病毒性肝炎树鼩动物模型研究与建模策略

病毒性肝炎是由肝炎病毒引起的肝脏疾病。在我国,病毒性肝炎高度流行,其中又以乙型肝炎病毒(Hepatitis B virus,HBV)和丙型肝炎病毒(Hepatitis C virus,HCV)危害较大。动物模型是研究疾病感染与发病机制,进行药物与疫苗研究的必要工具。目前病毒性肝炎实验动物模型的研究已取得长足的发展,主要集中于病毒在动物体内的感染特性及发病规律方面。本文仅就病毒性肝炎动物模型,尤其乙型、丙型肝炎树鼩动物模型的研究及建模策略进行综述。     

反义技术在抗HBV感染中的应用

反义技术是近些年来随着现代分子生物学技术的发展而产生的新的生物医学治疗技术。它采用反义核酸分子抑制、封闭或破坏靶基因组的技术手段,包括反义寡核苷酸、核酶及RNA干扰等。反义分子通过与靶基因异性互补配对结合,阻断靶基因的复制、转录或翻译过程,从而发挥抗病毒作用。针对乙型肝炎病毒的反义技术也有了广泛而深入的研究。根据反义技术在分子、细胞以及动物水平上的研究表明：反义技术能够高效、特异地抑制HBV的复制与表达。     

反义核酸技术应用难点及研究进展

反义核酸技术已被广泛用于治疗药物、药物靶点确认、探知病理基因的表达。目前对其作用原理的研究集中于其被吸收入细胞的机制、在细胞内的分布、反义核酸序列的最佳长度和性质,并针对体内可能抑制反义核酸活性的影响因素,采取了各种相应的反义核酸优化技术,如对反义核酸的化学修饰、联结高效的转运载体、确定最佳的反义结合位点等,通过这些技术来提高其体内稳定性、跨细胞转运的效率,识别靶序列的特异性,以获得更多更好的反义药物投入实用。     

载体疫苗,反义治疗及抗病育种的研究进展

载体疫苗、反义治疗及抗病育种的研究进展张德礼（北京军区后勤部军马防治检验所,１０００７１）近年来,国内外在利用载体技术制各重组疫８、利用反义技术治疗疾病以及通过转基因技术进行抗病育种方面的研究取得了重大进展。一、病毒载体疫苗载体疫苗是以病毒或细菌作载...     

miRNA-21反义核酸对人淋巴瘤Raji细胞的生长抑制作用研究

为研究以miRNA-21为靶标的反义核酸（AMO-miR-21）对淋巴瘤Raji细胞的生长抑制作用,使用LipofectamineTM 2000将化学合成的反义核酸转染Raji细胞,采用四甲基偶氮唑蓝（MTT）法和台盼蓝拒染法检测细胞生长抑制率;实时定量PCR技术检测细胞miRNA-21水平改变;PI和Annexin V双染,流式细胞仪检测细胞凋亡.结果显示转染Raji细胞48～72h,反义核酸组细胞生长受到明显抑制,反义核酸抑制细胞活性的最佳浓度是0.4μmol/L;细胞内miRNA-21的表达水平明显下调;细胞凋亡明显增加;此外,反义组细胞中抑癌基因Pdcd4的mRNA和蛋白质呈高水平表达.提示以miRNA-21为靶标的反义核酸是人淋巴瘤Raji细胞生长的有效抑制剂和凋亡诱导剂.miRNA-21可能是淋巴瘤治疗的潜在靶标,其通过下调抑癌基因Pdcd4的表达水平发挥抗肿瘤作用.     

丙型肝炎病毒分子生物学研究进展

丙型肝炎病毒(HCV)是经血源传播的一类肝炎病毒。1989年美国Chiron公司Choo等率先将HCV cDNA克隆成功,使HCV成为第一个利用分子生物学技术而发现的病毒。近两年来,HCV研究的进展十分迅速,已成为病毒性肝炎研究领域中的一个热点。本文就HCV分子生物学的研究进展作一综述。     

反义核酸在肿瘤研究中的应用

反义核酸研究已活跃于肿瘤研究及基因治疗领域,反义核酸通过碱基配对待异性地抑制基因表达,因此为研究肿瘤中癌基因和生长因子的功能及癌基因突变检测提供了更为有效的手段,并为肿瘤的基因治疗提供了可能途径.文章综述了反义核酸在基因治疗中所面临的问题及部分解决办法.     

HBV和HCV的合并感染

乙型肝炎病毒和丙型肝炎病毒合并感染是病毒性肝炎的常见形式。本文就合并感染的流行病学、病毒间相互作用、临床致病、治疗预防等研究进展进行简要综述。     

反义核酸的骨架修饰及其应用

反义核酸的发展经历了反义寡核苷酸,混合骨架寡核苷酸和多肽核酸等几个阶段。这３种不同类型的反义核酸均能与ＤＮＡ或ＲＮＡ结合,阻断目的基因的表达。３种反义核酸的结构有较大差异,各自的性质和反义作用机理也不尽相同。尽管作用机制还不十分明确,反义核酸已广泛应用于生物学和医学等领域,作为反义药物用于治疗癌症等疾病,或作为试剂研究生物大分子的功能。     

靶向C—Raf-1基因的反义核酸抗乙型肝炎病毒活性研究

目的：通过体内外实验验证靶向c-Raf-1基因的反义核酸是否具有抑制乙型肝炎病毒（HBV）的活性。方法：设计靶向c-Raf-1基因的反义核酸,并在细胞水平进行体外抗HBV活性筛选,通过RT-PCR检测c-Raf-1基因mRNA水平的变化,通过体内药效学实验进一步验证反义核酸的抗HBV效果。结果：经体外筛选,靶向c-Raf-1基因的反义核酸Raf-3145具有相对明显的抑制HBV表面抗原（HBsAg）的作用,并可剂量依赖性地抑制c-Raf-1基因的表达;体内药效学结果显示,反义核酸Raf-3145在30 mg/kg剂量下对HBsAg的表达具有一定的抑制作用。结论：经体内外活性评价,初步确定了靶向宿主基因c-Raf-1的反义核酸具有一定的抑制HBsAg表达的活性,也进一步验证了c-Raf-1基因可以作为抗HBV药物设计的候选靶点。     

Haemosporidian parasites depress breeding success and plumage coloration in female barn swallows Hirundo rustica

Parasites are major effectors of natural selection and also play a role in sexual selection processes. Haemosporidian blood parasites are common in vertebrates and have been shown to vary in their effects depending on both the parasite and host species, on the host trait investigated as well as on host condition and stage of infection. Here we investigated infection of adult barn swallows Hirundo rustica by Plasmodium, Leucocytozoon and Haemoproteus species during the chronic stage of infection and the consequences for host fitness traits. Prevalence was higher than 10% only for Plasmodium. Chronic stage infection by Plasmodium was associated with reduced female breeding success, but did not affect breeding dates. Infection did not affect the expression of male secondary sexual traits (tail length and melanin‐based plumage coloration), but was associated with paler coloration of females. Finally, we found a negative effect of infection by Plasmodium on feather growth rate in older but not in yearling individuals. Because feathers are moulted during wintering in sub‐Saharan Africa where infection of barn swallows by Plasmodium occurs, our results suggest that male secondary sexual traits have little potential to reveal acute‐stage infection whereas plumage coloration of females may advertise their infection status. In addition, these results suggest that infection by Plasmodium can influence the course of plumage moult. Thus, our results add to the observations of negative effects of haemosporidian infection on fitness traits in birds and provides evidence that these effects can vary among traits and in relation to age and sex.     

Laodelphax striatellus Atg8 facilitates Rice stripe virus infection in an autophagy-independent manner

Rice stripe virus(RSV)is the causative agent of rice stripe disease and is completely dependent on insect vectors for its plant-to-plant transmission.Laodelphax striatellus is the major insect vector for RSV.In this study,we explored the interactions be-tween RSV infection and L.striatellus autophagy,a potential intrinsic antiviral mechanism in insects.We found that L.striatellus autophagic activity did not affect RSV infection;however,the autophagy related-8(Atg8)gene significantly enhanced virus infection.Dur-ing RSV initial infection within the L.striatellus midgut,silencing of Atg8 expression significantly decreased the phosphorylation of c-Jun N-terminal kinase(p-JNK);however,when RSV infection is absent,silencing of Atg8 did not alter p-JNK levels.Thesc results indicated that Atg8 might activate the JNK machinery by allowing more virus infection into cells.We further revealed that Atg8-deficiency significantly decreased RSV accumu-lation on the surface of the insect midgut epithelial cells,suggesting a receptor trafficking function of the y-aminobutyric acid receptor-associated protein family.Using the RSV ovary entry as a model,in which vitellogenin receptor(V gR)mediates RSV cell entry,we clarified that Atg8-deficiency decreased the abundance of V gR localizing on the cytomem-brane and disturbed the attachment of RSV in the germarium zones.Collectively,these results revealed an autophagy-independent function of L.striatellus Atg8 that enhances RSV initial infection by increasing virus attachment on the infection sites.     

ARGONAUTE 2 increases rice susceptibility to rice black-streaked dwarf virus infection by epigenetically regulating HEXOKINASE 1 expression

ARGONAUTE (AGO) proteins play crucial roles in plant defence against virus invasion. To date, the role of OsAGO2 in rice antiviral defence remains largely unknown. In this study, we determined that the expression of OsAGO2 in rice was induced upon rice black-streaked dwarf virus (RBSDV) infection. Using transgenic rice plants overexpressing OsAGO2 and Osago2 mutants generated through transposon-insertion or CRISPR/Cas9 technology, we found that overexpression of OsAGO2 enhanced rice susceptibility to RBSDV infection. Osago2 mutant lines exhibited strong resistance to RBSDV infection through the elicitation of an early defence response, including reprogramming defence gene expression and production of reactive oxygen species (ROS). Compared to Nipponbare control, the expression level of OsHXK1 (HEXOKINASE 1) increased significantly, and the methylation levels of its promoter decreased in the Osago2 mutant on RBSDV infection. The expression profile of OsHXK1 was the opposite to that of OsAGO2 during RBSDV infection. Overexpression of OsHXK1 in rice also induced ROS production and enhanced rice resistance to RBSDV infection. These results indicate that OsHXK1 controls ROS accumulation and is regulated by OsAGO2 through epigenetic regulation. It is noteworthy that the Osago2 mutant plants are also resistant to southern rice black-streaked dwarf virus infection, another member of the genus Fijivirus. Based on the results presented in this paper, we conclude that OsAGO2 modulates rice susceptibility to fijivirus infection by suppressing OsHXK1 expression, leading to the onset of ROS-mediated resistance. This discovery may benefit future rice breeding programmes for virus resistance.     

Dynamic infection of Verticillium dahliae in upland cotton

• Verticillium wilt, an infection caused by the soilborne fungus Verticillium dahliae, is one of the most serious diseases in cotton. No effective control method against V. dahliae has been established, and the infection mechanism of V. dahliae in upland cotton remains unknown.
• GFP‐tagged V. dahliae isolates with different pathogenic abilities were used to analyse the colonisation and infection of V. dahliae in the roots and leaves of different upland cotton cultivars, the relationships among infection processes, the immune responses and the resistance ability of different cultivars against V. dahliae.
• Here, we report a new infection model for V. dahliae in upland cotton plants. V. dahliae can colonise and infect any organ of upland cotton plants and then spread to the entire plant from the infected organ through the surface and interior of the organ.
• Vascular tissue was found to not be the sole transmission route of V. dahliae in cotton plants. In addition, the rate of infection of a V. dahliae isolate with strong pathogenicity was notably faster than that of an isolate with weak pathogenicity. The resistance of upland cotton to Verticillium wilt was related to the degree of the immune response induced in plants infected with V. dahliae. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton. These results provide a theoretical basis for studying the mechanism underlying the interaction between V. dahliae and upland cotton.

     

The Lon-2 protease of Borrelia burgdorferi is critical for infection in the mammalian host

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. To date, the contribution of Lon-2 to B. burgdorferi fitness and infection remains unexplored. Herein, we showed that expression of lon-2 was highly induced during animal infection, suggesting that Lon-2 is important for B. burgdorferi infection. We further generated a lon-2 deletion mutant. Compared with that of wild-type (WT) strain, the infectivity of the mutant was severely attenuated in a murine infection model. Although no growth defect was observed for the mutant in normal BSK-II medium, resistance of the lon-2 mutant to osmotic stress was markedly reduced. In addition, when exposed to tert-Butyl hydroperoxide, survival of the lon-2 mutant was impaired. In addition, we found that the protein levels of RpoS and RpoS-dependent OspC were decreased in the mutant. All these phenotypes were restored to WT or near-WT levels when lon-2 mutation was complemented in cis. Taken together, these results demonstrate that Lon-2 is critical for B. burgdorferi to establish infection and to cope with environmental stresses. This study provides a foundation for further uncovering the direct link between the dual roles of Lon-2 in protein quality control and bacterial pathogenesis.     

Anaplasma phagocytophilum infection induces apoptosis in HL-60 cells

Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis, is an obligate intracellular bacterium that survives in neutrophils by delaying apoptosis. The human promyelocytic leukemia cell line HL-60 has been the ultimate choice for culturing Anaplasma in vitro. In this study, we assessed the various events of drug-induced apoptosis in A. phagocytophilum-infected HL-60 cells. Anaplasma infection reduced the cell viability and increased the apoptosis in HL-60 cells and staurosporine or etoposide-induced apoptosis was further exacerbated with Anaplasma infection. Altogether our results suggest that A. phagocytophilum infection is proapoptotic in HL-60 cells unlike in neutrophils where it is antiapoptotic.     

Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against influenza virus infection

Aims: To evaluate the protective effects of oral administration of milk fermented with a Lactococcus strain against influenza virus (IFV) infection in a mouse model. Methods and Results: Milk fermented with exopolysaccharide‐producing Lactococcus lactis subsp. cremoris (L. cremoris) FC was orally administered to BALB/c mice for 12 days. Mice were intranasally infected with IFV A/New Caledonia/20/99 (H1N1) on day 8, and survival was determined for 14 days after IFV infection. Survival rate and body weight loss after IFV infection in the L. cremoris FC fermented milk‐administered group were significantly improved compared with those in the control group. In the unfermented milk‐administered group, survival rate was not improved, whereas body weight loss was slightly improved compared with that in the control group. The mean virus titre in the lung of the L. cremoris FC fermented milk‐administered group 3 days after infection was significantly decreased compared with that in the control group. Conclusions: These results suggest that oral administration of milk fermented with L. cremoris FC protects mice against IFV infection. Significance and Impact of the Study: These results demonstrate that oral administration of milk fermented with exopolysaccharide‐producing Lactococcus strains might protect host animals against IFV infection.     

Distribution and occurrence of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in the predator beetle Rhizophagus grandis G: yll. (Coleoptera: Rhizophagidae)-rearing laboratories

The distribution and occurrence of the insect pathogenic algae Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) in the predator beetle Rhizophagus grandis (Coleoptera: Rhizophagidae)-rearing laboratories were studied and reported here for the first time. The insect pathogenic alga Helicosporidium sp. infection was observed in all R. grandis-rearing laboratories. The infection rate reached more than 20% which is significant among the samples in some R. grandis-rearing laboratories. The infection rates of the examined beetles showed noticeable differences between localities and years. There was no significant difference in the infection levels of male and female beetles. These results showed that Helicosporidium sp. is one of the factors that decrease efficiency of the R. grandis-rearing laboratories.