Human Cytomegalovirus Infection Of Embryo Mouse's Cerebral Cortex Cells And Its Molecule Mechanism

Objective To research HCMV proliferate infection of embryo mouse's cerebral cortex cells in vitro. To discuss the machanism of HCMV cross-species infection. To provide experimental supports for the HCMV infection of central nervous system for clinical research..Methods Firstly, HCMV AD169 was purified by PFU assay. The virus quantity was tested by PFU assay. The embryo mouse's cerebral cortex cells and human embryo lung cells were inoculated with HCMV AD169 of 4×105 PFU/ ml. On the 1^st ,3^rd ,5^th ,7^th day, the cell cultures and the supernatant were harvested and the following parameters were detected using different methods. (1) observing HCMV specific CPE and its characteristics of pathological changes of neonatal mouse's cerebral cortex cells daily by microscope; (2) detecting HCMV IE,UL54 and UL83 genes and their corresponding transcripts by PCR and RT-PCR and the changing characteristics and tendency of mRNA by half quantity RT-PCR throughβ-actin as reference in different time; (3) determining the infectious HCMV quantities by PFU assay when the HCMV specific CPE cells were about 75% or 100% ;(4)observing the ultrastructure and virus particles in neonatal mouse's cerebral cortex cells by transmission electron microscope.Result (1) On the 2^nd day, the HCMV specific CPE could be observed in neonatal mouse's cerebral cortex cells inoculated with purified HCMV AD169 of given dose of 4×10⁵ PFU/ ml. Some pathological changes could be found such as celluar swelling, more intracellular particles and so on. On the 3^rd, 5^th and 7^th day, the quantity of infected cells increased gradually and the CPE was more obvious. Axonotmesis, cathepsis and even cell shedding could be observed. While the HCMV specific CPE could be observed in human embryo lung cells on the 1^st day. The infection aggravated with the time prolonged until the same pathological changes as before were observed. Comparatively, HCMV was more sensitive to HEL; (2) The HCMV IE,UL83 and UL54 and their corresponding transcripts could be detectd in the cultures of neonatal mouse's cerebral cortex cells on the 1^st ,3^rd ,5^th ,7^th day. Through the analysis of gray scale by Bioscience image analysis system, the quantity of HCMV IE,UL83 and UL54 mRNA increased apparently as the infection time prolonged.. This suggested HCMV infection in neonatal mouse's cerebral cortex cells was proliferate infection; (3 )The round plaque could be observed in human embryo lung cells on the 7^th day by PFU assay. The plaques became larger and more as infection days increased. These changes occurred about in 14 days. The virus quantity could be calculated as 3.8×10⁵PFU/ml; (4)The structures of neonatal mouse's cerebral cortex cells were destroyed seriously and there were virus nucleic acid in the intranuclear and herpesvirus-like particles in the cytoplasm of nerve cells by transmission electron microscope.Conclusion : HCMV AD169 has the ability to proliferately infect neonatal mouse's cerebral cortex cells in vitro which proves HCMV cross-species infection and offers practical supports for the clinical research of central neural system infection with HCMV.