Research of evidence of persistent infection of respiratory syncytial virus in pathological models of respiratory syncytial virus-induced nephropathy rats

Abstract

Objective To study the evidence of persistent infection of respiratory syncytial virus (RSV) in SD rats pathological model of nephropathy which were infected by RSV. Methods A total of 62 cases of specific pathogen-free male SD rats with 2-month-old, 180-200 g of body weight were selected and randomly divided into RSV groups (n=32) and control groups (n=27) by randomized digital table method. ①RSV groups were set up by dealing with RSV fluid, pock forming unit (PFU)=6×106 (0.4 mL/d via intraperitoneal and 0.2 mL/d via nasal) for 3 days; control groups were set up by dealing with phosphate buffer saline (PBS) fluid (0.4 mL/d via intraperitoneal and 0.2 mL/d via nasal) for 3 days. The day before injection was regarded as 0 d, while the 1st day began when models were successfully set up. Ultrastructural changes of glomeruli in SD rats at 7th day and 60th day after being successfully set up of a pathological model of nephropathy were observed by electron microscopy, in order to judge whether the pathological models of nephropathy to SD rats were successfully built or not. ②A total of 35 cases of SD rats in RSV groups were randomly divided into 7 subgroups by randomized digital table method, such as RSV 0, 7, 15, 30, 60 d, 90, 120 d subgroups, 5 rats in each RSV subgroup; while 27 cases of SD rats in control groups were randomly divided into 7 subgroups by the same method, such as PBS 0, 7, 15, 30, 60, 90, 120 d subgroups, and 5, 5, 5, 3, 3, 3, 3 cases of SD rats in each PBS subgroup, respectively. Biochemical parameters including levels of 24 h urinary protein, serum albumin and serum creatinine were measured in each subgroup, and the parameters were analyzed by statistical methods. ③All SD rats in RSV and PBS subgroups were killed after biochemical parameters being measured, and then the inferior 2/3 of left kidneys, left lungs and spleens of each rat were harvested, fixed by 4% paraformaldehyde solution for 24 h, and embedded by paraffin to complete hematoxylin-eosin (HE) staining. Each slice was observed under light microscope to find out whether pathomorphological changes existed or not. ④With RSV G protein monoclonal antibody as primary antibody, immunohistochemical specimens of SD rats' kidneys, lungs and spleens in 7 RSV subgroups were made out to find evidence of RSV G protein under light microscope. ⑤With RSV G protein monoclonal antibodies as primary antibody combined with green fluorescence, blue stain nuclei by 4', 6-diamidino-2-phenylindole (DAPI) at the same time, all SD rats' kidneys in RSV subgroups were cut into fluoroimmunoassay slices to find out the evidence of RSV G protein and the relation between its location and the nucleus. ⑥Harvesting the remaining 1/3 of the left kidney, with 3% glutaraldehyde fixation before sent to the West China Hospital, Sichuan University, SD rat's glomerular ultrastructural pathology structure changes in each RSV subgroup were observed under electronic microscope, meanwhile RSV particles were searched. Results ①The pathological model of nephropathy SD rats in RSV group were duplicated successfully, as same as the model of SD rats in control group. ②Comparison of proteinuria in 24 h at the same point between RSV subgroups and PBS subgroups suggested that there were no statistical differences on 0 and 90th day (P>0.05), while the proteinuria in 24 h of RSV 7, 15, 30, 60, 120 d subgroups were all significantly higher than those in PBS subgroups at the same point (t=6.9, 3.1, 3.9, 2.2, 5.6; P 0.05). ③HE stains showed that pathological changes at different levels of kidneys, lungs and spleens were observed in RSV 7, 15, 30, 60, 90, 120 d subgroups compared with RSV 0 d subgroup. ④Immunohistochemical stains showed that RSV G protein expressed positively in RSV 7, 15, 30, 60, 90, 120 d subgroups' kidneys, lungs and spleens while RSV 0 d subgroup had no positive expression. Quantitative analysis of RSV G protein suggested that the expression of RSV G protein increased gradually before day 30th and maintained at a high level, and then decreased after day 60th. ⑤Fluorescence staining suggested that RSV G proteins were located in the RSV infected cells' nuclei. ⑥RSV particles were observed in RSV subgroups' kidneys under electronic microscopy and glomerular pathogenesis persisted even on day 120th. Conclusions The expression of RSV G protein from 7th to 120th day successfully demonstrate that RSV could persist on a long time in RSV nephropathy rats, which may caused by immune dysfunction with RSV and it's persistence. Key words: Minimal change nephritic syndrome; Respiratory syncytial virus infections; Persistent infection; Fluoroimmunoassay; Pathological model, nephropathy; Child