection. Society of General Microbiology Annual conference, Liverpool, UK
Year(s) Of Engagement Activity 2013,2014,2015
 
Description Innovate Guildford Outreach (12th March) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact With amazing exhibits, activities, workshops, talks and competitions, it's FREE to attend. Explore the four zones: Engineering, One Health, Digital Media and Future Living, experience interactive performances, hear inspiring speakers (including leading gaming pioneer Peter Molyneux and animal health pioneer Professor Nick Bacon) and take in an amazing atmosphere. Theatre, music and comedy will feature through the day, with everything from how technology might have affected Shakespeare's plays to risky recycling and photographic secrets.
Year(s) Of Engagement Activity 2017
URL https://www.guildford.gov.uk/innovateguildford