Rapid Internal Control Reference Recombinase-Aided Amplification Assays for EBV and CMV Detection / 生物医学与环境科学（英文）

Epstein-Barr virus (EBV) and cytomegalovirus (CMV), two of the most prevalent human herpesviruses, cause a wide spectrum of diseases and symptoms and are associated with serious health problem. In this study, we developed an internal control reference recombinase-aided amplification (ICR-RAA) assay for the rapid detection of EBV and CMV within 30 min. The assay had a sensitivity of 5 and 1 copies/test for EBV and CMV, respectively, with no cross reaction with other pathogens. In comparison with those of the commercial quantitative polymerase chain reaction (qPCR), the sensitivity of the EBV and CMV ICR-RAAs using extracted DNA was 93.33% and 84.84%, respectively; the specificity was 98.75% and 100.00%, respectively; and the Kappa values were 0.930 and 0.892 (

Role of TGF-β1 in hepatic fibrosis induced by echinococcus multilocular infection / 医学研究生学报

ObjectiveTo investigate the role of transforming growth factor (TGF-β1) in hepatic fibrosis induced by echinococcus multilocular infection and its possible mechanisms in this process.Methods Forty-five C57BL/6 mice were randomly divided into the model group（30）and the control group (15). Protoscolece suspension of echinococcus multilocular was infused through portal vein in the model group (4000/each). Mice in the control group was injected the same volume of normal saline solution. Six mice in the model group and 3 mice in the control group were sacrificed at 1, 2, 4, 8 and 12 weeks after infection. The liver tissues were observed the histopathological changes by using hematoxylin-eosin (H&E) staining. The fibrosis degree and glycogen synthesis function of liver tissue were observed by Sirius-red staining and Periodic acid schiff (PAS), respectively. The expression levels of TGF-β1 and a-smooth muscle actin (α-SMA) were measured by immunohistochemical staining.ResultsThe obvious abnormal changes were not observed in 1 week after model setup. The diffuse distribution of multiple white spots began to appear at 2 weeks, but the amount of white plaques decreased after 8 weeks. Meanwhile, forming small lesions were not obviously observed the boundary with the surrounding normal liver tissue. Clear echinococcal vesicles were seen at week 12. H&E staining showed that hepatic tissue structure of control group was normal. In the model group, the number of lesions with worms decreased gradually and amount of granulomas were increased. The inflammatory lesions did not change significantly. Sirius-red staining demonstrated that collagen deposition in the control group was mainly around the bile duct and blood vessels. However, the deposition in the model group was mainly around the lesion and the degree of fibrosis became more serious with time. PAS staining displayed that the content of glycogen in the liver tissues of the control group was rich, evenly distributed and stained uniformly. However, the glycogen staining positive area decreased with the time of infection and the staining became lighter in the model group. Immunohistochemical staining indicated that the positive expression of α-SMA and TGF- β1 in the control group were mainly found in the bile ducts and perivascular areas. The positive areas in the model group were mostly granulomatous areas around the metacercariae and fibroblasts. Expression of α-SMA and TGF- β1 increased over time after infection with the expression peak at 12 weeks(16.80±2.09、4.10±2.14).ConclusionThe degree of fibrosis in liver tissues at different time points was consistent with the expression trend of TGF- β1 and α-SMA. TGF-β1 may promote collagen deposition and lead to fibrosis by activating hepatic stellate cells.

Exhaustion of CD8 T cell immune functions in spleen of mice with different doses of Echinococcus multilocularis infections / 中国血吸虫病防治杂志

ObjectiveTo examine the changes in the immune functions of CD8+ T cells in the spleen of mice following Echinococcus multilocularis infections at various doses and at different time points. MethodsThe E. multilocularis protoscoleces were collected, and E. multilocularis infection was modeled in mice via the hepatic portal vein at doses of 50 (low-dose), 500 (medium-dose) and 2 000 protoscoleces (high-dose), while physiological saline served as controls. Mouse spleen was isolated 2 (earlystage), 12 (middle-stage) and 24 weeks post-infection (late-stage), and spleen lymphocytes were harvested. The phenotype of memory CD8+ T cells and 2B4 expression were quantified in the mouse spleen, and the secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-17A and IL-10 was measured. Results A central-memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the early stage of high-dose protoscolece infections, and the proportion of central-memory CD8+ T cells was significantly greater in the high-dose group than in the control group (35.50% ± 2.00% vs. 25.90% ± 2.46%, P < 0.01), while a effector- memory phenotype was predominant in the CD8+ T cells in the spleen of mice at the late stage of medium- and high-dose protoscolece infections, and the proportions of effector-memory CD8+ T cells were significantly greater in the medium- (25.70% ± 4.12%) and high-dose group (28.40% ± 4.12%) than in the control group (10.50% ± 6.45%) (P < 0.05). The proportions of the central-memory CD8+ T cells were significantly higher in the high-dose group than at middle and late stages than at the early stage (P < 0.01), and the proportion of effector-memory CD8+ T cells was significantly greater in the high-dose group at the late stage than at early and middle stages (P < 0.05). The secretion of IFN-γ and IL-17A by spleen CD8+ T cells was elevated in the low- and medium-dose groups at the early stage of infection, and high-dose protoscolece infection promoted the secretion of IFN-γ and TNF-α by spleen CD8+ T cells; however, the levels of IFN-γ and TNF-α were significantly lower at the late stage than at the early and middle stages (P < 0.05). In addition, high 2B4 expression was detected in spleen CD8+ T cells in the middle- and high-dose groups at the late stage of infection, and the 2B4 expression was significantly higher in the medium(4.73% ± 1.56%) and high-dose groups (4.94% ± 1.90%) than in the low-dose group (2.49% ± 0.58%) and the control group (2.92% ± 0.60%) (P < 0.05). Conclusions E. multilocularis may be killed and eliminated through the host immune responses at the middle and late stages of low- and medium-dose protoscolece infections, while high-dose protoscolece infections may trigger the upregulation of 2B4 expression in mouse spleen CD8+ T cells at the late stage, which leads to immune exhaustion and the resultant chronic infections.

Development of an Internally Controlled Reverse Transcription Recombinase-aided Amplification Assay for the Rapid and Visual Detection of West Nile Virus / 生物医学与环境科学（英文）

West Nile virus (WNV) causes West Nile fever and West Nile encephalitis. Because infection by WNV creates serious public health problems, its simple, rapid, and visual detection is very important in clinical practice, especially in resource-limited laboratories. We have developed a rapid, specific, and highly sensitive internally controlled reverse transcription recombinase-aided amplification (RTRAA) assay to detect WNV, using both real-time fluorescence and the lateral flow dipstick (LFD) at 39.0 °C for 30 min. The analytical sensitivity of the RT-RAA assay was 10 plasmid copies and 1.6 pfu per reaction with real-time fluorescence, and 1,000 plasmid copies per reaction with the LFD. No crossreaction with other control viruses was observed. Compared with the RT-qPCR assay, the RT-RAA assay demonstrated 100% sensitivity and 100% specificity for WNV.