卡维地洛对柯萨奇病毒B3感染小鼠儿茶酚胺及IL-6表达水平的影响

目的：研究卡维地洛对急性柯萨奇B3（CVB3）病毒性心肌炎小鼠的儿茶酚胺及炎症因子IL-6表达水平的影响。方法： 随机将80只雄性BALB/C小鼠分为3组：正常对照组（n=20）,心肌炎组（n=30）,卡维地洛组（n=30）。后两组经腹腔接种CVB3诱发急性病毒性心肌炎,感染 24 h 后卡维地洛组每日灌胃给予卡维地洛 10 mg/kg,连续 14 d。于接种第7和14天随机从各组抽取8只小鼠取血后处死并留取心脏等标本。比较干预组与对照组心肌病理改变,采用高效液相色谱 电化学法检测去甲肾上腺素含量,采用RT-PCR法检测心肌IL-6mRNA的表达,采用酶联免疫吸附实验方法检测心肌IL-6蛋白的表达。结果：与心肌炎组比较,卡维地洛组心肌病理损伤明显减轻。与正常组比较,病毒性心肌炎小鼠血浆去甲肾上腺素明显升高,卡维地洛干预后小鼠血浆去甲肾上腺素明显下降（P〈0．05）。与正常组比较,心肌炎组心肌IL-6表达明显上调（P〈0．05）。与心肌炎组比较,卡维地洛组IL-6表达明显下调（P〈0．05）。结论：卡维地洛通过抑制儿茶酚胺对心肌的毒性作用以及下调心肌IL-6表达水平,减轻心肌炎小鼠的心肌损害。     

Effect of prophylactically applied CpG ODN on the development of myocarditis in mice infected with Coxsackievirus B3

Coxsackievirus B3 was one of the major pathogens causing viral myocarditis. Toll-like receptor 9 activation contributed to the innate immune response in the process of CVB3-induced myocarditis. In order to find out how CpG oligodeoxynucleotide, known as a TLR-9 agonist, would affect the CVB3-induced myocarditis, we chose a C-type CpG oligodeoxynucleotide (YW002) injected to the mice one day before CVB3 challenge. On day 4 post CVB3 infection, 3 mice in each group were randomly sacrificed and their hearts were isolated to detect CVB3 replication. On day 10, the CVB3 neutralizing antibody and inflammatory change of the hearts were detected. The results indicated that the CVB3-induced myocarditis was aggravated with the declining body weight of mice, decreasing neutralizing antibody, and uncontrolling virus replication by injecting 20 μg YW002 per mouse. When adjusted the amount at 10 μg YW002 per mouse, there were no signs of aggravation in myocarditis. Plus, the mortality of the infected mice was reduced, the neutralizing antibody level was raised and the replication of virus was restrained. These results suggested that a proper amount of CpG oligodeoxynucleotide application could help to inhibit CVB3 infection.     

黄芪三萜皂苷对CVB3诱导病毒性心肌炎的保护作用研究

目的：研究黄芪三萜皂苷（Astragalus saponins,AST）对CVB3病毒诱导病毒性心肌炎的保护作用及机制。方法：利用CVB3病毒、原代心肌细胞及小鼠构建病毒性心肌炎模型并使用AST进行干预,在实验过程中记录小鼠的生存率和体质量变化,体外超声评价小鼠的心脏功能及检测心肌蛋白中LDH和CK-MB水平。心肌组织天狼星红染色评价纤维化水平及TUNNEL染色检测心肌凋亡情况。Western Blot检测心肌蛋白中Caspase-3和Fas表达情况以研究AST保护病毒性心肌炎的机制。结果：AST能够显著增加CVB3注射后的小鼠生存率、缓解CVB3诱导小鼠的体质量减轻。体外超声结果显示,AST能够显著改善CVB3诱导小鼠心脏的收缩功能障碍。CBV3诱导组小鼠心肌蛋白中LDH和CM-KB水平较正常小鼠显著增加,而AST对此有显著的抑制作用。心肌组织病理染色结果显示,AST对CVB3诱导的心肌扩张和纤维化具有显著的保护作用。CVB3诱导组小鼠心肌组织的凋亡水平显著高于正常小鼠,而AST对此有显著的抑制作用。在CVB3感染的心肌组织中,Caspase-3和Fas表达水平显著的升高,而AST能够显著的抑制CVB3诱导的二者在心肌组织中的表达。结论：AST通过提高小鼠的生存率、抑制心肌扩张、心肌组织纤维化和心肌细胞凋亡对CVB3诱导的病毒性心肌炎具有显著的保护作用,这种作用可能与抑制CVB3诱导的Caspase-3和Fas在心肌组织中表达有关。     

Treatment of mice with dextran sulfate sodium-induced colitis with human interleukin 10 secreted by transformed Bifidobacterium longum

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) the etiology of which has not yet been fully clarified. Cytokine interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascade in UC and is likely a candidate for therapeutic intervention. However, its intravenous administration is costly and inconvenient. Therefore, we established a novel IL-10 delivery system by transforming a hIL-10-containing plasmid into B. longum (BL-hIL-10) and investigated its effects on 5% dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and the possible underlying mechanism. Our results show that (1) hIL-10 was expressed and secreted into the culture supernatant of BL-hIL-10 after L-arabinose induction in vitro as examined by Western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR; (2) addition of BL-hIL-10 culture supernatant had no cytotoxic effect and morphological alteration, but significantly inhibited the enhancement of proinflammatory cytokines by lipopolysaccharide (LPS) in THP-1 cells; (3) oral administration of BL-hIL-10 alleviated colitis syndrome of the model mice, attenuated colitis-activated NF-κB pathway measured by DNA-binding assay and colitis-elevated expression of proinflammatory cytokines examined with CCK cytotoxic kits, and upregulated CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes measured by flow cytometry. In conclusion, BL-hIL-10 as a novel oral hIL-10 delivery system has been successfully established and oral administration of BL-hIL-10 alleviated inflammatory damage of colonic tissue in the model mice by blocking the colitis-activated NF-κB pathway and upregulating CD4+CD25+Foxp3+ Treg in blood and mesenteric lymph nodes in mice.     

The comparison of α-bromo-4-chlorocinnamaldehyde and cinnamaldehyde on coxsackie virus B3-induced myocarditis and their mechanisms

Early experiments showed cinnamaldehyde had obvious therapeutic effect on viral myocarditis, but cinnamaldehyde was unstable in vivo. To overcome this limitation, we used cinnamaldehyde as a lead compound to synthesize α-bromo-4-chlorocinnamaldehyde (BCC). In the present study, we compared the therapeutic effects of BCC with cinnamaldehyde on coxsackie virus B3 (CVB3)-induced viral myocarditis (VMC), as well as investigated the possible mechanism. The antiviral and cytotoxic effects in vitro were evaluated on HeLa cells infected by CVB3 and rat cardiomyocytes respectively. Our results showed that IC50 were 0.78±0.13 μM and 48.16±5.79 μM in BCC and cinnamaldehyde-treated cells. 50% toxic concentration (TC) in BCC-treated cells was 22-fold higher than in the cinnamaldehyde group. In vivo BALB/c mice were infected with CVB3 for establishing VMC models. The results demonstrated that BCC reduced the viral titers and cardiac pathological changes in a dose-dependent manner. Myocardial virus titers were significantly lower in the 50 mg/kg BCC-treated group than in cinnamaldehyde groups. In addition, BCC could significantly inhibit the replication of CVB3 mRNA and the secretion of inflammatory cytokines TNF-α, IL-β and IL-6 in CVB3-infected cardiomyocytes. We further observed that BCC suppressed CVB3-induced NF-κB activation, IκB-α degradation and phosphorylation in a concentration-dependent manner, and reduced Toll like receptor (TLR) 4 protein level in hearts. These results suggest that BCC had a promising therapeutic effect on VMC with a highly significant favorable effects and less toxicity than cinnamaldehyde. Furthermore, the effect of BCC on VMC might be through inhibition of inflammatory signaling.     

卡维地洛和美托洛尔对病毒性心肌炎小鼠IL-12与IFN-γ表达水平的影响

目的：对比研究卡维地洛和美托洛尔对柯萨奇B3（CVB3）病毒性心肌炎小鼠的作用。方法：随机将110只雄性BALB/C小鼠分为4组：正常对照组（n=20）,心肌炎组（n=30）,卡维地洛组（n=30）与美托洛尔组（n=30）。后3组经腹腔接种CVB3诱发急性心肌炎,感染24h后卡维地洛组与美托洛尔组分别每日灌胃给予卡维地洛10mg/kg、美托洛尔30mg/kg,直至第14天末,分别于接种第7天和第14天随机从各组抽取若干只小鼠取血后处死并留取心脏等标本。结果：卡维地洛组显著减轻心脏病理损伤,显著降低心脏重量/身体重量（HW/BW）比值,但美托洛尔组与心肌炎组比较无统计学差异;卡维地洛干预后第7天小鼠心肌IFN-γ与IL-12含量显著升高,MDA含量显著降低,但美托洛尔组与心肌炎组比较无统计学差异;干预后第14天卡维地洛组SOD含量显著增加、MDA含量显著降低,美托洛尔组SOD和MDA含量与心肌炎组比较无统计学差异。结论：卡维地洛通过增加心肌IFN-γ与IL-12表达以及抗氧化损伤的作用,减轻心肌炎小鼠的心肌损害,起到保护性作用,而美托洛尔无类似作用。     

Low-dose inorganic mercury increases severity and frequency of chronic coxsackievirus-induced autoimmune myocarditis in mice

Mercury is a widespread environmental contaminant with neurotoxic impacts that have been observed over a range of exposures. In addition, there is increasing evidence that inorganic mercury (iHg) and organic mercury (including methyl mercury) have a range of immunotoxic effects, including immune suppression and induction of autoimmunity. In this study, we investigated the effect of iHg on a model of autoimmune heart disease in mice induced by infection with coxsackievirus B3 (CVB3). We examined the role of timing of iHg exposure on disease; in some experiments, mice were pretreated with iHg (200 μg/kg, every other day for 15 days) before disease induction with virus inoculation, and in others, they were treated with iHg after the acute (viral) phase of disease but before the development of dilated cardiomyopathy (DCM). iHg alone had no effect on heart pathology. Pretreatment with iHg before CVB3 infection significantly increased the severity of chronic myocarditis and DCM compared with control animals receiving vehicle alone. In contrast, treatment with iHg after acute myocarditis did not affect the severity of chronic disease. The increased chronic myocarditis, fibrosis, and DCM induced by iHg pretreatment were not due to increased viral replication in the heart, which was unaltered by iHg treatment. iHg pretreatment induced a macrophage infiltrate and mixed cytokine response in the heart during acute myocarditis, including significantly increased interleukin (IL)-12, IL-17, interferon-γ, and tumor necrosis factor-α levels. IL-17 levels were also significantly increased in the spleen during chronic disease. Thus, we show for the first time that low-dose Hg exposure increases chronic myocarditis and DCM in a murine model.     

Cardiomyocyte-specific deletion of Senp2 contributes to CVB3 viral replication and inflammation

Viral myocarditis (VMC) is characterized by cardiac inflammation and excessive inflammatory responses after viral infection. SENP2, a deSUMO-specific protease, has been reported to regulate antiviral innate immunity. This study aimed to investigate whether SENP2 affects CVB3-induced VMC. We generated a CVB3-induced VMC mouse model in 6-week-old cardiomyocyte-specific Senp2 knockout mice. The mice were sacrificed at days 0, 2, 4 and 6 after CVB3 infection. The survival rate, body weight, myocardial histopathological changes, viral load, cytokine levels and antiviral gene expression in cardiac tissues of both groups were investigated. Our study indicated that the expression of Senp2 in primary cardiomyocytes was upregulated by CVB3 infection. Moreover, deletion of Senp2 in the heart exacerbated CVB3 infection-induced myocarditis, facilitated CVB3 viral replication and downregulated the expression of antiviral proteins. In conclusion, our findings suggest a protective role for SENP2 in CVB3-induced VMC.     

Oral administration of Bifidobacterium longum ameliorates influenza virus infection in mice

We investigated whether the oral administration of Bifidobacterium longum BB536 could ameliorate influenza virus (IFV) infection in a mice model. Mice were orally administrated BB536 or saline for 2 weeks and then infected with IFV. Orally administered BB536 significantly alleviated symptoms, reduced the loss of body weight, and inhibited viral proliferation in the lungs relative to the control group findings. Histopathological findings in the lungs were improved in the BB536 group compared to control group findings. There was no significant difference in the levels of interleukin-6 (IL-6), interferon-γ (IFN-γ), IL-10 and IL-12p40 in the lungs between the groups, but the levels of IL-6 and IFN-γ were lower (p=0.076, 0.103, respectively) in the BB536 group compared with those of control group. The levels of IL-6 and IL-10 correlated significantly with the values of weight loss, and the levels of IFN-γ correlated with the virus titers in the lungs. These results suggested the potential of the oral administration of BB536 in ameliorating IFV infection and the possible involvement of anti-inflammatory effects of BB536 in the anti-infection effects against IFV.     

蛋白酶体抑制剂MG-132对柯萨奇B3病毒性心肌炎小鼠的作用研究

目的：研究泛素蛋白酶体抑制剂MG-132对柯萨奇B3（CVB3）病毒性心肌炎小鼠的作用,探讨泛素蛋白酶体系统在病毒性心肌炎发病学中的作用机制。方法：随机将80只雄性BALB／C小鼠分为4组（,2—20）：正常对照组、心肌炎组、心肌炎＋处理组、正常＋处理组。腹腔接种CVB3诱发急性心肌炎,次日腹腔注射MG-132,0．75mg／kg;连续给药7d,对照组腹腔注射PBS。第8天小鼠取材,观察心脏病理变化,测定心肌CVB3病毒复制及血清肌钙蛋白、脑钠肽水平。结果：MG-132显著减轻心肌炎小鼠心脏病理损伤,显著降低心脏重量／身体重量比值,MG-132干预后第8天小鼠血清肌钙蛋白、脑钠肽水平显著降低,同时荧光定量PCR显示CVB3mR—NA复制水平显著降低。结论：MG-132通过抑制CVB3病毒复制,显著减轻心肌炎小鼠心脏病理损伤,起到保护心肌作用。     

Selenium and viral virulence

A mouse model of coxsackievirus-induced myocarditis is being used to investigate nutritional determinants of viral virulence. This approach was suggested by research carried out in China which showed that mice fed diets composed of low selenium ingredients from a Keshan disease area suffered more extensive heart damage when infected with a coxsackie B4 virus than infected mice fed the same diet but supplemented with selenium by esophageal intubation. Selenium deficiency in our mice increased the virulence of an already virulent strain of coxsackievirus B3 (CVB3/20) and also allowed conversion of a non-virulent strain (CVB3/0) to virulence. Such conversion of CVB3/0 was accompanied by a change in the viral genome to more closely match that of the virulent virus, CVB3/20. As far as the authors are aware, this is the first report of host nutrition influencing the genetic make-up of an invading pathogen. Nutritionists may need to consider this mechanism of increased viral virulence in order to gain a better understanding of diet/infection relationships.     

Coxsackievirus B3 infection promotes generation of myeloid dendritic cells from bone marrow and accumulation in the myocardium

Myocarditis is associated with increased number of CD4⁺ T cells in the myocardium after coxsackievirus B3 (CVB3) infection. Previous studies show that CD11c⁺ myeloid dendritic cells (mDC) loaded with myosin could induce myocarditis. This study aims to investigate the generation and accumulation of mDC in CVB3-induced myocarditis. The presence of mDC in myocardium was detected by immunohistochemisty. Bone marrow-derived mDC were generated from uninfected and CVB3-infected mice. The percentage of CD11c⁺ mDC on cultured cells and mean fluorescence index (MFI) of double positive cells (CD11c⁺CD40⁺, CD11c⁺CD80⁺) were measured by flow cytometry. The expression of chemokine receptors (CCR5, CCR7) on mDC and chemokines (CCL4, CCL19) in the myocardium was detected. The migration of mDC in response to CCL4 or CCL19 was measured by chemotaxis assay. Mature mDC were elevated in the myocardium from CVB3-infected mice. The percentage of mDC generated from CVB3-infected group was increased. The MFI of CD11c⁺CD40⁺ and CD11c⁺CD80⁺ was increased in CVB3-infected group. The mDC showed a down-regulation of CCR5 and unaffected CCR7 mRAN levels associated with elevated CCL4 and CCL19 in the myocardium in CVB3-infected group. Numbers of migrating bone marrow-derived mDC from CVB3-infected mice were increased in vitro. We conclude that CVB3 infection induced the greater generation of mDC from bone marrow and accumulation of mature mDC in myocardial tissues. This migration was associated with increased levels of both CCL4 and CCL19 in the heart tissue. These suggest that blocking the migration of mDC may provide a novel therapy for myocarditis.     

Phyllaemblicin B inhibits Coxsackie virus B3 induced apoptosis and myocarditis

Coxsackie virus B3 (CVB3) is believed to be a major contributor to viral myocarditis since virus-associated apoptosis plays a role in the pathogenesis of experimental myocarditis. In this study, we investigated the in vitro and in vivo antiviral activities of Phyllaemblicin B, the main ellagitannin compound isolated from Phyllanthus emblica, a Chinese herb medicine, against CVB3. Herein we report that Phyllaemblicin B inhibited CVB3-mediated cytopathic effects on HeLa cells with an IC₅₀ value of 7.75 ± 0.15 μg/mL. In an in vivo assay, treatment with 12 mg kg⁻¹ d⁻¹ Phyllaemblicin B reduced cardiac CVB3 titers, decreased the activities of LDH and CK in murine serum, and alleviated pathological damages of cardiac muscle in myocarditic mice. Moreover, Phyllaemblicin B clearly inhibited CVB3-associated apoptosis effects both in vitro and in vivo. These results show that Phyllaemblicin B exerts significant antiviral activities against CVB3. Therefore, Phyllaemblicin B may represent a potential therapeutic agent for viral myocarditis.     

白藜芦醇抗柯萨奇病毒感染性小鼠心肌炎的实验研究

目的：研究白藜芦醇抗柯萨奇病毒感染性小鼠心肌炎的干预作用并探计其作用机理。方法：对Balb/c小鼠采用柯萨奇病毒CVB3腹腔内注射染毒，制作病毒感染性心肌炎小鼠动物实验模型，采用黄芪煎液作为中药治疗对照，观察白藜芦醇饮水经口给药对模型小鼠的一般情况、生存率、肝脾大体及血清丙二醛(MDA)、一氧化氮(NO)等生化指标及心脏组织病理学的影响。结果：与黄芪煎液中药治疗对照相比，白藜芦醇对柯萨奇病毒感染性心肌炎小鼠的一般情况、生存率、心肝脾大体及血清丙二醛(MDA)、一氧化氮(NO)等生化指标及心脏组织病理学均有良好的改善作用。结论：白藜芦醇对柯萨奇病毒感染性小鼠心肌炎有良好的拮抗作用，对病毒性心肌炎的防治有一定意义。     

Treg responses are associated with PM2.5-induced exacerbation of viral myocarditis

Abstract

The adverse cardiovascular events induced by ambient fine particles (PM_2.5) are paid more attention in the world. The current study was conducted to explore the mechanisms of T regulatory cells (Treg) responses in PM_2.5-induced exacerbation of viral myocarditis. The male BALB/c mice were administered an intratracheal (i.t.) instillation of 10?mg/kg b.w. PM_2.5 suspension. Twenty-four hours later, the mice were injected intraperitoneally (i.p.) with 100?μl of coxsackievirus B3 (CVB3) diluted in Eagle's minimal essential medium (EMEM). Seven days after the treatment, serum, splenetic, and cardiac tissues were examined. The results showed that pre-exposure to PM_2.5 aggravated the cardiac inflammation in the CVB3-infected mice along with an increase of Treg cells in the spleen. The mRNA expressions of interleukin-6 (IL-6), TNF-α, transforming growth factor-β (TGF-β), and Foxp3 were up-regulated in the PM_2.5-pretreated mice than that in the CVB3-treated mice. Similar results were found in the sera. In addition, compared with the CVB3-treated mice, the cardiac protein expression of TGF-β increased in the PM_2.5-pretreated mice. These results demonstrated that preexposure to PM_2.5 exacerbated virus-induced myocarditis possibly through the depression of the immune response and increase of inflammation in myocardium through the Treg responses.     

MYOCARDITIS AS SYSTEMIC DISEASE: NEW PERSPECTIVES ON PATHOGENESIS

1. Myocarditis may be an early indicator of or may subsequently lead to dilated cardiomyopathy in humans. This hypothesis has evolved from research on viruses that induce myocarditis, wherein the coxsackie B group viruses (CVB) in the family Picornaviridae are the most common known viral infectants of heart muscle. 2. Many competing hypotheses exist as to the pathogenesis of CVB3-induced myocarditis, including direct virus-induced myocyte damage and immunopathological disease with autoimmune sequelae. Evidence to support the direct-damage and viral RNA-persistence hypothesis is derived from in situ hybridization and gene amplification studies. 3. Recent use of terminal deoxynucleotidyl transferase-mediated nick-end labelling indicates that this injury in target organs is largely non-apoptotic in nature. Most apoptotic bodies in cardiac tissue are derived from immune cells. 4. Beyond infection of heart muscle, CVB3 can also associate with, infect and persist in cells of immune origin. The CVB3 localizes to follicles in spleens and lymph nodes of the murine host and this particular localization may continue in mice susceptible to more aggressive myocarditis. Whether virus-immune cell association in these compartments is advantageous (or essential) to the host in the evolution of anti-viral immune responses or whether it is more advantageous to the virus in immunosuppression of the host is not known. 5. We suggest that CVB3 can directly perturb or alter the immune response, thereby delaying viral clearance from vulnerable systemic organs. Both host and viral genetic factors can influence susceptibility, persistance and disease progression. 6. Picornaviruses use a unique method for the initiation of translation, involving the internal binding of the ribosome on a sequence element of the 5′ untranslated region, termed an internal ribosome entry sit. (IRES). 7. The IRES of CVB3 is located at approximately stem loops G, H and I, spanning nucloetides 530 and 630. Arrest of host translation is also a feature of picornavirus infection. Such regulation of host cell translation machinery no doubt fosters viral replication at the expense of the host cell. 8. Differences between cell types in the mechanisms, along with those at other key steps in the viral life cycle and in signalling via kinase pathways, may determine viral tropism and cellular destruction and the physiological outcome of neighbourin. cells.     

Astragaloside IV exerts antiviral effects against coxsackievirus B3 by upregulating interferon-gamma

Coxsackievirus B3 (CVB3) is a major pathogen for viral myocarditis and dilated cardiomyopathy in children and young adults. The aim of this study was to determine the antiviral effects of astragaloside IV against CVB3, and the underlying mechanism. First, we evaluated antiviral effects of astragaloside IV in vitro by measuring the virus titers of CVB3 in primarily cultured myocardial cells infected with CVB3, and in vivo by assessing the morbidity, mortality, heart-to-body weight ratio (HW/BW), and virus titers in BALB/c mice infected with CVB3. Then, we performed serum pharmacological experiments by testing the effect of sera from SD rats treated with astragaloside IV on proliferation of CVB3 in primarily cultured myocardial cells. Finally, we determined the effect of astragaloside IV on IFN-gamma mRNA expression in the hearts of infected BALB/c mice. We observed that astragaloside IV decreased virus titers of CVB3 in primarily cultured myocardial cells. Morbidity, mortality, HW/BW, and virus titers all decreased, and necrosis and mononuclear cell infiltration were alleviated in CVB3-infected mice treated with astragaloside IV, compared with those infected mice without the treatment. In addition, proliferation of CVB3 was inhibited by the sera of rats treated with astragaloside IV. Moreover, we observed that IFN-gamma mRNA expression was increased in mice treated with astragaloside IV. Therefore, we conclude that astragaloside IV exerts antiviral effects against CVB3 by upregulating expression of IFN-gamma mRNA.     

The genus Bifidobacterium for cancer gene therapy

A fundamental obstacle in cancer gene therapy is the specific targeting of therapy to solid tumor; as yet, no systemic delivery system exists. Bifidobacterium longum, a strain of domestic bacteria that is non-pathogenic and anaerobic, selectively localized to and proliferated in solid tumors after systemic application. We propose a novel approach to cancer gene therapy in which anaerobic bacteria of the genus B longum are used to achieve tumor-specific gene delivery and enzyme-prodrug therapy.     

The human fatty acid synthase: a new therapeutic target for coxsackievirus B3-induced diseases?

Coxsackievirus is linked to a large variety of severe human and animal diseases such as myocarditis. The interplay between host factors and virus components is crucial for the fate of the infected cells. However, host proteins which may play a role in coxsackievirus-induced diseases are ill-defined. Two-dimensional gel electrophoresis of protein extracts obtained from coxsackievirus B3 (CVB3)-infected and uninfected HeLa or HepG2 cells combined with spot analysis revealed several proteins which are exclusively up-regulated in infected cells. One of these proteins was identified as the fatty acid synthase (FAS). By using cerulenin and C75, two known inhibitors of FAS we were able to significantly block CVB3 replication. FAS appears to be directly involved in CVB3-caused pathology and is therefore suitable as a therapeutic target in CVB3-induced diseases.     

Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs

Coxsackievirus B3 (CVB3) is a common pathogen of myocarditis. We previously synthesized a siRNA targeting the CVB3 protease 2A (siRNA/2A) gene and achieved reduction of CVB3 replication by 92% in vitro. However, like other drugs under development, CVB3 siRNA faces a major challenge of targeted delivery. In this study, we investigated a novel approach to deliver CVB3 siRNAs to a specific cell population (e.g. HeLa cells containing folate receptor) using receptor ligand (folate)-linked packaging RNA (pRNA) from bacterial phage phi29. pRNA monomers can spontaneously form dimers and multimers under optimal conditions by base-pairing between their stem loops. By covalently linking a fluorescence-tag to folate, we delivered the conjugate specifically to HeLa cells without the need of transfection. We further demonstrated that pRNA covalently conjugated to siRNA/2A achieved an equivalent antiviral effect to that of the siRNA/2A alone. Finally, the drug targeted delivery was further evaluated by using pRNA monomers or dimers, which carried both the siRNA/2A and folate ligand and demonstrated that both of them strongly inhibited CVB3 replication. These data indicate that pRNA as a siRNA carrier can specifically deliver the drug to target cells via its ligand and specific receptor interaction and inhibit virus replication effectively.