Molecular characterization of natural recombinant ALVS and their use as vectors for gene delivery into stable cell lines
Barbosa, Taylor Marcelo Correa
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Avian Leukosis viruses (ALV) previously found as contaminant of Marek’s Disease vaccines were molecularly characterized. All three isolates, named PDRC-1039, PDRC-3246 and PDRC-32-49, were found to be recombinant viruses. The mosaic viruses mostly consist of endogenous ALV sequences but, the surface protein (gp85) is of exogenous origin and very similar to ALV-A subgroup. Due to their high similarity to each other, the viruses were considered to be from a common source, despite being from different vaccine companies. One of the viruses, PDRC-1039, was used to construct a novel ALV based retroviral defective vector system for gene delivery, named pBZ system. Eight different vectors were constructed to allow versatility of vector utilization. All vectors are bicistronic with one drug resistant gene, which allow selection of transduced cells. All vectors were tested for transfection and transduction using Green Fluorescence Protein (GFP). The titers achieved with transduction were similar for all vectors. An avian fibroblastoid cell line (DF-1) was used for transduction and expression of GFP. Selected cells were positive for fluorescence during all passages tested, which varies from 5 to 25 depending on the vector design. One of the pBZ vectors, the pBZ3.0, was used to transduce the Infectious Laryngotracheitis Virus (ILTV) glycoprotein I (gI) into DF-1 cells. After transduction and drug selection with G-418, single colonies were tested for ILTV gI expression by immunoflourescence and westernblot assays. Two clones of DF-1 ILTV gI positive cells were expanded and used as antigen in an inactivated vaccine. SPF chickens were subcutaneously injected with the DF-1 ILTV gI vaccine. Birds were challenged with ILTV USDA reference strain at 28 days of age. All vaccinated chickens mounted a humoral response against the ILTV gI protein. Serum samples from birds vaccinated twice (1 and 10 days of age) had higher antibody levels. There was no difference in protection and viral load in the trachea among vaccinated and control birds. The pBZ system can be used to stably transduce DF-1 cells to express exogenous proteins, and these recombinant proteins can be used to induce humoral responses in chickens.