Effects of co-infections on Marek's disease in poultry and development of novel recombinant Marek's disease virus vector vaccines

Marek's disease (MD) causes paralysis and tumours in chickens. It is caused by serotype 1 strains of the Marek's disease virus (MDV-1) which is shed from skin of infected chickens and persists for many months in dust in contaminated poultry houses. It is highly contagious and spreads to other chickens by inhalation. MD is a major disease affecting poultry health, welfare, and productivity, with annual estimated loss to the global poultry industry of $2 billion. MD is endemic in UK poultry but is effectively controlled by live vaccine viruses, which are harmless relatives of MDV-1, and include CVI988, HVT, and MDV serotype 2 (MDV-2). MDV-2 vaccines are widely used in the Americas and Asia but not in the UK. However, by testing samples collected from poultry farms, we found MDV-2 is widespread in the UK. MDV-2 strains circulate freely and naturally at high levels and persist long-term in the flock, but little is known about them; are they derived from vaccine strains which 'escaped' from imported poultry, or are they naturally occurring strains? Chickens can be infected with any combination of MDV-1, MDV-2, and vaccine viruses at the same time (co-infection). We have found MDV-2 in healthy chicken flocks, as well as flocks that have MD. We would like to know whether co-infection with MDV-2 affects flock health and disease, and production parameters such as egg production and mortality, and whether certain MDV-2 strains could be used as effective recombinant vaccines against MD and other poultry diseases. Our objectives are to: (1) Investigate prevalence of naturally occurring MDV-2 infection in the field, and it's influence on flock productivity, immune responses and disease, (2) Characterise MDV-2 field isolates and (3) Exploit novel MDV-2 as potential viral vectors for novel recombinant vaccines.
The project is a partnership with poultry industry vets. We will select two MDV-2-positive and two MDV-2-negative flocks for two bird types (broiler-breeder, layer) for regular sampling to collect blood samples from chickens and dust from the housing sheds. We will also collect data on flock health and productivity. At Pirbright, we will test the samples by 'polymerase chain reaction' to detect the genetic material of MDV-1, MDV-2 and vaccine viruses to show the kinetics of MDV-2 infection and shedding, and the frequency of co-infection with MDV-1 field strains and vaccine viruses. Using mathematical modelling, we will also investigate dynamics of transmission of MDV-2 within flocks. We will determine variability of MDV-2 strains by sequencing the virus genetic material and comparing with known MDV-2 strains. We will study the characteristics of selected MDV-2 strains by growing these viruses in cell culture then using them to infect chickens under controlled laboratory conditions to examine replication, persistence, clinical signs and transmission of MDV-2. Most MDV-2 strains have characteristics which make them suitable as vaccines against MD: they do not cause disease, they grow well in the chicken and persist for many months, and they are easily transmitted between chickens to maintain a high level of exposure of the flock to vaccine virus. Furthermore, MDV-2 can be genetically engineered to carry genes from other important poultry viral pathogens, e.g., infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV); a recombinant 'vectored vaccine' like this could potentially protect chickens against IBD and ND as well as MD in a single vaccination. We will engineer an appropriate MDV-2 strain to create a 'rMDV2-IBD-ND' virus, then test its ability to protect chickens against these three diseases under controlled laboratory conditions.
This study is important to understand the effect of widespread MDV-2 infection on health and productivity of commercial poultry flocks. A new recombinant MDV-2 vaccine would be a useful addition to the set of live virus vaccines used to control MD and other poultry diseases.

Marek's disease (MD) is a contagious fatal endemic disease of poultry caused by serotype 1 strains of MD virus (MDV-1). MDV serotype 2 (MDV-2) is also widespread in UK poultry, in both presence and absence of MDV-1, and with or without pathology. Prevalence of MDV-2 infection and its effect on flock productivity and disease will be investigated by longitudinal sampling of blood and dust from MDV-2-positive and negative broiler-breeder and layer poultry farms and real-time qPCR to determine kinetics of replication and shedding of MDV-2 and frequency of co-circulation with MDV-1 field strains and vaccine viruses. Mathematical modelling will be used to investigate transmission dynamics of MDV-2. Flock health and production data will enable analysis of effects of MDV-2 co-infection on these parameters. MDV-2 will be isolated from four positive farms by culturing splenocytes. The isolates will be sequenced and a phylogenetic tree constructed to determine relatedness to known MDV-2 vaccine and field strains. Two strains will be chosen for controlled trials in SPF birds to examine replication kinetics, persistence, clinical signs and horizontal transmission to sentinel birds, in comparison with MDV-2 vaccine strain SB-1. One strain will be chosen to develop a novel MDV-2 recombinant vaccine using CRISPR/Cas9 gene editing to insert infectious bursal disease virus VP2 gene and Newcastle disease virus F gene. The resulting virus, rMDV2-IBD-ND, will be tested in a vaccine trial in SPF birds to investigate protection against MD following challenge with MDV-1, and generation of antibodies against the IBDV VP2 and NDV F proteins. The data will give an understanding of the effect of MDV-2 infection on poultry health and productivity, while development of a novel rMDV-2 vaccine could be a useful addition to the set of live vaccines used to control MD and other poultry diseases, with advantages of transmissibility and no interference with HVT-based recombinant vaccines.