| Objective: Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract infection, is responsible for severe illness both in new born and young children and in elderly people. Vaccination is the important measure to prevent virus infection. The vaccinated host often led to increased Th2 type response and lung immune pathology after RSV infection. This is the main obstacle for successful RSV vaccine. And RSV infection pathogenesis is unclear. For decades, clarifying pathogenesis of RSV infection and vaccine enhanced lung immune pathology is the most important in the RSV research field, and is the important basis of developing cutting-edge treatment measures and safe and effective RSV vaccine.The ideal experimental animal model is required to study pathogenesis. Pneumonia virus of mice (PVM), along with human RSV, are viruses of the family Paramyxoviridae, subfamily Pneumovirinae, is a natural mouse pathogen. Robust replication of PVM takes place in bronchial epithelial cells in response to a minimal virus inoculum. PVM IFNection in mice reproduces many of the clinical and pathologic features of the more severe forms of RSV IFNection in human infants. In this study, newborn 4-7 days and 6-9 weeks of adult female BALB / c mice were infected with PVM. Immune response, inflammatory response and lung pathological changes after PVM infection in neonatal mouse and grown mouse model were investigated, which may provide some data for future research using the PVM model.Methods:1 Preparate and titer PVM with BHK-21 cells2 Animal experiments4-7 days neonatal BALB / c mouse and 6-9 weeks grown BALB/c mouse were randomly divided into 4 groups(6 mice/group): (1) neonatal mouse infected group, (2) neonatal mouse control group, (3) grown mouse infected group, (4) grown mouse control group. The neonatal mice were infected with 375 TCID50 PVM, and grown mice infected with 500 TCID50 PVM.2.1 Weight of the mice infected were observed everyday2.2 The total RNA of the lung tissue in neonatal mouse infected and control group 1,3,5,7,9,11,14 and 18 days after infection was extracted. PVM SH gene was amplified by semi-quantitative RT-PCR2.3 3,7,14 days after infection, the lung lavage cells were stained with Switzerland - Giemsa, and all nucleated cells were counted2.4 3,7,14 days after the specimens from infected lung tissues of mouse piece of pathology, HE staining of inflammatory infiltrates in lung tissue and tissue injury2.5 Lung Th1-type cytokines IFN-γ, IL-12, Th2 type cytokines IL-5, IL-6, IL-13, interferon IFN-α, IFN-β, tumor necrosis factor TNF-α, chemokine MCP-1, MIP-1α, CCL5, MCP-3, and mucus factor gob5 were dectected at 1,3,5,9,14 days. After PVM infection with real-time quantitative PCR.2.6 Serum IgG antibody 14 days after infection was measured by ELISA method2.7 The grown groups were treated as neonatal groupsResult:1 Preparation of the experiment required PVM virus. PVM virus titers were determined TCID50=107.5/ml2 Weight Compared with the neonatal mouse control group, the weight of the neonatal mouse infected group has reduced 4 days after infection;7 days after infection, the weight reduced to the minimum, and then slowly increased. 14 days after infection,there is no difference between the neonatal mouse infected group and the neonatal mouse control group.Compared with the grown mouse control group, 3 days after infection, the weight of the grown mouse infected group rapidly reduced, 5 days after infection,weight reduced to the minimum. 7 days after infection,the weight slowly increased. 8 days after infection , the weight turned to normal . 3 PVM SH gene was amplified by semi-quantitative RT-PCRThe expression of SH gene of the neonatal mouse infected group appeared on the 1 day after infection and disappeared on the 18 days after infection . 5 days after infection , the expression of SH was up to the highest level.7 days and 9 days after infection,the expression of SH is lower, and disappeared on the 18 days.Like the neonatal mouse infected group, the expression of SH gene of the grown mouse infected group also appeared on the 1 day after infection, and disappeared on the 18 days after infection. 5 and 7days after infection, the level of SH was signigicantly higher than other time point. 4 HE staining of lung tissueCompared with the neonatal mouse and grown mouse control groups in which the structure of the alveolar walls was intacted without oedema, only with a few inflammatory cells. In the lungs of neonatal mouse and grown mouse infected groups, 3 days after infection , the number of inflammatory cells increased, and alveolar walls became thick. 7 days after infection, inflammatory cells,including lymphocytes,visible mononuclear-macrophages, neutrophils, eosino- phils, were more, and the alveolar wall was thicker with sever edema. 14 days after infection inflammation diminished, 3 days and 7 days after infection, grown mice infected appeared significant inflammatory cells infiltrating, severe alveolar walls congestion, oedema, alveolar interval fusion. 14 days after infection, inflammation attenuated. 5 The inflammatory cells in lung lavage fluidIn the grown mouse infected group 3 days and 7 days after infection, the total number of cells in lung lavage fluid was higher than the neonatal mouse infected group. The proportion of neutrophils was 53.25% and 67.00%, respectively. The proportion of neutrophils in neonatal mouse infected group was only 0.00 % and 27.00 %, respectively. These data suggest that PVM infection of grown mouse is more severe than neonatal mouse. 6 Serum IgG antibody was measured by ELISA method PVM infection in neonatal or grown mouse induced IgG antibody. infected group,IgG antibody titer in grown mouse infected (1/400) iwass 1/400,higher than that in the neonatal mouse infected group , IgG antibody titer is( 1/200).7 Cytokines were dectected with real-time quantitative PCRAfter 3 days infection, the expression levels of Th1 type cytokine IFN-γin lung tissue from neonatal mouse infected significantly increased compared with controls( P<0.05), and continued to increase up to 150-fold higher than controls 9 days after infection. The expression level of IL-12 in neonatal mouse infected was not significantly different compared with controls(P>0.05).The expression levels of IFN-γand IL-12 in lung tissue from grown mouse infected by PVM was significant increased after 1 day, 133-fold higher than controls 5 days, and started to decrease 9 days. The expression levels of IL-12 was 2-fold higher than controls in 5 days; After that no difference was between two groups(P<0.05).The expression levels of IL-5 and IL-13 from neonatal mouse infected by PVM has no significant difference compared with controls( P>0.05); The IL-5 expression levels from grown mouse has no difference with control( P>0.05); The expression levels of IL-13 from grown mouse was 4-fold higher than control after 5 days (P<0.05).IFN-αwas expressed significantly in early time after infection in neonatal and grown mice, then decreased sharply. In neonatal mice infected, the expression kinetics of IFN-βwas same as IFN-α. In grown mice infected, IFN-βbegan to be expressed after infection, and was reach to peak after 5 days.The expression levels of IL-6 from neonatal mouse was 17-fold higher than controls after 3 days, which reached to peak (P<0.05); The expression levels of IL-6 from grown mouse was 18-fold higher than controls after 5 days, which reached to peak (P<0.05).The expression levels of TNF-αfrom neonatal mouse was 30-fold higher than controls 9 days after infection (P<0.05); The expression levels of TNF-αfrom grown mouse was 76-fold higher than controls after 4h (P<0.05), after 1 day gradually decreased and had no differenc after 14 days(P>0.05).The expression levels of MCP-1,MCP-3,MIP-1αand CCL5 from neonatal mouse infected by PVM has significant difference compared with controls( P<0.05); MCP-1 expression level was 41- fold higher after 3 days and lower after 14 days compared with controls( P<0.05); MCP-3 expression level was 118- fold higher after 3 days and has no difference after 14 days compared with controls (P>0.05); MIP-1αexpression level was 21-fold higher after 5 days; CCL5 expression level was 2-fold higher after 3 days(P<0.05); The expression levels of MCP-1 from grown mouse infected by PVM was 120-fold higher than controls(P<0.05);After 14 days still higher; MCP-3 expression level was 195-fold higher after 5 days(P<0.05); After 14 days still higher; MIP-1αexpression level was 50-fold higher after 1 days(P<0.05); After 14 days still higher(P<0.05); CCL5 expression level was 5.5-fold higher after 1 days than controls (P<0.05).The expression levels of mucus factor gob5 in lung tissue from both neonatal and grown mouse have significant difference with controls(P<0.05); 6-fold higher than controls after 9 days from neonatal mouse; 16-fold and 11-fold higher after 5 and 9 days from neonatal mouse than controls; disappeared after 14 days.Conclusion: PVM infection in both neonatal and grown BALB/c mouse elicited inflammatory pathologic responses in lung tissue. Grown mice infected by PVM exhibited stronger inflammation, which may be same as infants infected by RSV. |
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