BACKGROUND:The Chinese tree shrew (Tupaia belangeri chinesis) is a rising experimental animal and has been used for studying a variety of human diseases, such as metabolic and viral infectious diseases.METHODS:In this study, we established an immortalized tree shrew hepatic cell line, ITH6.1, by introducing the simian virus 40 large T antigen gene into primary tree shrew hepatocytes (PTHs).RESULTS:The ITH6.1 cell line had a stable cell morphology and proliferation activity. This cell line could be infected by enterovirus 71 (EV71), but not hepatitis C virus (HCV), although the known HCV entry factors, including CD81, SR-BI, CLDN1 and OCLN, were all expressed in the PTHs and ITH6.1 of different passages. Comparison of the transcriptomic features of the PTHs and different passages of the ITH6.1 cells revealed the dynamic gene expression profiles during the transformation. We found that the DNA replication- and cell cycle-related genes were upregulated, whereas the metabolic pathway-related genes were downregulated in early passages of immortalized hepatocytes compared to the PTHs. Furthermore, expression of hepatocytes function-related genes were repressed in ITH6.1 compared to that of PTHs.CONCLUSION:We believe these cellular expression alterations might cause the resistance of the ITH6.1 cell to HCV infection. This tree shrew liver cell line may be a good resource for the field.KEY POINTS:• A tree shrew hepatic cell line (ITH6.1) was established. • ITH6.1 cells could be infected by EV71, but not HCV. • ITH6.1 had an altered expression profiling compared to the primary hepatocytes.

The pathological manifestations of fatal cases of human hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) are characterized by inflammatory damage to the central nervous system (CNS). Here, the dynamic distribution of EV71 in the CNS and the subsequent pathological characteristics within different regions of neonatal rhesus macaque brain tissue were studied using a chimeric EV71 expressing green fluorescence protein. The results were compared with brain tissue obtained from the autopsies of deceased EV71-infected HFMD patients. These observations suggested that the virus was prevalent in areas around the blood vessels and nerve nuclei in the brain stem and showed a preference for astrocytes in the CNS. Interestingly, infected astrocytes within the in vivo and in vitro human and macaque systems exhibited increased expression of excitatory neurotransmitters and cytokines that also stimulated the neuronal secretion of the excitatory neurotransmitters noradrenalin and adrenalin, and this process most likely plays a role in the pathophysiological events that occur during EV71 infection.

Background: Mumps, a communicable, acute and previously well-controlled disease, has had recent and occasional resurgences in some areas.Methods: A randomized, double-blind, controlled and multistep phase I study of an F-genotype attenuated mumps vaccine produced in human diploid cells was conducted. A total of 300 subjects were enrolled and divided into 4 age groups: 16-60 years, 5-16 years, 2-5 years and 8-24 months. The groups were immunized with one injection per subject. Three different doses of the F-genotype attenuated mumps vaccine, A (3.5 +/- 0.25 logCCID(50)), B (4.25 +/- 0.25 logCCID(50)) and C (5.0 +/- 0.25 logCCID(50)), as well as a placebo control and a positive control of a licensed A-genotype vaccine (S79 strain) were used. The safety and immunogenicity of this vaccine were compared with those of the controls.Results: The safety evaluation suggested that mild adverse reactions were observed in all groups. No serious adverse event (SAE) was reported throughout the trial. The immunogenicity test showed a similar seroconversion rate of the neutralizing and ELISA antibody in the 2- to 5-year-old and 8- to 24-month-old groups compared with the seroconversion rate in the positive control. The GMT of the neutralizing anti-F-genotype virus antibodies in the vaccine groups was slightly higher than that in the positive control group.Conclusions: The F-genotype attenuated mumps vaccine evaluated in this clinical trial was demonstrated to be safe and have effective immunogenicity vs. control.

Enterovirus 71 (EV71) is the major pathogen responsible for fatal hand, foot and mouth disease (HFMD). Our previous work reported on an EV71-infected rhesus monkey infant model that presented with histo-pathologic changes of the central nervous system (CNS) and lungs. This study is focused on the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs) from EV71-infected rhesus monkey infants. The expression of more than 500 functional genes associated with multiple pathways was modulated. The expression of genes associated with immune inflammatory responses was up-regulated during the period from days 4 to 10 post-infection. The expression of two genes (TAC1 and IL17A), which play major roles in inflammatory reactions, was remarkably up-regulated during the infection period. Furthermore, a higher expression level of the TAC1 gene was identified in the CNS compared to the lungs, but a high expression level of the IL-17A gene was observed in the lungs and not in the CNS. The results of this study suggest at least two facts about EV71 infection, which are that: the TAC1 gene that encodes substance P and neurokinin-A is present in both PBMCs and the hypothalamus; and the up-regulation of IL-17A is sustained in the peripheral blood.

Preliminary studies of the major pathogen enterovirus 71 (EV71), a member of the Picornaviridae family, have suggested that EV71 may be a major cause of fatal hand, foot and mouth disease cases. Currently, the role of the pathological changes induced by EV71 infection in the immunopathogenic response remains unclear. Our study focused on the interaction between this virus and immunocytes and indicated that this virus has the ability to replicate in CD14(+) cells. Furthermore, these EV71-infected CD14(+) cells have the capacity to stimulate the proliferation of T cells and to enhance the release of certain functional cytokines. An adaptive immune response induced by the back-transfusion of EV71-infected CD14(+) cells was observed in donor neonatal rhesus monkeys. Based on these observations, the proposed hypothesis is that CD14(+) cells infected by the EV71 virus might modulate the anti-EV71 adaptive immune response by inducing simultaneous T-cell activation.

Enterovirus 71 (EV71) has been demonstrated as the major pathogen causing circulating hand-foot-mouth disease (HFMD) and leading to fatal cases in the Asian-Pacific region. However, it remains unclear about the mechanisms of EV71 infecting the nervous system and inducing complications of the central nervous system. Based on the previous work about EV71 infecting astrocytes, with a small interfering RNA (siRNA) library containing 212 phosphatase genes being screened, the expression of some phosphatase genes in astrocytes derived from rhesus monkey brains were down-regulated by siRNA, the proliferation characteristic of EV71 in the cells was compared, and further analysis of the phosphatases was conducted, which can directly effect the EV71 proliferation by varying its expression level and activity. The results demonstrated that there are a number of tyrosine phosphatases and Serine/Threonine phosphatases related to intracellular signal regulation pathway and they play a regulating role in EV71 proliferation. Furthermore, the virus also has effect on the immunoreactivity of infectious astrocytes through the interaction with these phosphotases.

Although clinical trials for the enterovirus type 71 (EV71) inactivated vaccine have been progressing, the potential mechanism of EV71 infection and its associated pathogenesis are not well-characterized in terms of comprehensive analysis of the induced immune response, which is generally recognized as an important indicator of the safety of vaccines. To investigate the Th1/Th2 response following viral challenge in neonatal rhesus monkeys immunized with different doses of EV71 inactivated vaccines, the variety of different Th1 and Th2 cytokines in the organs or tissues of the monkeys were identified. The results suggest that depending on the viral challenge, the Th1/Th2 reaction induced by different doses of EV71 inactivated vaccine varies. More specifically, there is an enhanced immune response in 80EU- and 1280EU-immunized monkeys, whereas 320EU immunization induces a mild response. Although there is no direct impact on the variation in immune protection induced by the vaccine, the Th1 reaction functions in T-cell cytotoxicity, which will aid further investigation of the pathogenic characteristics of small pathological changes in the central nerves system (CNS) likely induced by the Th1 response.

To find an optimal method to split influenza virus by Triton-X100,different concentrations of TritonX-100 were applied to split 3 different influenza virus,which were recommended by WHO as the 2007—2008 influenza vaccine production strains.The effect of split was observed by an electronic microscope.The immunity effect can detected after using the antigen that purified by sucrose density gradient centrifuge to immune the animal.It was showed that influenza B could be completely split by 1% Triton X-100,while influenza A-H1N1 and H3N2,could be completely split by 2% Triton X-100,which indicated that for different influenza strains,Triton X-100 performed different effect.Because the influenza vaccine production strains change every year,to insuring the quality of influenza ytic vaccine,it is necessary to investigate the different split concentrations for newly added strains,.

Objective To prepare Vero cell-adapted influenza H1N1 virus strain by reassortment and lay a foundation of production of influenza vaccine using Vero cells as a substrate.Methods SPF chick embryo and Vero cells were co-infected with Vero cell-adapted influenza virus strain A / Yunnan / 1 / 2005Va(H3N2)and strain A / Caledonia / 20 / 99(H1N1)recommend by WHO for production of influenza vaccine in the Northern Hemisphere during 2007～2008.The reassortants were screened with antibody against strain A / Yunnan / 1 / 2005Va(H3N2)subcultured in Vero cells for 12 passages and identified for subtype by HI test and RT-PCR.The viruses before and after reassortment were cultured in a large quantity in chick embryos and Vero cells respectively, and inactivated with formaldehyde.ICR mice were immunized with the prepared inactivated influenza vaccines to observe the im-munogenicities.Results A Vero cell-adapted influenza H1N1 virus strain was obtained, of which the HI titer was maintained at 512 after subculture for 9 passages, and the immunogenicity showed no significant difference with that before reassortment.Conclusion The Vero cell-adapted epidemic strain of influenza virus may be obtained by reassortment of Vero cell-adapted and epidemic strains.