Genome-Wide Gene Expression Analysis Identifies the Proto-oncogene Tyrosine-Protein Kinase Src as a Crucial Virulence Determinant of Infectious Laryngotracheitis Virus in Chicken Cells

Li, Hai; Wang, Fengjie; Han, Zongxi; Gao, Qi; Li, Huixin; Shao, Yuhao; Sun, Nana; Liu, Shengwang

Virus-induced cell death results in extensive tissue damage during the late stages of a virulent virus infection. We sought to The percentages of ILTV-infected LMH cells at 1 dpi and 2 dpi were measured by flow cytometry using a polyclonal antibody against glycoprotein I of ILTV, which was produced in SPF rabbits, followed by a fluorescein isothiocyanate-conjugated anti-rabbit second antibody. The level of background staining was determined using normal SPF rabbit serum as a control. Data are presented as the mean Ϯ SD (n ϭ 3). *, P Ͻ 0.05; N.S., no significant difference. determine whether Src had any effect on ILTV-induced cell death using two strategies. First, the depletion of Src halted ILTV-induced Src phosphorylation at Tyr 416 (Fig. 3A) , resulting in the promotion of the CPE (Fig. 3A) . This effect was also evident in LMH cells that were pretreated with a well-characterized Src-spe-cific chemical inhibitor, SU6656 (48) (Fig. 3B) , which interrupts Src phosphorylation upon ILTV infection (Fig. 3B) . FACS analysis of PI-stained cells found that ILTV infection in combination with the Src inhibitor resulted in levels of cell death at 2 dpi that were 4-fold higher than those in cells infected with ILTV alone (Fig. 3C) . A further detailed analysis using annexin V-PI staining revealed a significant enhancement of necrosis and apoptosis in ILTV-infected cells upon either siRNA-mediated Src depletion or the administration of SU6656 (Fig. 3D and E) , thereby demonstrating that inhibition of Src promoted LMH cell death during ILTV infection. The reduced threshold of ILTV-induced cell death by Src inhibition suggests that Src prolongs the survival of LMH cells in response to ILTV infection. Along with greater ILTV virulence, inhibition of host Src by either siRNA-mediated Src depletion or the administration of SU6656 resulted in Ͼ60% reductions in virus replication ( Fig. 3F and G, right) , as determined by qPCR when all virus-infected cells were dead ( Fig. 3F and G, left) . were validated in ovo using 9-day-old SPF chicken embryos that were inoculated through the allantoic cavity with 1 ϫ 10 4 TCID 50 of ILTV. Under our experimental conditions, inoculated embryo deaths occurred by 9 dpi, and all inoculated embryos were dead by 12 dpi (see Fig. S2 in the supplemental material). We first inoculated embryos with ILTV in combination with a serial dilution of the Src inhibitor SU6656. For the entire observation period (5 dpi), dead embryos were not observed at the lowest dose of SU6656 tested (25 g). A dose of 50 g of SU6656 resulted in the deaths of inoculated embryos by 2 dpi, with the mortality rate approaching 50% at 5 dpi (Fig. 4A) . The highest dose of SU6656 (250 g) resulted in the deaths of inoculated embryos by 1 dpi; all inoculated embryos were dead by 3 dpi (Fig. 4A) . During the observation period, there were no embryo deaths in eggs treated with ILTV alone or in uninoculated eggs treated with the highest dose of SU6656 alone (Fig. 4A) . Thus, the in ovo CPE that we observed was due to the combined effects of ILTV infection and SU6656 administration. Virus replication in allantoic fluids or CAMs was quantified by qPCR assays. Virus replication was reduced by SU6656 in a dose-dependent manner in allantoic fluids and CAMs ( Fig. 4B and C) .