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

The biological characteristics of ILTV-LSJ09 infection in LMH cells, including cell growth, cell death, virus replication, virus release, and virulence, were examined. ILTV infection significantly reduced the number of adherent cells that were negative for trypan blue staining by the third day postinfection (3 dpi), while cells in the control cultures continued to grow (Fig. 1A) . Consistent with the reduction in the proportion of living cells following ILTV infection in our system (Fig. 1A) , the proportion of apoptotic cells significantly increased from 2 dpi compared with that of control cells (Fig. 1B) . The CPE of ILTV on LMH cells occurred by 2 dpi and became more severe as time progressed (Fig. 1C ). In parallel with the induction of apoptosis and the occurrence of the CPE, the results of the qPCR assay showed that ILTV replicated exponentially in LMH cells from 2 dpi, whereas viral replication was not significant at 1 dpi compared with that at 0 dpi (Fig . 1C ; the P values for replication at 1 and 2 dpi were 0.127 and 0.022, respectively). In addition, a concomitant, continuous increase in the copy number of the viral genome was observed in the medium from 2 dpi (Fig. 1C) , which possibly explains the occurrence of the ILTV CPE at 2 dpi (Fig. 1C) . The results of the qPCR assays were confirmed by TCID 50 assays (Fig. 1D ). No significant CPE was observed until 2 dpi, after which the virus titer increased continually in the cells and medium (Fig. 1D ). The proportion of ILTVinfected cells was determined by FACS, and it was approximately 10% at 2 dpi but only about 1.5% at 1 dpi (Fig. 1E ).