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Objective To establish a method for the isolation of Zika virus and to gather experi-ences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples posi-tive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nu-cleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The se-quences of NS5 gene were analyzed by using MEGA6. 0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle thresh-old (Ct) values of 24. 4 and 25. 3, respectively. The second generation of Zika virus was identified in suck-ling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus ( GDZ16002 strain) be-longed to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage.
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Research on laboratory animals is an important issue in biomedicine.Children are a special drug-using population.The selection of suitable experimental animals is a key issue to ensure the scientific quality of research for pediatric drugs.Based on the review of a large number of literature, the authors summarized the application of suckling mice in the pharmacological research and toxicological evaluation of pediatric drugs for the treatment of common diseases in children.We also summarized the existing problems in pediatric toxicology and proposed solutions for providing a reference of test animal application in pediatric drug research.
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Interactions between FMDV and cardiac cells are multifaceted and complex ,these interactions leads to pro-teins alterations in cardiac cells inevitably .To understand the pathogenesis of myocarditis after FMDV infection in mice ,the suckling mouse model for myocarditis induced by foot-and-mouth disease virus (FMDV) was established in this study .Suckling mice within 3 days old was selected to infect by FMDV .Myocarditis caused by FMDV in suckling mice was confirmed with clinical symptom monitor .The observation of Hematoxylin and eosin stain (H&E stain) and transmission electron microscopy (TEM) were performed after samples processing .According to conventional polymerase chain reaction (PCR) methods ,prim-ers of VP1 gene was designed ,synthesised and specific FMDV VP1 gene was amplified from the heart muscle of suckling mice . The results indicated that suckling mice appeared low spirit condition ,dyspnea ,and dull reaction within 36 hours after chal-lenge with FMDV .Infiltration of inflammatory cells and dissolution of myocardial fibers were observed with H&E stain and TEM .Special target gene of FMDV was amplified from the heart of infected group .Obvious inflammation in the heart of suck-ling mice caused by FMDV was observed .It's suggested that suckling mouse model for myocarditis induced by FMDV was es-tablished successfully ,which would lay the foundation for researches of myocarditis mechanism in young cloven-hoofed ani-mals .
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Objective To establish an animal model of human rotavirus(HRV)-induced diarrhea. Methods Sixty ICR suckling mice were randomly divided into two groups, and each was further divided into 3 subgroups(A, B, C for group Ⅰ, and D, E, F for group Ⅱ);group B, C, E and F were assigned as experimental groups. while group A and D were controls. Mice in group B and C were inoculated with3 × 10~5 PFU and 3×10~6 PFU HRV Wa strain fluid respectively when they were 4-day old. Mice in group E and F were inoculated with 3×10~5 PFU and 3×10~6 PFU HRV Wa strain fluid respectively when they were10-day old. The symptoms, fecal viral excretion, intestinal histopathologies and ultrastructures of animals were observed. One-way ANOVA was used to compare the differences among the groups. Results There were significant differences in the duration of diarrhea and viral excretion, jejunal villous height, the weight at d4 and d7 after inoculation among three subgroups in group Ⅰ(F=204.38, 86.60, 7.18, 18.41 and10.08, P<0.01). Diarrhea was not observed in group Ⅱ, and the differences in jejunal villous height and the weight at d4 and d7 after inoculation among three subgroups were not significant(F=0.16, 0.13 and 1.03, P>0.05). Compared with the group D, the duration of viral excretion was longer in group E and F(F=8. 34, P<0.01). Conclusion Animal model of HRV diarrhea can be established in 4-day-old ICR suckling mice infected with 3×10~6PFU HRV.
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Objective To investigate the protective effect of Qiweibaizhu Powder against HRV infection of intestinal mucosal epithelial cells of suckling mice.Methods The 5-day-old NIH mice were made HRV diarrhea model by oral infection,and randomly divided into six groups:normal group(NG),model group(MG),Qiweibaizhu Powder group(BG),ribavirin group(ZG),suckling mice in each group were instilled corresponding drugs 3 times/d(NG and MG with normal saline) through the mouth.5 d after treatment,suckling mice were killed,small intestine stool was taken to test HRV,and pathological changes in small intestinal mucosa were observed by HE staining.Results Intestinal faeces HRV clearance of BG was significantly better than MG(P
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Objective To study the pathogenesis of rotavirus(RV) diarrhea.Methods Simian rotavirus(SA-11) was grown in cultured MA-104 cell.The viral titers of the culture supernatant were determined by plaque forming assay.KM mice aged 7 days were inoculated with the viral supernatant via feeding tube(gavage).Histological and ultramicrostructure changes of the small intestines were observed under light microscope and electron microscope.The values of crypt depth and villi height were measured with software(image pro plus 5.1,IPP5.1).The distribution of the RV antigen in small intestine and the filamentous actin of the small intestine chorioepithelium were observed with immunohistochemical techniques.The apoptosis of the small intestine epithelium cells was observed with an in situ apoptosis detection kit.Results There were mild hyperemia,dropsy and extensive vacuolar degeneration of small intestine villi under light microscope.Plenty of lipid droplet-like structure at the top of the villi,microvilli malalignment or defluxion and enterocyte defluxion could be seen by electron microscope,but no obvious structure changes at the cell junctions were seen.The RV antigen mainly distributed at the top of the villi.The quantity of small intestine filamentous actin decreased and enterocyte apoptosis increased after RV infection.Conclusion RV mainly infects the mature villous epithelium.The presentation of RV diarrhea relates to the lesion of cytoskeleton,the microvilli lesion of the small intestine,enterocyte apoptosis and defluxion,villi atrophy,etc.,but may have no relationship with the structural changes of cell junctions in the small intestine epithelium.