营养与病毒感染性疾病动物模型

当前新现病毒性疾病的研究最大的＂瓶颈＂在于没有胜任动物模型。建立在非人灵长类动物基础上的模型,虽然可以部分复制人类疾病特征,但其经济性欠佳且与动物权益的保护有所冲突;而啮齿类动物对新现病毒的易感性往往较低,也不能很好地复制人类疾病。本文对营养、免疫及疾病易感性关系研究的进展进行文献回顾,以发现解决当前难题的线索。     

中蜂囊状幼虫病毒RdRp基因dsRNA干扰的研究

中蜂囊状幼虫病毒(Chinese sacbrood virus,CSBV)可以引起中蜂幼虫死亡,给中蜂的养殖造成严重的威胁。其RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)是病毒复制中必不可少的中心酶,控制着病毒的复制和翻译过程。本研究以CSBV RdRp基因为靶标,选取两个干扰区域RdRp-1和RdRp-2,并构建相应的dsRNA表达载体,获取dsRNA后进行RNAi实验,通过qRT-PCR检测CSBV RdRp基因的表达情况。实验结果显示:干扰片段RdRp-1不能显著下调RdRp基因的转录,而干扰片段RdRp-2可显著性下调RdRp表达并具有剂量依赖性,当添食2μg dsRdRp-2时,在72 h RdRp基因表达下调了85%,CSBV的衣壳蛋白VP1基因下调表达78%,幼虫死亡率降低60%,表明RdRp基因可以作为RNA干扰的靶标用于CSBV防治,本研究为后期在养蜂场进行蜜蜂病毒病的防治奠定了基础。     

细胞水平狂犬病病毒感染的RNA干扰

以质粒pSilencer2.1-U6 Hygro为基础,设计并构建了针对狂犬病病毒(RV)糖蛋白和核蛋白mRNA的共9个siRNA表达载体,转染BHK-21细胞系后,在潮霉素-B的筛选压力下,获得9个稳定转录相关siRNA的BHK-21细胞株。1000TCID50的RV分别感染24孔板内的上述9株细胞,48h后以直接免疫荧光法检测各株细胞上RV的增殖,结果显示,在经不同siRNA表达载体转染的BHK-21细胞中,RV增殖水平有不同程度下降,RV增殖水平最低者为对照细胞的1%,即RNA干扰效应最高可阻断99%RV的感染。针对其中阻断水平超过90%的靶序列G69和N19,人工合成其双链siRNA,瞬时转染BHK-21细胞后,仍可达到80%以上的感染阻断率。本试验为有效阻断RV早期感染提供了新选择,为通过RNAi研究RV的基因组功能提供了新的依据。     

Recent Advances in Animal Models of Zika Virus Infection

An infection by Zika virus(ZIKV), a mosquito-borne flavivirus, broke out in South American regions in 2015, and recently showed a tendency of spreading to North America and even worldwide. ZIKV was first detected in 1947 and only 14 human infection cases were reported until 2007. This virus was previously observed to cause only mild flu-like symptoms.However, recent ZIKV infections might be responsible for the increasing cases of neurological disorders such as GuillainBarre′ syndrome and congenital defects, including newborn microcephaly. Therefore, researchers have established several animal models to study ZIKV transmission and pathogenesis, and test therapeutic candidates. This review mainly summarizes the reported animal models of ZIKV infection, including mice and non-human primates.     

裸鼠呼吸道合胞病毒感染的动物模型

呼吸道合胞病毒(RSV)是全世界婴幼儿下呼吸道感染的首位病毒病原体,免疫缺陷个体容易发生严重感染,目前尚无理想RSV感染动物模型用于研究.我们用细胞免疫缺陷裸鼠感染RSV,旨在建立理想的动物模型,为RSV感染的防治研究奠定基础.裸鼠滴鼻感染RSV后肺组织分离到病毒,直接免疫荧光检测到支气管肺泡灌洗液RSV抗原阳性,空斑形成实验检测肺组织病毒滴度在感染后第3天达高峰,并持续到第9天仍能检测到病毒.免疫组化检测RSV抗原主要分布在细支气管、毛细支气管和肺泡上皮细胞胞浆内.肺组织病理学显示RSV感染导致裸鼠淋巴细胞浸润为主的肺间质性炎症,电镜分析超微结构可见到细胞内病毒颗粒和气血屏障的破坏.支气管肺泡灌洗液白细胞计数显示裸鼠RSV感染炎症高峰在感染后第9天.裸鼠RSV感染的病毒复制和病理改变特点与人相似,病毒持续高水平复制,是客观而实用的评价抗RSV制剂效果的小鼠模型.     

裸鼠呼吸道合胞病毒感染的动物模型

呼吸道合胞病毒(RSV)是全世界婴幼儿下呼吸道感染的首位病毒病原体,免疫缺陷个体容易发生严重感染,目前尚无理想RSV感染动物模型用于研究。我们用细胞免疫缺陷裸鼠感染RSV,旨在建立理想的动物模型,为RSV感染的防治研究奠定基础。裸鼠滴鼻感染RSV后肺组织分离到病毒,直接免疫荧光检测到支气管肺泡灌洗液RSV抗原阳性,空斑形成实验检测肺组织病毒滴度在感染后第3天达高峰,并持续到第9天仍能检测到病毒。免疫组化检测RSV抗原主要分布在细支气管、毛细支气管和肺泡上皮细胞胞浆内。肺组织病理学显示RSV感染导致裸鼠淋巴细胞浸润为主的肺间质性炎症,电镜分析超微结构可见到细胞内病毒颗粒和气血屏障的破坏。支气管肺泡灌洗液白细胞计数显示裸鼠RSV感染炎症高峰在感染后第9天。裸鼠RSV感染的病毒复制和病理改变特点与人相似,病毒持续高水平复制,是客观而实用的评价抗RSV制剂效果的小鼠模型。     

RNA干扰在柯萨奇病毒致心肌炎小鼠模型中的抗病毒研究

为了研究慢病毒介导的shRNA(Short hairpin RNA,shRNA)在柯萨奇B组3型病毒(Coxsackievirus B3,CVB3)导致的心肌炎小鼠模型中的抗病毒作用,合成针对CVB3基因组3753～3771区域的慢病毒Lenti-sh3753,感染HeLa细胞后感染CVB3病毒,通过荧光显微镜观测shRNA的表达和病毒致细胞病变效应,并测定培养上清中的病毒滴度,将慢病毒Lenti-sh3753感染BALB/c小鼠后感染CVB3病毒,观察小鼠的存活率,心脏组织中的病毒滴度和病理变化。结果发现Lenti-sh3753能在HeLa细胞中表达shRNA,并能有效抑制细胞中病毒RNA的复制。在小鼠模型上,Lenti-sh3753能提高小鼠的存活率,降低心脏中的病毒含量,从而减轻病理反应。这些结果提示,Lenti-sh3753在细胞和动物模型中能针对性地降解CVB3病毒RNA,明显降低病毒滴度,有效控制病毒感染。     

建立H5N1流感病毒感染雪貂动物模型

目的建立甲型H5N1流感病毒感染雪貂动物模型[1-3]。方法 H5N1流感病毒株A/Vietnam/1203/2004病毒以103和104 TCID50滴度分别感染雪貂。对4～10月龄去势雪貂经兽用氯胺酮轻度麻醉后进行滴鼻感染,每个稀释度接种3只雪貂,感染后第5天安乐处死。感染后每天记录雪貂一般临床变化。感染前0 d采集鼻甲骨活检,感染后1～5 d鼻甲骨活检检测病毒载量和病毒滴度。处死时取雪貂气管、肺、心、肝、脾、肾、小肠、脑组织作病毒滴度检测和病理检查。结果 H5N1103和104 TCID50的病毒分别感染雪貂,雪貂死亡都率在33%。103TCID50和104 TCID50病毒分别感染雪貂,动物都出现持续3 d体温升高,104 TCID50组出现超过20%的体重下降。上呼吸道排毒呈现上升趋势,并可在除呼吸系统以外的组织器官中分离到病毒。感染的雪貂病理表现为重度肺炎。结论雪貂感染H5N1病毒株后在临床表现、病毒学、分子生物学、病理学方面的检测都可以证实雪貂感染H5N1病毒动物模型已建立,104 TCID50病毒滴度是一个建立感染动物模型比较合适的剂量。     

Absence of Interference During High-Multiplicity Infection by Clonally Purified Vesicular Stomatitis Virus

Stocks of vesicular stomatitis virus free of defective interfering particles were produced by serial clonal isolation. High-multiplicity infections with these stocks led to no interference or formation of defective interfering particles. Defective interfering particles were generated by three successive passages at high multiplicity.     

Prevention of Chinese Sacbrood Virus Infection in Apis cerana using RNA Interference

Chinese sacbrood virus (CSBV) is the pathogen of Chinese sacbrood disease, which poses a serious threat to honeybee Apis cerana, and tends to cause bee colony and even the whole apiary collapse. Here we report on prevention of CSBV infection by feeding second instar larvae of A. cerana with specific sequences of CSBV double-stranded RNA (dsRNA). Protection of the bee larvae from CSBV by ingestion of CSBV-derived dsRNA was further demonstrated by quantitative real-time PCR (qRT-PCR) and northern blot analysis. The result provides a potential method to protect A. cerana from CSBV infection.     

乙型肝炎病毒动物模型的研究现状

讨论了目前乙肝病毒动物模型建立的基本原理和方法,同时比较了各种模型的用途与优缺点,为研究者选择合适的动物模型提供了依据。     

Enhanced Gene Silencing in Cells Cured of Persistent Virus Infection by RNA Interference

We compared HEp-2-derived cells cured of persistent poliovirus infection by RNA interference (RNAi) with parental cells, to investigate possible changes in the efficiency of RNAi. Lower levels of poliovirus replication were observed in cured cells, possibly facilitating virus silencing by antiviral small interfering RNAs (siRNAs). However, green fluorescent protein (GFP) produced from a measles virus vector and also GFP and luciferase produced from plasmids that do not replicate in human cells were more effectively silenced by specific siRNAs in cured than in control cells. Thus, cells displaying enhanced silencing were selected during curing by RNAi. Our results strongly suggest that the RNAi machinery of cured cells is more efficient than that of parental cells.Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a natural biological phenomenon regulating a wide range of cellular pathways (8, 20). RNAi-based therapies with siRNAs or small hairpin RNAs (shRNAs) have been developed against several viral infections, and a reduction of the viral yield by several orders of magnitude has frequently been obtained (4, 9). However, virus clearance from cells and the complete cure of persistent virus infections have only rarely been reported (24, 25). We have developed several models of persistent virus infection by using poliovirus (PV), a positive-strand RNA virus of the Picornaviridae family (5, 7, 16, 21). We previously studied the effects of antiviral siRNAs applied months after the infection of HEp-2 cells with a persistent PV mutant (7, 25). We used a mixture (“the Mix”) of two synthetic siRNAs targeting the viral RNA genome in the 5′ noncoding (NC) region and the 3D polymerase (3D^pol) (siRNA-5′NC and siRNA-3D^pol, respectively; synthesized by Sigma-Proligo). When repeated transfections with the Mix were performed in persistently PV-infected cultures, most cultures stopped producing virus (25). Here, we investigate the important issue of changes in RNAi efficacy following siRNA treatment, 2 to 5 months after the cure. The efficiency of gene silencing in cells was stable during this period.We used the HEp-Q4 and -Q5 cell lines, which were cured of persistent PV infection after transfections with the Mix (25). The cured cells and their parental cell line, HEp-2, had similar growth rates (data not shown). To compare PV silencing efficiencies in the three cell lines, they were transfected either with the Mix or with an irrelevant siRNA (siRNA-IRR) in the presence of Lipofectamine 2000 (Invitrogen) in 24-well plates as previously described (25). Treated and mock-treated cells were infected 16 h posttransfection with PV strain Sabin 3, at a multiplicity of infection (MOI) of 1 50% infectious dose (ID₅₀) per cell. The viral progeny was titrated 24 h postinfection, as previously described (16). HEp-Q4 and HEp-Q5 were permissive to PV infection, although viral yields were about 1 log lower in these cells than in HEp-2 cells (Fig. ​(Fig.1A).1A). Virus silencing was observed in all three cell lines treated with the Mix; however, silencing was significantly more efficient in HEp-Q4 (≈2.2 times more efficient; P = 0.013, Student''s t test) and HEp-Q5 (≈5.6 times more efficient; P = 0.015) than in HEp-2 cells (Fig. 1A and B). Similar results were obtained with an shRNA (Thermo Scientific) targeting the same region as the siRNA-5′NC (data not shown).Open in a separate windowFIG. 1.Efficiency of enterovirus silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A) Yield of progeny virus produced by cells infected at an MOI of 1 ID₅₀, 16 h posttransfection with the antiviral Mix containing two anti-PV siRNAs (20 pmol), the irrelevant siRNA-IRR (20 pmol), or no siRNA. Samples were harvested 24 h postinfection. Each bar represents the mean value ± SEM of six infected cultures from three independent experiments. (B to E) For each cell line, silencing efficiency is expressed as the ratio of infectious virus yield (titer in ID₅₀/ml) in the presence of the irrelevant siRNA-IRR to infectious virus yield (titer in ID₅₀/ml) in the presence of the antiviral siRNAs in cured cells, normalized with respect to the silencing efficiency in HEp-2 cells. S2, PV strain Sabin 2. (F) GFP silencing efficiency for each cell line is expressed as a ratio [1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] in cured cells, normalized with respect to the efficiency of silencing in HEp-2 cells. Each bar represents the mean value ± SEM of at least four cultures from two independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.We investigated whether the differences in silencing efficacies between the three cell lines were due to differences in siRNA transfection efficiency by transfecting HEp-2, HEp-Q4, and HEp-Q5 cells with fluorescein isothiocyanate-conjugated siRNA (siRNA-FITC; 20 pmol/well; Cell Signaling) and testing them between 4 and 48 h posttransfection. The fluorescence of transfected cells was measured with a FACScan flow cytometer (Becton Dickinson), and data were analyzed with CellQuest software (Becton Dickinson). The percentages of siRNA-FITC-positive cells were similar for all cell types (Fig. ​(Fig.2A).2A). The mean fluorescence per positive cell and the percentage of cells displaying fluorescence peaked 16 and 24 h posttransfection, respectively, and decreased thereafter (Fig. ​(Fig.2).2). These findings suggest both that the presence of siRNAs in cells was similarly transient in the three cell types, as previously reported (27), and that the high silencing efficiencies in cured cells were not a consequence of higher transfection efficiencies. All subsequent experiments were performed between 16 and 40 h posttransfection.Open in a separate windowFIG. 2.Transfection efficiencies of fluorescein-conjugated siRNAs in HEp-2, HEp-Q4, and HEp-Q5 cells. A fluorescent siRNA-FITC (20 pmol) was used to transfect each of the three cell lines in the presence of Lipofectamine 2000. Fluorescent cells were analyzed 4 to 48 h posttransfection by using a FACScan flow cytometer (Becton Dickinson). The percentage of fluorescent cells (A) and the mean fluorescence per positive cell, in arbitrary units (B), are shown. Each bar represents the mean value ± SEM. (C) Representative FACS plots (cell granularity versus cell size), showing the similarities between the three cell populations.Fluorescence-activated cell sorting (FACS) plots for granularity versus cell size were very similar for the three cell lines (Fig. ​(Fig.2C),2C), as were those for cell numbers versus fluorescence (not shown), suggesting highly related cell populations. Although highly probable, it remains to be confirmed that the cured cells originated from a subpopulation of HEp-2 cells.Virus silencing was also investigated in cured cells infected with Sabin 2 or coxsackievirus A17 (CAV17) strain 67591 (22) or in cells transfected with Sabin 2 RNA. The experimental conditions used for Sabin 2 and CAV17 were identical to those for Sabin 3, except that only the 3D polymerase was targeted by siRNAs. Sabin 2 RNA (1 μg) was prepared as previously described (12) and used with siRNA-3D^pol (20 pmol/well) for the cotransfection of cells in the presence of Lipofectamine 2000. Virus yields were determined 7.5 h after transfection. In all cases, virus silencing was more effective in HEp-Q4 and -Q5 cells than in HEp-2 cells (Fig. 1C to E). Additional experiments were performed with a PV replicon encoding the green fluorescent protein (GFP), PV-eGFP (28) (2 μg/well), which was used with siRNA-eGFP (20 pmol/well; Ambion) for cotransfection. GFP fluorescence was measured by flow cytometry, 16 h after transfection. As for PV, a higher silencing efficiency was observed in cured cells than in HEp-2 cells (Fig. ​(Fig.1F1F).We then investigated whether the lower level of viral multiplication in HEp-Q4 and -Q5 cells in the absence of siRNAs involved an entry or postentry step. We quantified the expression of the PV receptor (CD155) at the surface of cells. We used flow cytometry after indirect immunofluorescence labeling with anti-CD155 antibodies, as previously described (16). More than 98.4% ± 2% (mean ± standard error of the mean [SEM]) of cured cells, like HEp-2 cells, tested positive for CD155 (data not shown). In the absence of siRNAs, a decrease in viral replication was also observed in HEp-Q4 and -Q5 cells infected with the Sabin 2 PV strain in cells, in which the early stages of the viral cycle were bypassed by transfection with Sabin 2 RNA, and in cells infected with the CAV17 virus, which uses a cell receptor other than CD155 (12) (data not shown). Together, these results suggest that PV multiplication is reduced at a postentry step, probably at replication, in cured cells.We investigated whether PV silencing was also enhanced in other HEp-derived cells in which Sabin 3 PV multiplication was reduced by using HEp-S31 (cl18) cells that had been cured of persistent PV infection by growth at a supraoptimal temperature rather than by RNAi (2). PV yield was ≈1.6 logs lower in HEp-S31 (cl18) cells than in HEp-2 cells (data not shown). Sabin 3 PV silencing in HEp-S31 (cl18) cells was 1.7 ± 0.9 times more effective (mean of six experiments) than that in HEp-2 cells (relative efficacy of 1) (data not shown), but this difference was not significant. However, these results do not exclude the possibility that reduced PV replication facilitates PV silencing by the Mix in cured cells. We therefore pursued our work with a different virus.We investigated whether the high silencing efficiency in HEp-Q4 and -Q5 cells was specific to enteroviruses by using a measles virus expressing GFP, MV-eGFP (26), and siRNA-eGFP to silence GFP expression. Cells were transfected with either siRNA-eGFP or siRNA-IRR, infected with MV-eGFP (1 ID₅₀ per cell, 16 h posttransfection), and the GFP silencing efficiency was determined 40 h posttransfection by flow cytometry. For each cell line, silencing efficiency was expressed as a percentage {[1 − (percentage of siRNA-eGFP-transfected cells expressing GFP)/(percentage of siRNA-IRR-transfected cells expressing GFP)] × 100}. GFP silencing was significantly stronger in HEp-Q4 cells (≈14%; P = 0.048) and HEp-Q5 cells (≈17%; P = 0.010) than in HEp-2 cells (Fig. ​(Fig.3A).3A). There was no significant difference in the silencing efficiency of GFP between HEp-Q4 and -Q5 cells (Fig. ​(Fig.3A).3A). The anti-PV Mix did not silence GFP expression (data not shown), indicating that the silencing of GFP was not due to anti-PV siRNAs persisting in cured cells months after the initial treatment.Open in a separate windowFIG. 3.Efficiency of GFP and luciferase silencing in HEp-2, HEp-Q4, and HEp-Q5 cells after transfection with specific siRNAs. (A and B) GFP silencing, expressed as a percentage calculated for each cell line as follows: {[1 − (GFP expression in the presence of siRNA-eGFP)/(GFP expression in the presence of the irrelevant siRNA-IRR)] × 100}. (A) Cells were infected 16 h posttransfection with a measles virus encoding eGFP (MV-eGFP [26]) at an MOI of 1 ID₅₀/cell, and fluorescent cells were analyzed 24 h after infection (40 h posttransfection). Each bar represents the mean value ± SEM of three independent experiments. (B) Cells were cotransfected with pEGFP-C1 and siRNA-eGFP or siRNA-IRR and analyzed 40 h later. Each bar represents the mean value ± SEM of four independent experiments. (C) Luciferase silencing efficiency for each cell line, expressed as the ratio of luciferase activity in the presence of the irrelevant siRNA-IRR to luciferase activity in the presence of the specific siRNAs in cured cells, normalized with respect to silencing efficiency in HEp-2 cells. Relative efficiencies are shown as in Fig. ​Fig.11 for luciferase, because the enzymatic reaction amplified the signal. Each bar represents the mean value ± SEM of triplicates from three independent experiments. *, P < 0.05 based on Student''s t test comparing HEp-Q4 and HEp-Q5 with HEp-2 cells.To test whether the high silencing efficiency in HEp-Q4 and -Q5 cells was dependent on viral infection, plasmid vectors pEGFP-C1 (Clontech Laboratories) and pRL-CMV (Promega) were used to generate GFP (6) and Renilla luciferase (18), respectively. These plasmids do not replicate in human cells. Cells (10⁶) were cotransfected with pEGFP-C1 (1 μg) and siRNAs (20 pmol) in the presence of Lipofectamine 2000, as recommended by the manufacturer. GFP fluorescence was analyzed by flow cytometry 40 h posttransfection. Silencing efficiencies were expressed as a percentage {[1 − (mean GFP levels in the presence of siRNA-eGFP)/(mean GFP levels in the presence of siRNA-IRR)] × 100)}. Mean silencing efficiency was significantly higher in HEp-Q4 (≈15%; P = 0.003) and HEp-Q5 (≈15%; P = 0.002) cells than in HEp-2 cells (Fig. ​(Fig.3B).3B). The efficiency with which the GFP encoded by pEGFP-C1 was silenced was similar in HEp-Q4 and -Q5 cells.The efficacy of siRNAs was then assessed with pRL-CMV, which encodes the Renilla luciferase and Silencer Renilla luciferase (AM4630; Ambion). Cells (10⁶) were cotransfected with the plasmid (100 ng) and either specific or irrelevant siRNA (7 pmol) in the presence of Lipofectamine 2000. Luciferase assays were performed with a Dual-Glo luciferase assay system (Promega), as recommended by the manufacturer at 40 h posttransfection, and luminescence was measured with a luminometer (Centro LB960; Berthold). The results of the sensitive luciferase assays confirmed that the relative efficiency of silencing was significantly higher in cured than in parental cells (Fig. ​(Fig.3C).3C). By contrast, results obtained in HEp-S31 (cl18) cells, cured without siRNAs, were not significantly different from those obtained in control HEp-2 cells (data not shown), strongly suggesting that the treatment of HEp-Q4 and -Q5 cells with specific siRNAs selected cells in which siRNAs mediated silencing more efficiently than in parental cells.The difference in silencing efficiency between cured and HEp-2 cells may be due to differences in the abundance and/or efficacy of cellular factors involved in gene silencing. Some major actors of the RNAi pathway, particularly those associated with the RNA-induced silencing complex (RISC), have been identified (3, 10, 13, 19). The active endonucleolytic core of the RISC includes the guide strand of the siRNA and a slicer protein called Argonaute 2 (Ago2) (17). We used Western blotting to study Ago-2 and other factors contributing to the function of RISC (3, 10, 11, 14, 19, 23): the endonuclease Dicer, the transactivation response RNA binding protein (TRBP), the protein activator of double-stranded RNA-dependent protein kinase (PACT), and the RNA helicase A (RHA) (Fig. ​(Fig.4).4). Exportin 5, which plays a role upstream from the dicing process in the export of small RNA precursors (29), was included as a control.Open in a separate windowFIG. 4.Comparative analysis of proteins involved in RNAi in HEp-2, HEp-Q4, and HEp-Q5 cell lines. Whole-cell lysates were tested for Exportin 5 (A), Dicer (B), Ago-2 (C), the helicase RHA (D), TRBP (E to H) and PACT (I) by Western blotting with the corresponding specific antibodies. Blots were subsequently stripped and reprobed with antiactin antibodies to confirm equal protein loading. (E and F) TRBP levels in HEp-Q4 and HEp-Q5 cells were determined by densitometry and are plotted in arbitrary units, as ratios relative to the level of actin and to the level of TRBP in HEp-2 cells. In panel F the symbols correspond to TRBP levels determined in nine different experiments. (G) TRBP levels in HEp-2 cells transfected with pcDNA-TRBP (14) and in cells cotransfected with pcDNA-TRBP and siRNA-TRBP. (H) TRBP levels were compared in human IMR5 cells, HEpS31 (cl18) cells previously cured of persistent PV infection by growth at a supraoptimal temperature, and the control HEp-2 cell line. TRBP/actin densitometry and PACT/actin densitometry results are indicated in arbitrary units in the histograms below the corresponding Western blot results shown in panels H and I.Proteins (30 to 50 μg) from each cell line were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10 to 20% Tricine gels; Invitrogen) and transferred to nitrocellulose membranes (Amersham Biosciences) as previously described (1). The membranes were incubated with one of the following primary antibodies (1): anti-Ago2 monoclonal antibody (MAb; Abcam), anti-RHA MAb (Abcam), and anti-TRBP2 MAb (Santa Cruz Biotechnology); rabbit antibodies against Dicer (Santa Cruz Biotechnology); anti-PACT MAb (Santa Cruz Biotechnology), and anti-Exportin 5 MAb (Abcam). The antiactin MAb (AC-40; Sigma-Aldrich) was used to check for equal protein loading. Membranes were then washed and treated with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 2 h at room temperature. Protein bands were detected with an enhanced chemiluminescence detection kit (ECL⁺; Amersham Biosciences) and a G:box (Syngene).Exportin 5, Dicer, Ago-2, and RHA were similarly abundant in all three cell lines (Fig. 4A to D), suggesting that quantitative differences in protein levels were unlikely to be responsible for the enhanced silencing in HEp-Q4 and -Q5 cells. There was significantly more TRBP in HEp-Q4 (≈21%; P = 0.026) and HEp-Q5 (≈28%; P = 0.016) cells than in HEp-2 cells, as indicated by the results of nine experiments (Fig. 4E and F). The specificity of the anti-TRBP antibody was checked on extracts of HEp-2 cells transfected with a plasmid encoding TRBP, pcDNA-TRBP (14), with and without silencing by siRNA-TRBP (Fig. ​(Fig.4G).4G). GFP silencing was not enhanced in HEp-2 cells overproducing TRBP, and it was not decreased by downregulating TRBP gene expression with siRNA-TRBP (data not shown). These results suggest that the high levels of TRBP in the cured cell lines are not the cause of the enhanced silencing in these cells.There was less TRBP protein in HEp-S31 (cl18) cells (2) than in HEp-2 and other control cells (IMR5) (Fig. ​(Fig.4H),4H), indicating that high levels of TRBP are not necessarily selected in cells persistently infected with PV. PACT was slightly downregulated in the cured cells (Fig. ​(Fig.4I).4I). Moreover, PACT is unlikely to be involved in the enhanced silencing in cured cells, because we used synthetic siRNAs and PACT functions principally during siRNA production by Dicer (14). We did not investigate the activities or subcellular distributions of the various factors involved in RNAi in the three cell lines, and they may differ. It is also possible that other factors, not tested here, contribute to the efficacy of siRNAs in cured cells. The molecular details of the mechanism involved remain to be determined.Overall, our results suggest that both a decrease in viral replication and the enhancement of gene silencing contributed to the mechanism by which cells persistently infected with poliovirus were cured by RNAi. Our results also indicate that cells displaying enhanced silencing may be selected during treatment with siRNAs. This may result in profound changes to cell phenotype, because RNAi plays an essential role in the regulation of cellular gene expression (15).     

Conformational Plasticity of Hepatitis C Virus Core Protein Enables RNA-Induced Formation of Nucleocapsid-like Particles

Many of the unanswered questions associated with hepatitis C virus assembly are related to the core protein (HCVcp), which forms an oligomeric nucleocapsid encompassing the viral genome. The structural properties of HCVcp have been difficult to quantify, at least in part because it is an intrinsically disordered protein. We have used single-molecule Förster Resonance Energy Transfer techniques to study the conformational dimensions and dynamics of the HCVcp nucleocapsid domain (HCVncd) at various stages during the RNA-induced formation of nucleocapsid-like particles. Our results indicate that HCVncd is a typical intrinsically disordered protein. When it forms small ribonucleoprotein complexes with various RNA hairpins from the 3′ end of the HCV genome, it compacts but remains intrinsically disordered and conformationally dynamic. Above a critical RNA concentration, these ribonucleoprotein complexes rapidly and cooperatively assemble into large nucleocapsid-like particles, wherein the individual HCVncd subunits become substantially more extended.     

Rabbit as a Novel Animal Model for Hepatitis E Virus Infection and Vaccine Evaluation

Background

The identification of hepatitis E virus (HEV) from rabbits motivated us to assess the possibility of using rabbits as a non-human primate animal model for HEV infection and vaccine evaluation.

Methodology/Principal Findings

First, 75 rabbits were inoculated with seven strains of genotypes 1, 3, 4, and rabbit HEV, to determine the appropriate strain, administrative route and viral dosage. Second, 15 rabbits were randomly divided into three groups and vaccinated with 0 µg (placebo), 10 µg and 20 µg of HEV candidate vaccine, HEV p179, respectively. After three doses of the vaccination, the rabbits were challenged with 3.3×10⁵ genome equivalents of genotype 4 HEV strain H4-NJ703. The strain of genotype 1 HEV was not found to be infectious for rabbits. However, approximately 80% of the animals were infected by two rabbit HEV strains. All rabbits inoculated with a genotype 3 strain were seroconverted but did not show viremia or fecal viral shedding. Although two genotype 4 strains, H4-NJ153 and H4-NJ112, only resulted in part of rabbits infected, another strain of genotype 4, H4-NJ703, had an infection rate of 100% (five out of five) when administrated intravenously. However, only two out of fifteen rabbits showed virus excretion and seroconversion when inoculated orally with H4-NJ703 of three different dosages. In the vaccine evaluation study, rabbits vaccinated with 20 µg of the HEV p179 produced anti-HEV with titers of 1∶10⁴–1∶10⁵ and were completely protected from infection. Rabbits vaccinated with 10 µg produced anti-HEV with titers of 1∶10³–1∶10⁴ and were protected from hepatitis, but two out of the five rabbits showed virus shedding.

Conclusions/Significance

Rabbits may be served as an alternative to the non-human primate models for HEV infection and vaccine evaluation when certain virus strains, appropriate viral dosages, and the intravenous route of inoculation are selected.     

Interference of Hepatitis B Virus with Cellular Signaling

The presence of hepatitis B virus (HBV) proteins leads to changes in the cellular gene expression. As a consequence, the cellular signaling processes are influenced by the actions of HBV proteins. It has been shown that HBV nucleocapsid protein and the amino-terminal part of polymerase termed as terminal protein (TP) could inhibit interferon signaling. Further, the global gene expression profiles differ in hepatoma cells with and without HBV gene expression and replication. The expression of interferon (IFN) stimulated genes (ISGs) was differently regulated in cells with HBV replication and could be modulated by antiviral treatments. The HBV TP has been found to modulate the ISG expression and enhance the HBV replication. The modulation of the cellular signaling processes by HBV may have significant implications for pathogenesis.