Pretreatment with a grape seed proanthocyanidin extract downregulates proinflammatory cytokine expression in airway epithelial cells infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) infections are associated with significant morbidity and mortality. Inflammation is mediated by cytokine secretion from RSV­infected airway epithelial cells. Grape seed proanthocyanidin extract (GSPE) exhibits potent antioxidant capacity, as well as anti­bacterial, anti­viral, anti­carcinogenic, anti­inflammatory and anti­allergic actions. However, few studies have explored the anti­inflammatory effects of GSPE on airway epithelial cells infected with RSV. Airway epithelial A549 cells were pretreated with GSPE and its effects on cytokine production during RSV infection were investigated. A549 cells were infected with RSV, with or without GSPE pretreatment, and cultured for 24, 48 and 72 h. The expression of interleukin (IL)­1ß, IL­6 and IL­8, were measured by reverse transcription­quantitative polymerase chain reaction, ELISA and western blotting. RSV infection induced significant increases in proinflammatory cytokine expression. However, GSPE pretreatment decreased the mRNA and protein expression levels of IL­1ß, IL­6 and IL­8. GSPE regulated the immune response by reducing the RSV­induced transcription of proinflammatory cytokines in airway epithelial cells, suggesting that GSPE helps to prevent RSV­induced airway disease.

Modeling of fusion inhibitor treatment of RSV in African green monkeys.

Respiratory syncytial virus (RSV) is a respiratory infection that can cause serious illness, particularly in infants. In this study, we test four different model implementations for the effect of a fusion inhibitor, including one model that combines different drug effects, by fitting the models to data from a study of TMC353121 in African green monkeys. We use mathematical modeling to estimate the drug efficacy parameters, εmax, the maximum efficacy of the drug, and EC50, the drug concentration needed to achieve half the maximum effect. We find that if TMC353121 is having multiple effects on viral kinetics, more detailed data, using different treatment delays, is needed to detect this effect.

Qingkailing Injection () for Treatment of Children Pneumonia Induced by Respiratory Syncytial Virus: A Meta-Analysis of Randomized Controlled Trials.

OBJECTIVE: To evaluate the efficacy and safety of Qingkailing Injection (, QKL) for treatment of children pneumonia caused by respiratory syncytial virus (RSV). METHODS: Randomized clinical trials (RCTs) comparing QKL with ribavirin injection in the treatment of children pneumonia induced by RSV were searched in PubMed, Science Direct, Cochrane Library, Chinese VIP database, CNKI and Wanfang databases from their inception to March 2014. Meta-analyses were performed using RevMan 5.2 software. The methodological quality of the selected RCTs was evaluated by the Modified Jadad Score. The primary outcome measures were effective rate and the secondary outcomes were relief time of fever and cough. RESULTS: Seven RCTs with 992 cases published from 2008 to 2013 were identified. The meta-analysis results indicated that QKL was more effective in cure rate [risk ratios (RR)=1.32, 95% CI (1.17, 1.50), P<0.01], total effective rate [RR=1.07, 95% CI (1.02, 1.13), P=0.009] and less fever clearance time [mean difference=-0.73, 95% CI (-1.22,-0.23), P=0.004], compared with ribavirin injection in the treatment of RSV-induced children pneumonia. No dead case was reported in all trials. There were 3 trials mentioned adverse events, 2 reported no obvious adverse event occurred while 1 reported adverse events described as skin hypersensitivity, elevation of ALT, a mild abnormal of hepatic and renal function in both QKL and ribavirin group. CONCLUSIONS: QKL was an effective and relatively safe option for the treatment of RSV-induced children pneumonia. These therapeutic effects were promising but need to be interpreted with caution due to variations in the treatment and methodological weakness in the studies.

Grape Seed Proanthocyanidin Inhibits Mucin Synthesis and Viral Replication by Suppression of AP-1 and NF-κB via p38 MAPKs/JNK Signaling Pathways in Respiratory Syncytial Virus-Infected A549 Cells.

Airway epithelial cells are often infected by respiratory syncytial virus (RSV), one of the most common causes of asthma, bronchiolitis, chronic obstructive pulmonary disease, and pneumonia. During the infection process, excessive mucins instigate airway inflammation. However, the mechanism underlying RSV-induced airway hyper-responsiveness and inflammation is poorly understood. Furthermore, no reliable vaccines or drugs for antiviral therapy are available. In this study, the effect of the natural compound grape seed proanthocyanidin (GSP) on RSV-infected human airway epithelial cells A549 was evaluated. After pretreatment of the cells with or without exposure to RSV with 5-10 µg GSP/mL, the expression of various mucins (MUC1, MUC2, MUC5AC, MUC5B, and MUC8) was evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting, as well as confocal microscopy. We found that GSP significantly decreased RSV-induced mucin synthesis at the mRNA and protein levels. In addition, GSP suppressed the RSV-induced signaling pathways, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, together with nuclear factor kappa B (NF-κB) and activating protein-1 family members (c-Jun and c-Fos). Concomitantly, GSP inhibited the replication of RSV within A549 cells. Taken together, all our results suggest that GSP could be a potent therapeutic agent to suppress excessive mucus production and viral replication in RSV-induced airway inflammatory disorders.

Watsonianone A from Rhodomyrtus tomentosa Fruit Attenuates Respiratory-Syncytial-Virus-Induced Inflammation In Vitro.

Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens. Immoderate inflammation plays a great role in causing RSV-induced diseases. In the present study, watsonianone A, isolated from the fruit of Rhodomyrtus tomentosa (Ait.) Hassk, was found to show a good inhibitory effect on RSV-induced NO production, with a half-maximal inhibitory concentration of 37.2 ± 1.6 µM. Enzyme-linked immunosorbent assay and fluorescence quantitative polymerase chain reaction analyses indicated that watsonianone A markedly reduced both mRNA and protein levels of tumor necrosis factor α, interleukin 6, and monocyte chemoattractant protein 1 in RSV-infected RAW264.7 cells. Mechanistically, watsonianone A inhibited nuclear factor κB (NF-κB) activation by suppressing IκBα phosphorylation. Further analysis revealed that watsonianone A activated the thioredoxin system and decreased intracellular reactive oxygen species (ROS) levels, which are closely associated with NF-κB activation in RSV-infected cells. These results reveal that watsonianone A can attenuate RSV-induced inflammation via the suppression of ROS-sensitive inflammatory signaling.

A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Effect of Gubenfangxiao decoction on respiratory syncytial virus-induced asthma and expression of asthma susceptibility gene orosomucoid 1-like protein 3 in mice.

OBJECTIVE: To investigate the effect of Guben-fangxiao decoction (GBFXD) on respiratory-syncytial-virus (RSV)-induced asthma and the expression of asthma susceptibility gene, orosomucoid 1-like protein 3 (ORMDL3) in mice. METHODS: Seventy-two female BALB/c mice were randomly assigned to normal, model, GBFXD high dose, GBFXD moderate dose, GBFXD low dose and montelukast groups. An asthma model was induced via intraperitoneal injection and aerosol inhalation of ovalbumin (OVA) and repeated intranasal instillation of RSV in all mice, except those in the normal group. All treatments were administered at the first onset of asthma (within 8 weeks of model establishment) and the mice were euthanized after 28 days of treatment. The levels of transforming growth factor-ß (TGF-ß) and interleukin-6 (IL-6) in bronchoalveolar lavacie fluid (BALF) of the mice were measured and the expression of asthma susceptibility gene ORMDL3 in lung tissue was determined using real-time polymerase chain reaction (RT-PCR) and western blotting. RESULTS: Expression of ORMDL3 and levels of TGF-ß and IL-6 were significantly higher in the model group (P < 0.05, P < 0.01) compared with the normal mice. Levels of ORMDL3, TGF-ß and IL-6 were significantly lower in all three GBFXD treated groups (P < 0.05) compared with the model group. However, the levels in the GBFXD treatment groups did not differ significantly from the montelukast group. CONCLUSION: GBFXD had a therapeutic effect in this experimental model. The functional mechanism of GBFXD may involve multiple factors, including alleviation of airway inflammation, down-regulation of asthma susceptibility gene ORMDL3 and inhibition of airway remodeling.

Tangeretin from Citrus reticulate Inhibits Respiratory Syncytial Virus Replication and Associated Inflammation in Vivo.

Human respiratory syncytial virus (RSV) is a common pathogen that causes pneumonia and bronchiolitis in infants and young children. Our previous study showed that tangeretin from Citrus reticulate possessed potent in vitro anti-RSV effects comparable to that of ribavirin. Therefore, in this study, we investigated the in vivo anti-RSV activity of tangeretin in 3-week-old male BALB/c mice. A plaque reduction assay and fluorescence quantitative polymerase chain reaction (FQ-PCR) showed that tangeretin inhibited RSV replication in the lung of mice. Moreover, a luminex assay indicated tangeretin relieved RSV-induced lung inflammation by attenuating interleukin (IL)-1ß secretion. Possible anti-inflammatory mechanisms of tangeretin were preliminarily explored using a RSV-infected macrophage model. A FQ-PCR, enzyme-linked immunosorbent assay (ELISA), and luciferase assay revealed that tangeretin inhibited RSV-induced inflammation by suppressing nuclear factor-κB (NF-κB) activation. This study demonstrates that tangeretin inhibited RSV replication and RSV-induced lung inflammation in vivo and may be useful in preventing and treating RSV infections and inflammation.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

Antiviral activity of ginseng extract against respiratory syncytial virus infection.

Panax ginseng has been known to have a number of immuno-modulatory effects. In this study, we investigated whether Panax Korean red ginseng extract (KRGE) has in vitro and in vivo antiviral effects on respiratory syncytial virus (RSV) infection. KRGE improved the survival of human lung epithelial cells against RSV infection and inhibited RSV replication. In addition, KRGE treatment suppressed the expression of RSV-induced inflammatory cytokine genes (IL-6 and IL-8) and the formation of reactive oxygen species in epithelial cell cultures. Oral administration of mice with KRGE resulted in lowering lung viral loads after RSV infection. Additionally, the in vivo effects of KRGE showed an enhanced level of interferon-γ (IFN-γ) producing dendritic cells subsequent to RSV infection. Taken together, these results suggested that KRGE has antiviral activity against RSV infection.

BuShenYiQi Formula strengthens Th1 response and suppresses Th2-Th17 responses in RSV-induced asthma exacerbated mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of allergic asthma has been increased rapidly in recent years. About 20% of all these sufferers have experienced asthma exacerbation. Although corticosteroids and ß-agonists therapy improves serious asthma symptoms, they can׳t completely cure these allergic diseases. BuShenYiQi Formula (BSYQF) has been widely used to treat bronchial asthma and its exacerbation for decades in Huashan Hospital of Fudan University, China. Nevertheless, the mechanisms of BSYQF' anti-asthmatic effects haven׳t been fully elucidated. In this study, we evaluated the involvement of Th1, Th2 and Th17 cells in the anti-asthmatic effects of BSYQF in Respiratory Syncytial Virus (RSV)-induced asthma exacerbated mice. MATERIALS AND METHODS: BALB/c mice were challenged with ovalbumin (OVA), followed by RSV infections for establishment of asthma exacerbated model. Airway hyperresponsiveness (AHR) was examined by direct airway resistance analysis. Bronchoalveolar lavage fluid (BALF) was assessed for inflammatory cell counts and secreted levels of cytokines. Lung tissues were detected for inflammatory cell infiltration and mucus hypersecretion. Subsequently, CD4(+)T cells and alveolar macrophages were sorted and purified from mice lungs in different groups. CD4(+)T cell subpopulations including the expression levels of important transcription factors in T lymphocyte polarization were examined. In asthma exacerbation group, the purified CD4(+)T cells and macrophages were co-cultured, and the changes of co-cultured cells with BSYQF treatment were further analyzed in vitro. RESULTS: BSYQF significantly attenuated airway hyperresponsiveness and inhibited inflammatory cell infiltration, especially for excessive infiltration of eosinophils and neutrophils. Histopathological analysis showed that BSYQF could suppress airway inflammation and RSV replication. The decreases of antigen-specific IgE, IL-4, IL-5, IL-6, IL-17a and increases of IFN-γ, IL-12 were observed in BALF, lung homogenate or serum after BSYQF treatment. We further confirmed that BSYQF could down-regulate Th2-Th17 cell proportions with lower expressions of GATA3, STAT6 and RORγT, and up-regulate Th1 cell proportion with higher expression of T-bet. And as a result of strengthened Th1-response, activated macrophages were also observed by remarkable enhancement of signature gene expressions and phagocytosis. CONCLUSIONS: BSYQF can significantly attenuate RSV-induced asthma exacerbation. These effects may be mediated at least partially by regulating the balance between Th1 and Th2-Th17 responses.

Development of a high-throughput replicon assay for the identification of respiratory syncytial virus inhibitors.

Respiratory syncytial virus (RSV) drug discovery has been hindered by the lack of good chemistry starting points and would benefit from robust and convenient assays for high-throughput screening (HTS). In this paper, we present the development and optimization of a 384-well RSV replicon assay that enabled HTS for RSV replication inhibitors with a low bio-containment requirement. The established replicon assay was successfully implemented for high-throughput screening. A validation screen was performed which demonstrated high assay performance and reproducibility. Assay quality was further confirmed via demonstration of appropriate pharmacology for different classes of RSV replication tool inhibitors. RSV replicon and cytotoxicity assays were further developed into a multiplexed format that measured both inhibition of viral replication and cytotoxicity from the same well. This provided a time and cost efficient approach to support lead optimization. In summary, we have developed a robust RSV replicon assay to help expedite the discovery of novel RSV therapeutics.

Cytokine profiles, signalling pathways and effects of fluticasone propionate in respiratory syncytial virus-infected human foetal lung fibroblasts.

To examine cytokine production in response to RSV infection, we assessed the levels of 29 cytokines released from RSV-infected human foetal lung fibroblasts. We also examined the relationships between the effects of fluticasone propionate and various signalling pathways in the cells. Twenty-four hours after infection (1MOI), RSV-infected cells released cytokines, for example proinflammatory cytokines (IL-1ß, IL-6 and TNF-α), anti-inflammatory (IL-1ra), Th1 (IFN-γ, IFN-λ1a, IL-2 and IL-12), Th2 (IL-4, IL-5, IL-10 and IL-13), granulopoiesis-inducing (G-CSF and GM-CSF), eosinophil recruitment-inducing (eotaxin and RANTES) and neutrophil recruitment-inducing cytokines (IL-8, IP-10, MCP-1 and MIP-1α). Aberrant release of most was significantly suppressed by fluticasone propionate. Twelve hours after RSV infection, increased phosphorylation of Akt, p38 MAPK, ERK1/2 and IκB-α was noted. Fluticasone propionate suppressed the phosphorylation of Akt, p38 MAPK, and ERK1/2, but not IκB-α, in virus-infected cells. TLR-4 expression was unchanged in control and RSV-infected cells, and TLR-3 and RIG-I expression was not detected. The results indicate that RSV infection induces aberrant production and release of certain cytokines through these signalling pathways in human lung fibroblasts. Overproduction and imbalance of these cytokines may be associated with the pathophysiology of RSV-induced excessive and allergic inflammation.

Bioenergetics of murine lungs infected with respiratory syncytial virus.

BACKGROUND: Cellular bioenergetics (cellular respiration and accompanying ATP synthesis) is a highly sensitive biomarker of tissue injury and may be altered following infection. The status of cellular mitochondrial O(2) consumption of the lung in pulmonary RSV infection is unknown. METHODS: In this study, lung fragments from RSV-infected BALB/c mice were evaluated for cellular O(2) consumption, ATP content and caspase activity. The disease was induced by intranasal inoculation with the RSV strain A2 and lung specimens were analyzed on days 2-15 after inoculation. A phosphorescence O(2) analyzer that measured dissolved O(2) concentration as a function of time was used to monitor respiration. The caspase-3 substrate analogue N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspases. RESULTS: O(2) concentration declined linearly with time when measured in a sealed vial containing lung fragment and glucose as a respiratory substrate, revealing its zero-order kinetics. O(2) consumption was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. Cellular respiration increased by 1.6-fold (p<0.010) and ATP content increased by 3-fold in the first week of RSV infection. Both parameters returned to levels found in uninfected lungs in the second week of RSV infection. Intracellular caspase activity in infected lungs was similar to uninfected lungs throughout the course of disease. CONCLUSIONS: Lung tissue bioenergetics is transiently enhanced in RSV infection. This energy burst, triggered by the virus or virus-induced inflammation, is an early biomarker of the disease and may be targeted for therapy.

Inactivation of respiratory syncytial virus by zinc finger reactive compounds.

BACKGROUND: Infectivity of retroviruses such as HIV-1 and MuLV can be abrogated by compounds targeting zinc finger motif in viral nucleocapsid protein (NC), involved in controlling the processivity of reverse transcription and virus infectivity. Although a member of a different viral family (Pneumoviridae), respiratory syncytial virus (RSV) contains a zinc finger protein M2-1 also involved in control of viral polymerase processivity. Given the functional similarity between the two proteins, it was possible that zinc finger-reactive compounds inactivating retroviruses would have a similar effect against RSV by targeting RSV M2-1 protein. Moreover, inactivation of RSV through modification of an internal protein could yield a safer whole virus vaccine than that produced by RSV inactivation with formalin which modifies surface proteins. RESULTS: Three compounds were evaluated for their ability to reduce RSV infectivity: 2,2'-dithiodipyridine (AT-2), tetraethylthiuram disulfide and tetramethylthiuram disulfide. All three were capable of inactivating RSV, with AT-2 being the most potent. The mechanism of action of AT-2 was analyzed and it was found that AT-2 treatment indeed results in the modification of RSV M2-1. Altered intramolecular disulfide bond formation in M2-1 protein of AT-2-treated RSV virions might have been responsible for abrogation of RSV infectivity. AT-2-inactivated RSV was found to be moderately immunogenic in the cotton rats S.hispidus and did not cause a vaccine-enhancement seen in animals vaccinated with formalin-inactivated RSV. Increasing immunogenicity of AT-2-inactivated RSV by adjuvant (Ribi), however, led to vaccine-enhanced disease. CONCLUSIONS: This work presents evidence that compounds that inactivate retroviruses by targeting the zinc finger motif in their nucleocapsid proteins are also effective against RSV. AT-2-inactivated RSV vaccine is not strongly immunogenic in the absence of adjuvants. In the adjuvanted form, however, vaccine induces immunopathologic response. The mere preservation of surface antigens of RSV, therefore may not be sufficient to produce a highly-efficacious inactivated virus vaccine that does not lead to an atypical disease.

[In vitro inhibitory mechanism of respiratory syncytial virus with solution prescription of zhidanhuayu].

OBJECTIVE: To study the effective part of solution prescription of Zhidanhuayu (ZDHY) against respiratory syncytial virus (RSV) in vitro. METHODS: Observe the pathology of RSV to Hep-2 under the condition of different concentrations and each effective part of ZDHY. RESULTS: The concentration limit causing celluar toxicity of ZDHY is 5.5 mg/ml. The ZDHY failed to block the absorption of RSV to Hep-2 within this concentration, and consequently the cell fell into the full pathological changes. During the concentration of 2.75-5.50 mg/ml, the ZDHY directly destroyed virus array,meanwhile, the infected cells that treated by the medicine kept healthy also. CONCLUSION: ZDHY could not defend the infection of RSV, but is able to destroy the RSV directly and inhibit the RSV inhabiting in the cell.

Taking a Toll on human disease: Toll-like receptor 4 agonists as vaccine adjuvants and monotherapeutic agents.

Toll-like receptor (TLR) agonists are being developed for use as vaccine adjuvants and as stand-alone immunomodulators because of their ability to stimulate innate and adaptive immune responses. Among the most thoroughly studied TLR agonists are the lipid A molecules that target the TLR4 complex. One promising candidate, monophosphoryl lipid A, which is a derivative of lipid A from Salmonella minnesota, has proven to be safe and effective as a vaccine adjuvant in > 120,000 human doses. A new class of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates (AGPs), have been engineered specifically to target human TLR4 and are showing promise as vaccine adjuvants and as monotherapeutic agents capable of eliciting nonspecific protection against a wide range of infectious pathogens. In this review, the authors provide an update of the preclinical and clinical experiences with the TLR4 agonists, MPL (Corixa Corporation) adjuvant and the AGPs.

Requirement of cysteines and length of the human respiratory syncytial virus M2-1 protein for protein function and virus viability.

The M2-1 protein of human respiratory syncytial virus (hRSV) promotes processive RNA synthesis and readthrough at RSV gene junctions. It contains four highly conserved cysteines, three of which are located in the Cys(3)-His(1) motif at the N terminus of M2-1. Each of the four cysteines, at positions 7, 15, 21, and 96, in the M2-1 protein of hRSV A2 strain was individually replaced by glycines. When tested in an RSV minigenome replicon system using beta-galactosidase as a reporter gene, C7G, C15G, and C21G located in the Cys(3)-His(1) motif showed a significant reduction in processive RNA synthesis compared to wild-type (wt) M2-1. C96G, which lies outside the Cys(3)-His(1) motif, was fully functional in supporting processive RNA synthesis in vitro. Each of these cysteine substitutions was introduced into an infectious antigenomic cDNA clone derived from hRSV A2 strain. Except for C96G, which resulted in a viable virus, no viruses were recovered with mutations in the Cys(3)-His(1) motif. This indicates that the Cys(3)-His(1) motif is critical for M2-1 function and for RSV replication. The functional requirement of the C terminus of the M2-1 protein was examined by engineering premature stop codons that caused truncations of 17, 46, or 67 amino acids from the C terminus. A deletion of 46 or 67 amino acids abolished the synthesis of full-length beta-galactosidase mRNA and did not result in the recovery of viable viruses. However, a deletion of 17 amino acids from the C terminus of M2-1 reduced processive RNA synthesis in vitro and was well tolerated by RSV. Relocation of the M2-1 termination codon upstream of the M2-2 initiation codons did not significantly affect the expression of the M2-2 protein. Both rA2-Tr17 and rA2-C96G did not replicate as efficiently as wt rA2 in HEp-2 cells and was restricted in replication in the respiratory tracts of cotton rats.

Inhibitory effect of ferulic acid and isoferulic acid on the production of macrophage inflammatory protein-2 in response to respiratory syncytial virus infection in RAW264.7 cells.

We investigated the effect of ferulic acid (FA) and isoferulic acid (IFA), which are the main active components of the rhizoma of Cimicifuga heracleifolia (CH), an anti-inflammatory drug used frequently in Japanese traditional medicine, on the production of macrophage inflammatory protein-2 (MIR-2) in a murine macrophage cell line, RAW264.7, in response to respiratory syncytial virus (RSV) infection. Following the exposure of cells to RSV for 20h, the MIP-2 level in condition medium was increased to about 20 ng/ml, although this level in mock-infected cells was negligible. In the presence of either FA or IFA, RSV-infected cells reduced MIP-2 production in a dose-dependent manner. These data suggest that FA and IFA might be responsible, at least in part, for the anti-inflammatory drug effect of CH extract through the inhibition of MIP-2 production.