Dr Byung-Whi Kong, working at the University of Arkansas, is aiming to develop an immortalised chicken cell substrate for the propagation of the ILT virus for vaccine production. The research project is sponsored by the US Poultry and Egg Association.

In the report, Dr Kong explains that ILT is an acute respiratory tract disease mainly in chickens caused by the herpesvirus ILT (ILTV), and that it can result in significant economic losses in poultry-producing areas worldwide.

Current chicken embryo origin (CEO) vaccines available commercially can result in severe vaccinal laryngotracheitis by transmission from vaccinated to unvaccinated birds. Thus, the major objective of this proposal is to develop a virus-free, non-tumour causing, immortalised chicken cellular substrate for ILTV propagation for production of a genetically attenuated vaccine.

The specific objectives of the work were:

1. Isolation of the most suitable chicken primary cell substrates from various organs or tissues for ILTV infection and propagation
2. Immortalisation of the chicken cell substrate propagating ILTV using DNA fragments to modulate the cell cycle, and
3. ILTV recombination to detect virulent genes to produce recombinant ILTV vaccine using the most efficient immortal (capable of unlimited numbers of replications) chicken cell substrate.

Numerous chicken embryo primary cells were isolated from lung, trachea, liver, heart, muscle, intestine, kidney and bursal tissue, in addition to embryonic fibroblastic (CEF) cells. Of these, primary cells derived from kidney, liver and lung tissues propagated ILTV (Objective 1).

Through a variety of transfections using ectopic expression constructs and/or siRNAs for cell cycle regulatory genes, three liver cell lines, named CELisi-p53; CELi-Vector; and CELi-im, were immortalised and have continued to grow for more than one year. Currently, cells keep dividing at 0.7 to 0.9 PD per day growth rate. All three cell lines were tested for ILTV infection and propagation resulting in weak permissiveness and only low virus titres (around 10¹ pfu/ml) produced.

For the further optimisation of ILTV propagation in the newly immortalised liver cell lines, various conditions of ILTV infection such as initial cell density, number of virus infectious units inoculated, ILTV stocks and incubation time, are currently being compared to reach the highest ILTV titres, up to 10⁵ pfu per ml levels (Objective 2).

At least one of the three immortal liver cell lines is expected to serve as a stable cellular substrate for further production of a genetically attenuated ILTV vaccine.

For the genetic attenuation for ILTV, various efforts were tried for producing mutant strains of ILTV using homologous recombination between transfer vectors containing flanking sequences of target viral genes and ILTV genomic DNA (or live ILTV virus). To date, deletion of ILTV mutants have not been successful due to the potential loss of infectivity of stock ILTV (Objective 3).

In the near future, genetically attenuated ILTV, which can be utilised as a new type of live vaccine should come from continued research.

Currently, there is a heavy reliance upon avian eggs and primary avian cell cultures as substrates for vaccine production. Cultured primary cells can be highly variable and can be potentially contaminated with undesirable endogenous viruses. In addition, the source of primary cultures can be limited due to the availability of layer or other animal stocks and expensive due to the necessity of maintaining them in a specific pathogen-free state. Therefore, a certifiable and well characterized continuously growing (immortal) cell line is of primary importance to the vaccine industry for reducing or eliminating the need for primary animal tissue and subsequent establishment of cell cultures.

Newly established immortal chicken liver cell lines can serve an alternative substrate for ILTV propagation for further vaccine production. In addition, these cell lines can be applicable to propagate other avian infectious viruses, such as Marek's disease virus, avian pneumovirus and avian influenza virus for further vaccine production.

Dr Kong concludes that once a genetically attenuated ILTV vaccine can be developed that would not cause vaccinal ILT, it could become a potentially optimal vaccine to control ILT disease.

Currently, the control of ILTV in Arkansas is estimated to cost the in-state poultry industry more than $3 million annually (from a casual survey of industry and academic veterinarians). The proper control of this important field virus could protect poultry production systems from losses caused from the actual outbreak and resulting restrictions on international export of poultry.

December 2009