Scutellaria baicalensis Inhibits Coxsackievirus B3-Induced Myocarditis Via AKT and p38 Pathways.

Scutellaria baicalensis Georgi has been widely used in China for treatment of various diseases. This study investigated the effect of Scutellaria baicalensis Georgi extracts (SBE) against Coxsackievirus B3 (CVB3)-induced myocarditis in vitro and in vivo. In vitro, Hela cells and primary myocardial cells were infected with CVB3 and treated with SBE (50-800 µg/ml) and ribavirin (200 µM) for 48 h and then determined by CCK8 assay. Real-time PCR and western blotting assays were performed. In vivo, a myocarditis model was induced in male BALB/c mice by injecting CVB3 suspension intraperitoneally for three times, followed by treatment with SBE (400 and 200 mg/kg) and ribavirin (100 mg/kg) for 28 days. SBE ameliorated the cytotoxicity of CVB3 in Hela cells, especially at 400 µg/ml (39.93% vs 65.67%, p < 0.05) without influencing cell growth and also significantly reduced CVB3 replication in primary myocardial cells. The levels of AKT, ERK, and p38 were increased after CVB3 infection. SBE could downregulate the expressions of AKT and p38. In vivo, the mortality rate from CVB3 reached to 66.67%, while 10.00% and 23.33% of this came after 400 and 200 mg/kg SBE treatment, respectively (p < 0.05). The CVB3 replication was obviously reduced after SBE administration from day 5. Similarly, the levels of AKT, ERK, and p38 mRNAs and proteins were increased, and SBE suppressed the expression of AKT and p38. Our study indicates that SBE is a promising potent antiviral agent against CVB3-induced myocarditis by inhibition of virus replication via depressing AKT and p38 expressions.

A neonatal mouse model of coxsackievirus A10 infection for anti-viral evaluation.

Epidemiological data indicate that coxsackievirus A10 (CVA10) has become one of the main causative agents of hand, foot and mouth disease (HFMD) and in recent years has often been found to co-circulate with other enteroviruses, which poses a challenge for the prevention and control of HFMD. Although most CVA10-associated HFMD cases present mild symptoms, severe manifestations and even death can also occur. However, the study of the pathogenesis and the development of drugs and vaccines for CVA10 infection are still far from complete. In this study, we established a neonatal mouse model for anti-viral evaluation and characterized the pathology of CVA10 infection. To develop the mouse model, both inbred and outbred mouse strains were used to compare their sensitivity to CVA10 infection; then, one-day-old BALB/c mice were selected and inoculated intraperitoneally with a CVA10 clinical strain, CVA10-FJ-01. Clinical symptoms, such as wasting, hind-limb paralysis and even death were observed in the CVA10-infected mice. Moreover, pathological examination and immunohistochemistry staining showed that severe myonecrosis with inflammatory infiltration was observed in CVA10-infected mice, indicating that CVA10 exhibited strong tropism to muscle tissue. Using real-time PCR, we also found that the viral load in the blood and muscle was higher than that in other organs/tissues at different time points post-infection, suggesting that CVA10 had a strong tropism to mice muscle and that viremic spread may also contribute to the death of the CVA10-infected mice. Additionally, to evaluate the neonatal mouse model of CVA10 infection, female mice were immunized with formalin-inactivated CVA10 and then allowed to mate after the third immunization. The results showed that maternal antibodies could protect mice against CVA10 infection. In summary, the results demonstrated that the neonatal mice model was a useful tool for evaluating the protective effects of CVA10 vaccines and anti-viral reagents.

Glycyrrhizic acid as the antiviral component of Glycyrrhiza uralensis Fisch. against coxsackievirus A16 and enterovirus 71 of hand foot and mouth disease.

ETHNOPHARMACOLOGICAL RELEVANCE: The radices of Glycyrrhiza uralensis Fisch. and herbal preparations containing Glycyrrhiza spp. have been used for thousands of years as an herbal medicine for the treatment of viral induced cough, viral hepatitis, and viral skin diseases like ulcers in China. Glycyrrhizic acid (GA) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity. AIM: The present study attempt to validate the medicinal use of Glycyrrhiza uralensis for hand, foot and mouth disease (HFMD) and further to verify whether GA is an active antiviral component in the water extract of Glycyrrhiza uralensis. MATERIALS AND METHODS: Radices of Glycyrrhiza uralensis Fisch. were extracted with hot water. The chemical contents of the extract were profiled with HPLC analysis. The antiviral activity of the extract and the major components was evaluated against infection of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) on Vero cells. The cytopathic effect caused by the infection was measured with MTT assay. Infectious virion production was determined using secondary infection assays and viral protein expression by immunoblotting analysis. RESULTS: The extract at 1000 µg/ml suppressed EV71 replication by 1.0 log and CVA16 by 1.5 logs. The antiviral activity was associated with the content of GA in the extract since selective depletion of GA from the extract by acid precipitation resulted in loss of antiviral activity. In contrast, the acid precipitant retained antiviral activity. The precipitant at a concentration of 200 µg/ml inhibited EV71 and CVA16 replication by 1.7 and 2.2 logs, respectively. Furthermore, GA dose-dependently blocked viral replication of EV71 and CVA16. At 3 mM, GA reduced infectious CVA16 and EV71 production by 3.5 and 2.2 logs, respectively. At 5mM, CVA16 production was reduced by 6.0 logs and EV71 by 4.0 logs. Both EV71 and CVA16 are members of Enterovirus genus, time-of-drug addition studies however showed that GA directly inactivated CVA16, while GA anti-EV71 effect was associated with an event(s) post virus cell entry. CONCLUSIONS: This study validated the medicinal usefulness of radices Glycyrrhiza uralensis against the etiological agents of HFMD. In addition to the identification of GA as the antiviral component of Glycyrrhiza uralensis against EV71 and CVA16 infection, this study also reveals that GA inhibits EV71 and CVA16 with distinct mechanisms.

Comparative uptake of enteric viruses into spinach and green onions.

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon(®). HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.

Ching-fang-pai-tu-san inhibits the release of influenza virus.

ETHNOPHARMACOLOGICAL RELEVANCE: Ching-fang-pai-tu-san (CFPTS) is a Chinese herbal decoction that is used as a cure for the common cold, fever, headache, and poor circulation. However, no previous studies have investigated the mode of action of CFPTS against influenza virus infections. To investigate the antiviral mechanism of CFPTS, we examined viral entry, transcription, translation, viral glycoprotein hemagglutinin (HA) transport, and budding of the influenza virus. MATERIALS AND METHODS: The antiviral activity of nontoxic concentrations of CFPTS against influenza virus A/WSN/33 was examined by assaying (neutralization assay) its inhibition of the virus-induced cytopathic effects. The mode of CFPTS action was first examined with a time-of-addition assay of synchronized infections, followed by monitoring HA transport by immunofluorescence microscopy. Viral endocytosis was evaluated with attachment and penetration assays. The inhibition of viral replication was measured by quantitative real-time PCR, immunoblotting, and immunofluorescence microscopy. We also performed assays related to the inhibition of viral entry, such as neuraminidase activity and hemagglutinin activity assays. RESULTS: Based on the inhibition of the virus-induced cytopathic effect in Madin-Darby canine kidney cells, the EC(50) of CFPTS was about 1.44 ± 0.22 mg/mL against influenza virus A/WSN/33. CFPTS displayed a broad spectrum of inhibitory activities against different strains of influenza A virus, as well as some enteroviruses. However, this extract proved less effective against clinical oseltamivir-resistant strains and influenza B viruses. CFPTS did not suppress viral RNA or protein synthesis. According to a time-of-addition assay, the antiviral mechanism of CFPTS may involve viral budding or intracellular viral glycoprotein transport. A plaque reduction assay showed that CFPTS reduced both the plaque size and plaque quantity. The intracellular transport of viral glycoprotein hemagglutinin was blocked by CFPTS by immunofluorescence microscopic analysis. Thus, it is possible that the antiviral mechanism of CFPTS might inhibit the assembly of progeny virions and/or their subsequent release. CONCLUSIONS: Our results give scientific support to the use of CFPTS in the treatment of influenza virus infections. CFPTS has potential utility in the management of seasonal pandemics of influenza virus infections, like other clinically available drugs.

A water extract of Pueraria lobata inhibited cytotoxicity of enterovirus 71 in a human foreskin fibroblast cell line.

Enterovirus 71 (EV71) can cause brain encephalitis and mortality. However, effective vaccines or chemotherapeutic agents are not yet available. We tested the hypothesis that Pueraria lobata could inhibit the cytotoxic effect of EV71 in a human foreskin fibroblast cell line by the XTT method. Our results showed that the water extract of P. lobata could inhibit cytopathy induced by EV71 when given before (p < 0.0001), simultaneously with (p < 0.0001), or after viral infection (p < 0.0001). Water extract of P. lobata was effective and its minimal concentration that inhibited 50% of the cytopathic effect (IC50) was 0.028 microg/mL. P. lobata was also safe with a selectivity index greater than 107,000. Water extract of P. lobata appeared to inhibit viral attachment (p < 0.0001) and penetration (p < 0.0001). The anti-EV71 activity of the water extract of P. lobata was not mediated by interferons. In conclusion, the water extract of P. lobata was effective in the management of the disease induced by EV71 infection.

Antiviral effects of Salvia miltiorrhiza (Danshen) against enterovirus 71.

In this study, the antiviral activities of seven different extracts of Salvia miltiorrhiza (danshen) were determined. The first two extracts, SA1 and SA2, isolated at room temperature by ethyl acetate and water extraction, respectively, neutralized the enterovirus 71-induced cytopathic effect in Vero, rhabdomyosarcoma and MRC-5 cells. The other five crude extracts, extracted with warm water (60-70 degrees C) or organic solvents, did not have any protective activity. The 50% inhibitory concentrations for neutralizing the enterovirus 71-induced cytopathic effect were 0.742 +/- 0.042 mg/ml for SA1 and 0.585 +/- 0.018 mg/ml for SA2 in Vero cells. No antiviral activity was observed in the other viruses tested. Antiviral activity was more efficient in cultures treated with SA1 or SA2 during viral infection compared to the cultures treated before or after infection, suggesting that these danshen extracts could interfere with viral entry. SA1 and SA2 were able to inhibit viral RNA synthesis in the infected cells and to abate the apoptotic process in enterovirus 71-infected Vero cells. We conclude that danshen extracts possess antiviral activity and have potential for the development as an anti-enterovirus 71 agent.

Mechanism of increased coxsackie and adenovirus receptor gene expression and adenovirus uptake by phytoestrogen and histone deacetylase inhibitor in human bladder cancer cells and the potential clinical application.

Coxsackie and adenovirus receptor (CAR) is known as a principal receptor for adenovirus commonly used as a gene delivery vector. Down-regulation of CAR is often detected in several cancer types. Epigenetic modifiers such as histone deacetylase inhibitor FK228 (depsipeptide) have been shown to increase CAR expression as well as the uptake of adenovirus in bladder cancer in vivo and in vitro, indicating that altered transcriptional regulation of CAR is the key mechanism responsible for the decreased CAR levels in this cancer. In this study, we screened agents that could induce CAR expression in bladder cancer cells. Fifty-eight drugs with various chemical properties were tested. Ipriflavone and plant isoflavones were found to exhibit the ability to induce CAR gene expression in combination with FK228. Genistein, the natural isoflavone found in soybean, when combined with FK228, exerts a synergistic effect on CAR gene and protein expression in bladder cancer cells. Chromatin immunoprecipitation results showed an increased histone acetylation in the CAR promoter gene, which is due to the suppression of histone deacetylase activity by both agents. Also, our data indicated that combination treatment is a potent chemotherapeutic regimen for bladder cancer cells and the subsequent administration of recombinant adenovirus could further eliminate the remaining cells. Taken together, our results provide a strong rationale for combining chemotherapeutic and gene therapeutic agents to enhance the therapeutic efficacy in bladder cancer.

Host nutritional status: the neglected virulence factor.

The emergence of new infectious diseases and old diseases with new pathogenic properties is a burgeoning worldwide problem. Severe acute respiratory syndrome (SARS) and acquired immune deficiency syndrome (AIDS) are just two of the most widely reported recent emerging infectious diseases. What are the factors that contribute to the rapid evolution of viral species? Various hypotheses have been proposed, all involving opportunities for virus spread (for example, agricultural practices, climate changes, rainforest clearing or air travel). However, the nutritional status of the host, until recently, has not been considered a contributing factor to the emergence of infectious disease. In this review, we show that host nutritional status can influence not only the host response to the pathogen, but can also influence the genetic make-up of the viral genome. This latter finding markedly changes our concept of host-pathogen interactions and creates a new paradigm for the study of such phenomena.

Antioxidants and viral infections: host immune response and viral pathogenicity.

Malnutrition has long been associated with increased susceptibility to infectious disease. The increase in severity from and susceptibility to infectious disease in malnourished hosts is thought to be the result of an impaired immune response. For example, malnutrition could influence the immune response by inducing a less effective ability to manage the challenge of an infectious disease. Work in our laboratory has demonstrated that not only is the host affected by the nutritional deficiency, but the invading pathogen is as well. Using a deficiency in selenium (Se) as a model system, mice deficient in Se were more susceptible to infection with coxsackievirus, as well as with influenza virus. Se-deficient mice develop myocarditis when infected with a normally benign strain of coxsackievirus. They also develop severe pneumonitis when infected with a mild strain of influenza virus. The immune system was altered in the Se-deficient animals, as was the viral pathogen itself. Sequencing of viral isolates recovered from Se-deficient mice demonstrated mutations in the viral genome of both coxsackievirus and influenza virus. These changes in the viral genome are associated with the increased pathogenesis of the virus. The antioxidant selenoenzyme, glutathione peroxidase-1, was found to be critically important, as glutathione peroxidase knockout mice developed myocarditis, similar to the Se-deficient mice, when infected with the benign strain of myocarditis. This work points to the importance of host nutrition in not only optimizing the host immune response, but also in preventing viral mutations which could increase the viral pathogenicity.

Effect of gold(I) compounds on the virulence of an amyocarditic strain of coxsackievirus B3.

Coxsackieviruses, especially B strains (CVB), are known etiological agents of myocarditis. Both amyocardititc and myocarditic strains exist and at least one amyocarditic strain, CVB3/0, can convert to virulence when passaged through selenium or vitamin E-deficient mice. Gold(I)-containing compounds, such as aurothiomalate (ATM) and aurothioglucose (ATG), can act as selenium antagonists. In this study, we examined the effect of intraperitoneal administration of equal doses of ATM or ATG on the virulence of CVB3/0. ATM but not ATG increased mortality in CVB3/0-infected mice. CVB3/0-infected mice treated with ATM had total necrosis of the pancreatic exocrine tissue. Heart damage also occurred in ATM-treated mice but did not correlate with mortality. Increased viral titers and persistence were observed in ATM-treated mice and, to a lesser extent, in ATG-treated mice. Thus, under our conditions, only ATM increased the virulence of CVB3/0, whereas ATG did not. On the other hand, both ATG and ATM inhibited thioredoxin reductase activity in heart and pancreas, but neither affected glutathione peroxidase activity. In contrast, dietary selenium deficiency reduces both enzyme activities. Thus, it is unlikely that these compounds affect virulence by acting as selenium antagonists.

Viral isolation from cases of epidemic neuropathy in Cuba.

OBJECTIVE: To investigate the possibility of a viral agent in the central nervous system of patients with epidemic neuropathy. DESIGN: Virus isolation attempts, in cell cultures and suckling mice, from cerebrospinal fluid (CSF) of neuropathy patients and controls undergoing lumbar puncture for unrelated reasons. Serologic studies in patients, contacts, and controls. SETTING: An epidemic of optic and peripheral neuropathy affected more than 50,000 people in Cuba in 1991 through 1993. Illness was associated with dietary limitations and increased physical demands accompanying the shortages of food and fuel experienced in Cuba since 1989. Most patients responded to parenteral vitamin therapy, and the epidemic began to subside when oral vitamin supplementation was begun for the entire Cuban population. RESULTS: Coxsackievirus A9 (five isolates) and a similar, less cytopathic virus (100 isolates) were recovered from 105 (84%) of 125 CSF specimens from neuropathy patients. The strains with light cytopathic effect were antigenically related to Coxsackieviruses A9 and B4 by cross-neutralization and immunoblotting assays. Virus persisted in CSF of some patients for 1 to 12 months. Cerebrospinal fluid from patients and both types of virus from cell culture produced illness, including complete posterior flaccid paralysis, in newborn mice, and virus was reisolated from the mice. Mouse tissues and sural nerve biopsy specimens from patients were stained by immunoperoxidase and colloidal gold techniques using hyperimmune rabbit antisera against the virus with light cytopathic effect. CONCLUSIONS: Coxsackievirus A9 or an antigenically related agent with a light cytopathic effect was present in CSF of 84% of 125 patients with epidemic neuropathy. The role of these agents, probably in combination with nutritional factors, in the pathophysiology of the disease requires further investigation.

[Anti-virus effect of aralosides].

The result showed that the anti-virus effect of aralosides on infections with poliovirus II, ECHO delta virus, adenovirus II, herpes simplex virus I, coxsackie B3 virus and coxsackie A16 virus was remarkable. Aralosides could inhibit the development of cytopathic effect (CPE) and protect cultural cells from being infected with the above viruses.