Autophagy represents a new host-pathogen interface for identification of infectious bronchitis virus proteins that determine virulence

Infectious bronchitis is an important endemic disease of chickens and caused by the avian coronavirus infectious bronchitis virus (IBV). Poultry meat is an important food source and during the course of a year approximately 40 x 109 chickens are reared globally. The increasing demand for poultry meat has lead to the introduction intensive farming methods but productivity is often limited by infectious diseases which spread rapidly through high density chicken populations. Importantly for this proposal, a report sponsored by the UK government published in 2005 (http://www.defra.gov.uk/science/Project_Data/Document Library/ZZ0102/ZZ0102 1215_FRP.doc) revealed that the number one cause of economic loss in the UK poultry industry resulting from infectious diseases of chickens was caused by IBV. The virus is not only responsible for respiratory disease, but also causes damage to the kidneys and to egg producing organs of hens, affecting both the production and quality of eggs. Despite the availability of live and inactivated vaccines, IBV continues to be a major problem. The virus causes high morbidity, is ubiquitous world wide, and endemic in the UK, and shows extensive antigenic variation and short lived immunity. These factors lead to high rates of infection and poor cross-protection following infection or vaccination. Most vaccines are given to poultry by spray or in drinking water. Both approaches are rather hit-and-miss. The 'holy grail' of vaccine developers is to have vaccines that can be given by robotic machine to chicks before they hatch. Unfortunately, no existing IB vaccine can be given in ovo because the viruses stop the chicks hatching. One means of controlling IBV is to have a systematic way of generating live attenuated vaccines that provide protection against virulent strains. The reverse genetics necessary for the modification or removal of genes associated with virulence from IBV has been developed by the coronavirus group at the IAH Compton. Together with DEFRA and Intervet International, a major commercial vaccine developer, the IAH coronavirus group are manipulating the genes of IBV to get an optimum balance between attenuation of virulence and capacity to induce immunity. To apply this technology to the rational design of live vaccines it is now necessary to identify genes that contribute to IBV virulence, and understand how they function. Importantly for this proposal, recent work on mammalian coronaviruses, and work on the avian IBV coronavirus at Compton, suggests that virulence may be determined by the way in which cells control virus replication. We have produced IBVs that do not make a series of small proteins called 3a, 3b, 5a and 5b. These viruses grow normally in cell culture and allow chicks to hatch after inoculation in ovo. This shows that we can attenuate IBV by removing non-essential genes, but this is an empirical process because we do not know how the 3(ab) and 5(ab) proteins function in the context of virulence. The purpose of the present grant application to BBSRC is to establish the science behind the empirical observations that we are making. Experiments underpinning this proposal have shown that virulence of IBV may be associated with proteins that control replication, and that these proteins associate with membranes in cells that have the potential to destroy the virus before it can leave the cell. We now want to understand how the replicase proteins avoid destruction, and in this way determine virulence. This will enable us to fine tune our mutants to make viruses that survive long enough to infect chicks 'in ovo', and induce an immune response, but are too weak to harm the chicks and prevent them from hatching.

Infectious Bronchitis virus (IBV) is a coronavirus that causes an economically important disease of poultry. This project seeks to identify viral genes that contribute to IBV virulence, and understand how they function. Work at the IAH has shown that virulence is associated with replicase proteins encoded by ORF1. Coronavirus infection induces the formation of autophagosome membranes which provide a platform for assembly of replicase proteins. Interestingly, autophagosomes are intermediates in a cell stress pathway called autophagy that provides a defence against infection. It is possible that IBV virulence is linked to replicase proteins able to modulate autophagosomes during replication. We will generate a library of chimaeric viruses where defined replicase genes have been exchanged between virulent and avirulent IBV strains and use organ culture to see if virulence segregates with individual or specific combinations of replicase proteins. We will also determine the effects of autophagy on IBV replication and virulence. If autophagy enhances IBV replication and virulence, for example by providing membranes necessary for replication, we argue that virulence will be determined by replicase proteins that stimulate autophagy. Conversely, if autophagy provides a defence against IBV, replicase proteins would need to suppress production of autophagosomes. To study this we will determine if the IBV replicase physically associates with autophagosomes, and if IBV replicase proteins modulate autophagosome production. A link between autophagy and virulence will be made if we can establish that virulence correlates with sensitivity of viruses to autophagy, and demonstrate that replicase proteins that are able to confer virulence, are able to modulate autophagy. This will allow us to determine how genome variation in IBV affects the ability of the virus to subvert autophagosomes during replication, and how this affects the severity and outcome of infection.