应用Vero细胞分离新型布尼亚病毒

目的从不明原因发热伴血小板减少综合征患者血液中分离新型布尼亚病毒。方法应用Vero细胞培养法分离病毒,并用间接免疫荧光检测法(IFA)、实时荧光聚合酶链反应(Real-time PCR)技术进行鉴定。结果从24份不明原因发热患者血液标本中分离出6株发热伴血小板减少综合征布尼亚病毒。结论应用Vero细胞培养法可以直接从不明原因发热患者血液中分离发热伴血小板减少综合征布尼亚病毒。     

辽宁省肾综合征出血热沙鼠肾细胞、地鼠肾细胞灭活疫苗免疫效果评价

选择ＨＦＲＳ姬鼠型疫区盘锦大洼县、家鼠型疫区葫芦岛市，每个疫区选一试区与对照区，于每一试区分别接种ＨＦＲＳ长爪沙鼠肾细胞（Ⅰ型）和地鼠肾细胞（Ⅱ型）灭活疫苗各５０００人份。经过一个流行周年观察，显示两种疫苗安全，近期效果明显。沙鼠苗的中强反应率为２．１３％（５１／３５６），荧光抗体阳转率免前为６．２％（隐性感染），３针基础免疫后为１００％，一年后为５４．７６％，加强针后为１００％；抗体ＧＭＴ滴度分别为：３１．７、７９８、１６、５５７；中和抗体４次阳转率分别为０，７０．３７％、０、７３．３３％，抗体ＧＭＴ滴度分别为０、１０．７、０、１４．６。地鼠苗的中强反应率为３．９７％（５１／１２８４），荧光抗体４次阳转率分别为９．８８％（隐性感染）、１００％、３６．３６％、１００％；抗体ＧＭＴ滴度分别为３３．６、２３２、２５、３８７；中和抗体４次阳转率分别为０、５０％、１７．６５％、１００％，抗体ＧＭＴ滴度分别为０、９．３、２０、１５。但疫苗免疫针次、抗体持续时间、疫苗的远期流行病学效果等，有待进一步研究。     

Adapted focal experimental autoimmune encephalomyelitis to allow MRI exploration of multiple sclerosis features

We aimed to determine an optimal protocol for inducing a focal inflammatory lesion within the rat brain that could be large enough for an easier MRI monitoring while still relevant as a multiple sclerosis (MS) like lesion. We adapted a two-hit model based on pre-sensitization of the Lewis rat with myelin oligodendrocyte protein (MOG) followed by stereotaxic injection of pro-inflammatory cytokines (TNFα + IFNγ) within the internal capsule. We compared the following two strategies to increase focal lesion development for an easier MR translation: (1) a higher sensitization step (MOG50) or (2) a higher cytokine step with lower sensitization (MOG25). Control animals were administered only cytokines without MOG pre-sensitization. Animals were followed with T2, diffusion and T1 post gadolinium weighted images at 1, 3 and 7 days following cytokine injection. Immunostaining was performed at the same time points for macrophages (ED1), myelin (MBP and Luxol Fast Blue) and blood brain barrier integrity (IgG). At day 1, the focal lesions depicted with T2-weighted images were very similar among groups and related to vasogenic edema (high apparent diffusion coefficient (ADC), gadolinium enhancement and IgG extravasation) induced by cytokines irrespective of the pre-sensitization step. Then, at day 3, MOG50 rats developed statistically larger T2 lesions than MOG25 and control rats that were correlated with inflammatory cell accumulation. At day 7, MOG50 rats also showed larger T2 lesions than MOG25 and control rats, together with loss of anisotropy that were correlated with demyelination. In contrast, MOG25 and control rats developed similar MR lesions decreasing over time and almost undetectable at day 7. We conclude that with a high pre-sensitization step, the focal lesion can be monitored by MRI whose signal reflects some features of a MS-like lesion, i.e. edema, inflammatory cell accumulation and later demyelination.     

The distribution of DEN infected people in Dushan and Xingyi area of Yunnan-Guizhou Plateau, China

The dengue viruses （DEN, genus Flavivirus, family Flaviviridae） are mosquito borne and have caused 100 million cases of dengue fever each year in most tropical and subtropical areas of the world. However, in the Southwest area of Yunnan-Guizhou Plateau, China, the previous work demonstrated that different geographic strains of Aedes albopictus were susceptible to dengue virus. In this study, we collected 456 sera samples from patients with fever and 994 sera samples from healthy population in Dushan and Xingyi area of Yunnan-Guizhou Plateau, China. All sera samples were tested for dengue IgG by enzyme-linked immunosorbent assay （ELISA）. Patients＇ sera samples were tested for dengue IgM and DEN antigen was checked in the sera of 6 from 456 samples with which C6/36 cell in culated by IFA. The results indicate that these patients with fever were infected with DEN-2 and suggest that DEN infection had existed in Dushan and Xingyi area of Yunnan-Guizhou Plateau, China.     

Anti-influenza virus activity of the ethanolic extract from Peperomia sui

Ethnopharmacological relevance

Peperomia sui Lin and Lu (Peperomia sui), a well-known Taiwanese folk medicine, has a broad range of biological effects, especially in treatment of upper respiratory tract diseases. However, no previous study has explored the activity of Peperomia sui against influenza virus infections. This study was carried out to evaluate the anti-influenza virus activity and the potential virucidal effect of the ethanolic extract of Peperomia sui (PSE).

Methods

The anti-H6N1 avian influenza viral activity of PSE against the influenza virus A/Chicken/TW/0518/2011 (H6N1) in chicken fibroblast DF-1 cells was evaluated by cell viability assay, hemagglutination assay, neuraminidase activity assay, indirect immunofluorescence assay and quantitative RT-PCR assay.

Results

PSE significantly increased the viability of cells that were infected by the H6N1 virus. PSE also suppressed the synthesis of viral nucleoprotein (NP), and inhibited the growth of the virus in DF-1 cells. Further, PSE inhibited the neuraminidase activity of H6N1 virus.

Conclusions

The findings of this study provide important information for the exploitation and utilization of Peperomia sui in treatment of influenza infection.     

Functionally fused antibodies--a novel adjuvant fusion system

Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.     

Human papillomavirus 8 E6 disrupts terminal skin differentiation and prevents pro-Caspase-14 cleavage

Expression of the betapapillomavirus (betaPV) E6/E7 genes has been shown to impair both keratinocyte differentiation and apoptosis. Especially late-terminal keratinocyte differentiation shares certain aspects with apoptosis, such as fragmentation of DNA and activation of caspases. Here we investigated the disruption of keratinocyte differentiation in organotypic skin (raft) cultures of primary (PHK) and immortalized (N/TERT) human keratinocytes, in particular by human papillomavirus (HPV)8.Immunohistochemical analysis of HPV5 and HPV8 E6/E7-expressing PHK revealed thickening of the rafts and complete absence of stratum corneum formation, even after 18 days of culture. This phenotype was confirmed in N/TERT raft cultures. When expressed separately, the aberrant morphology was observed only in rafts expressing E6, not E7. Immunofluorescence analysis of HPV8 E6 PHK rafts showed an increase in number and size of Filaggrin- and Caspase-14-positive cells in the granular layer. In raft lysates analyzed by western-blot, the presence of pro-Caspase-14 in the differentiated keratinocytes was confirmed, but in the HPV8 E6 rafts none of the Caspase-14 subunits were detected.In conclusion, in the raft system, HPV8 E6 prevented late-terminal keratinocyte differentiation resulting in an accumulation of Filaggrin and pro-Caspase-14-positive cells in the absence of stratification. This differentiation arrest was accompanied by the failure to express Caspase-14 subunits, suggesting absence of Caspase-14 activation and probable abrogation of Filaggrin maturation in HPV8 E6-expressing keratinocytes.     

猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒(SFV)抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。