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Antiviral Research (v.74, #1)
Inhibitory effect of cinnamaldehyde, derived from Cinnamomi cortex, on the growth of influenza A/PR/8 virus in vitro and in vivo by K. Hayashi; N. Imanishi; Y. Kashiwayama; A. Kawano; K. Terasawa; Y. Shimada; H. Ochiai (pp. 1-8).
We have investigated the inhibitory effect of trans-cinnamaldehyde (CA), one of the principal constituents of essential oil derived from Cinnamomi cortex, on the growth of influenza A/PR/8 virus in vitro and in vivo. When 1-h drug treatment was initiated at various times post-infection (p.i.) in Madin–Darby canine kidney cells using a fixed dose of CA (40μM), the maximum inhibitory effect (29.7% virus yield of control) was obtained when drug treatment was started at 3h p.i. Under the same treatment schedule, CA inhibited the virus growth in a dose-dependent manner (20–200μM), and, at 200μM, the virus yield was reduced to an undetectable level. RT-PCR and SDS-PAGE analyses showed that CA inhibited viral protein synthesis at the post-transcriptional level. In mice infected with the lung-adapted PR-8 virus, inhalation (50mg/cage/day) and nasal inoculation (250μg/mouse/day) of CA significantly increased survival rates on the 8 days to 100% and 70%, respectively, in contrast to a survival rate of 20% in the untreated control group. Importantly, inhalation of CA caused virus yield reduction by 1log in bronchoalveolar lavage fluid on day 6 after infection, compared with that of the untreated control group. These findings might provide further support to the empirical indication of Cinnamomi cortex-containing Kampo medicines for acute respiratory infectious diseases.
Keywords: Cinnamaldehyde; Influenza; Protein synthesis; Survival rate
Enhanced potency and efficacy of 29-mer shRNAs in inhibition of Enterovirus 71 by Eng Lee Tan; Theresa May Chin Tan; Vincent Tak Kwong Chow; Chit Laa Poh (pp. 9-15).
Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It has been associated with severe neurological complications and has caused significant mortalities in large-scale outbreaks in Asia. In this study, we demonstrated an enhanced silencing of EV71 through the use of chemically synthesized 29-mer shRNAs. The 29-mer shRNAs were designed to target three highly conserved regions of EV71 genome. Transfection of rhabdomyosarcoma (RD) cells with the 29-mer shRNAs significantly inhibited EV71 replication in a dose-dependent manner as demonstrated by reduction of viral RNA, VP1 protein and plaque forming units. The inhibitory effects were more potent and were achieved at 10-fold lower concentrations when compared to 19-mer siRNAs reported previously [Sim, A.C.N., Luhur, A., Tan, T.M.C., Chow, V.T.K., Poh, C.L., 2005. RNA interference against Enterovirus 71 infection. Virology 341, 72–79]. The viral inhibitory effects lasted 72h post-infection and there was no adverse off-target silencing effect. Gene silencing by 29-mer shRNAs targeted at the 3Dpol region (sh-3D) was the most effective, achieving 91% viral inhibition. Further evaluation found that no enhanced inhibitory effects were observed when sh-3D was cotransfected with each of the other two candidates. This study showed an improvement in triggering RNAi using the more potent 29-mer shRNAs, indicating its therapeutic potential against EV71.
Keywords: Enterovirus 71; Hand, foot and mouth disease (HFMD); RNA interference; 29-mer shRNAs; Enhanced silencing effects; Potential antiviral therapy
Anti-hepatitis B virus activity of wogonin in vitro and in vivo by Qinglong Guo; Li Zhao; Qidong You; Yong Yang; Hongyan Gu; Guoliang Song; Na Lu; Jian Xin (pp. 16-24).
The traditional Chinese medicine Scutellaria radix has been used for thousands of years, mainly for the treatment of inflammatory conditions including hepatitis. The major active constituent, wogonin (WG), isolated from S. radix has attracted increasing scientific attention in recent years due to its potent biological activities. However, pharmacologic studies have primarily been focused on wogonin's anti-inflammatory and anti-cancer activities. In this study, we have investigated wogonin's anti-hepatitis B virus (HBV) activity both in vitro and in vivo. In the human HBV-transfected liver cell line HepG2.2.15, wogonin effectively suppressed the secretion of the HBV antigens with an IC50 of 4μg/ml at day 9 for both HBsAg and HBeAg. Consistent with the HBV antigen reduction, wogonin also reduced HBV DNA level in a dose-dependent manner. Duck hepatitis B virus (DHBV) DNA polymerase was dramatically inhibited by wogonin with an IC50 of 0.57μg/ml. In DHBV-infected ducks wogonin dosed i.v. once a day for 10 days reduced plasma DHBV DNA level with an ED50 of 5mg/kg. The in vivo anti-HBV effect of wogonin in ducks was confirmed by Southern blotting of DHBV DNA in the liver. Histopathological evaluation of the liver revealed significant improvement by wogonin. In addition, in human HBV-transgenic mice, wogonin dosed i.v. once a day for 10 days significantly reduced plasma HBsAg level. Immunohistological staining of the liver confirmed the HBsAg reduction by wogonin. In conclusion, our results demonstrate that wogonin possesses potent anti-HBV activity both in vitro and in vivo. Currently, wogonin is under early development as an anti-HBV drug candidate.
Keywords: Wogonin; Anti-HBV activity; Human HBV cells; DHBV; Human HBV-transgenic mice
Induction of potent and long-lasting CD4 and CD8 T-cell responses against hepatitis C virus by immunization with viral antigens plus poly(I:C) and anti-CD40 by Aintzane Zabaleta; Laura Arribillaga; Diana Llopiz; Javier Dotor; Juan J. Lasarte; Jesús Prieto; Francisco Borrás-Cuesta; Juan I. Esteban; Josep Quer; Francesc Vayreda; Pablo Sarobe (pp. 25-35).
Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.
Keywords: Hepatitis C; Vaccination; Adjuvant; Dendritic cell; T-cell
Effective inhibition of porcine transmissible gastroenteritis virus replication in ST cells by shRNAs targeting RNA-dependent RNA polymerase gene by Jun-fang Zhou; Xiu-guo Hua; Li Cui; Jian-guo Zhu; De-nian Miao; Yong Zou; Xi-zhong He; Wan-guo Su (pp. 36-42).
Transmissible gastroenteritis virus (TGEV) is identified as one of the most important pathogenic agents during swine enteric infection, leading to high mortality in neonatal pigs and severe annual economic loss in swine-producing areas. Up to date, various vaccines developed against TGEV still need to be improved. To exploit the possibility of using RNA interference (RNAi) as a strategy against TGEV infection, two shRNA-expressing plasmids (pEGFP-U6/P1 and pEGFP-U6/P2) targeting the RNA-dependent RNA polymerase (RdRp) gene of TGEV were constructed and transfected into swine testicular (ST) cells. The cytopathic effect (CPE) and MTS assays demonstrated that both shRNAs were capable of protecting cells against TGEV invasion with very high specificity and efficiency. A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with the two plasmids were reduced by 95.2% and up to 100%, respectively. Our results suggest that RNAi might be a promising new strategy against TGEV infection.
Keywords: TGEV; RNA interference (RNAi); Short hairpin RNA (shRNA); RdRp; ST
An inhibitor of the epidermal growth factor receptor function does not affect the ability of human papillomavirus 11 to form warts in the xenografted immunodeficient mouse model by Tanya Parkinson; Mary K. Howett; Patricia A. Welsh; Susan D. Patrick; Elizabeth B. Neely; Neil Flanagan; Vincent A. Pollack; Leslie R. Pustilnik; Jim Moyer; Manos Perros (pp. 43-50).
Epidermal growth factor receptor (EGFr) has been shown to be induced and activated in cells infected with HPV, suggesting that it may play a physiological role in viral replication or in the formation or maintenance of warts. To investigate this possibility, human foreskin tissue was infected with HPV11 and transplanted onto the renal capsule and the dermis of immunodeficient mice. The animals were treated orally or topically with the potent EGFr inhibitor CP-545130, with treatment starting either immediately following graft attachment, or following a 70 day period to allow development of warts. The rate of appearance of warts, wart size and number were monitored. In addition, we measured intra-lesional HPV replication levels and examined the morphology of the graft tissues. Analysis of the results showed no significant difference between placebo and compound-treated groups, despite high levels of compound present in the graft tissue. We conclude that EGFr kinase activity is not required for the development and maintenance of HPV-11-induced warts in this model.
Keywords: HPV; EGFR; Papillomavirus; Epidermal growth factor
Biophysical evidence of two docking sites of the carboxyl heptad repeat region within the amino heptad repeat region of gp41 of human immunodeficiency virus type 1 by Ding-Kwo Chang; Chang-Sheng Hsu (pp. 51-58).
Two HIV-1 gp41-derived peptide fusion inhibitors, T-20 and T-649, were synthesized and their binding profiles of the N-heptad repeat region (HR1) were compared to examine the molecular basis of the differential antiviral potency and viral resistance. Turbidity clearance experiments based on the overlapping 15-mer peptides derived from HR1 revealed a major binding site at the LLSGIV segment for both T-20 and T-649. Additionally, another docking site was found at the sequence encompassing the hydrophobic pocket of HR1 for T-649. Concordant results were observed from the surface plasmon resonance measurements. The binding affinity profile exhibited a major maximum around the LLSGIV motif for the two peptide fusion inhibitors while a less prominent docking region was located near the hydrophobic pocket for T-649. This bi-modal model deduced from T-20 and T-649 interaction with HR1 peptides could rationalize the failure of emergence of the fusion inhibitor-resistant virus with simultaneous mutations in each of the two binding regions, as well as the generally higher potency of T-649 against most viral strains.
Keywords: Heptad repeat region; Helix bundle; Fusion inhibitor; HIV-1; gp41 hydrophobic pocket; Turbidity clearance; Surface plasmon resonance; Bi-modal binding
Protocatechuic aldehyde inhibits hepatitis B virus replication both in vitro and in vivo by Zhe Zhou; Yi Zhang; Xiao-Ran Ding; Su-Hong Chen; Jing Yang; Xue-Jun Wang; Gao-Long Jia; Hong-Shan Chen; Xiao-Chen Bo; Sheng-Qi Wang (pp. 59-64).
Natural compounds provide a large reservoir of potentially active anti-hepatitis B virus (HBV) agents. We examined the direct effects of protocatechuic aldehyde (PA; derived from the Chinese herb, Salvia miltiorrhiza) on HBV replication in HepG2 2.2.15 cell line and duck hepatitis B virus (DHBV) replication in ducklings in vivo. The extracellular HBV DNA, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA, respectively. DHBV in duck serum was analyzed by dot blot. PA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner at concentrations between 24 and 48μg/mL. PA (25, 50, or 100mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. We provide the first evidence that PA, a novel anti-HBV substance derived from traditional Chinese herb S. miltiorrhiza, can efficiently inhibits HBV replication in HepG2 2.2.15 cell line in vitro and inhibit DHBV replication in ducks in vivo. PA therefore warrants further investigation as a potential therapeutic agent for HBV infections.
Keywords: Abbreviations; DHBV; duck hepatitis B virus; HBV; hepatitis B virus; PA; protocatechuic aldehyde; RT; reverse transcription; 3TC; lamivudine; ELISA; enzyme-linked immunosorbent assay; PCR; polymerase chain reaction; HBeAg; hepatitis B e antigen; HBsAg; hepatitis B surface antigen; pgRNA; pregenomic RNAProtocatechuic aldehyde; Duck hepatitis B virus; Hepatitis B virus; Salvia miltiorrhiza
Antiviral treatment of Coxsackie B virus infection in human pancreatic islets by Anna-Karin Berg; Annika Olsson; Olle Korsgren; Gun Frisk (pp. 65-71).
Enterovirus infections of the pancreatic islets are believed to trigger or precipitate the near total destruction of β-cells that constitutes type 1 diabetes (T1D). This study investigated the ability of an anti-picornaviral compound, pleconaril, to block the replication of two β-cell tropic Coxsackie B4 virus (CBV-4) strains in isolated human islets. The two strains, VD2921 and V89 4557, with demonstrated abilities to cause non-lytic persistence or lytic infection, respectively, in islets, represented two different potential mechanisms behind virus-induced T1D. The virus replication in the islets was studied with and without addition of pleconaril. In addition, islet morphology was studied every day. To test the effects of pleconaril and/or DMSO on the β-cells’ insulin secretion, glucose perifusions were performed on treated and untreated islets. Virus titrations showed a clear reduction of the replication of both strains after pleconaril treatment. The VD2921 strain was inhibited to undetectable levels. The V89 4557 strain, however, showed an initial reduction of titers but virus titers then increased despite the addition of a second dose of pleconaril. This incomplete inhibition of viral replication suggested the existence of a resistant subtype within this strain. Pleconaril treatment reduced the β-cells’ insulin secretion in response to glucose stimulation in some experiments and induced slight morphological changes to the islets compared to untreated controls. In summary, pleconaril reduced the replication of the two β-cell tropic CBV-4 strains in human islets. However, genetic differences between these strains influenced the effectiveness of pleconaril treatment. This stresses the importance of using multiple viral strains in antiviral tests.
Keywords: β-Cell; Islet of langerhans; Enterovirus; Picornavirus; Pleconaril; Type 1 diabetes
HIV1 protease inhibitors selectively induce inflammatory chemokine expression in primary human osteoblasts by Andrea P. Malizia; Mihai H. Vioreanu; Peter P. Doran; William G. Powderly (pp. 72-76).
HIV-infected patients are at increased risk of decreased bone mineral density. Several studies have implicated antiretroviral therapy as a contributor to the decreased bone mineral density seen in treated HIV-1 patients. Whilst the exact molecular mechanisms underlying decreased bone density remain to be elucidated, inflammation has been postulated to be an important pathogenomic mechanism. In this study, we have explored primary human osteoblast gene expression in response to protease inhibitors (PIs), by oligonucleotide microarray analysis. A list of dysregulated genes, correlated with the inflammatory response, increased significantly after NFV and RTV exposure. Analysis of gene and protein expression determined a selectively increase of the pro-inflammatory cytokines monocyte chemoattractant protein (MCP)-1 and interleukin-8 (IL-8) following exposure to a pharmacological concentration of NFV and RTV. These data suggested that generation of local inflammatory cascades may contribute to the development of decreased bone mineral density in highly active antiretroviral therapy (HAART)-treated HIV patients.
Keywords: HAART; Osteoblasts; Inflammation; MCP-1; IL-8; Nelfinavir; Ritonavir
Inhibition of avian metapneumovirus (AMPV) replication by RNA interference targeting nucleoprotein gene (N) in cultured cells by Helena Lage Ferreira; Fernando Rosado Spilki; Renata Servan de Almeida; Márcia M.A.B. Santos; Clarice Weis Arns (pp. 77-81).
Avian metapneumovirus (AMPV) is the primary causative agent of severe rhinotracheitis in turkeys. It is associated with swollen head syndrome in chickens and is the source of significant economic losses to animal food production. In this study, we designed specific short interfering RNA (siRNA) targeting the AMPV nucleoprotein (N) and fusion (F) genes. Three days post-virus infection, virus titration, real time RT-PCR, and RT-PCR assays were performed to verify the effect of siRNA in AMPV replication. A marked decrease in virus titers from transfected CER cells treated with siRNA/N was observed. Also, the production of N, F, and G mRNAs in AMPV was decreased. Results indicate that N-specific siRNA can inhibit virus replication. In future studies, a combination of siRNAs targeting the RNA polymerase complex may be used as a tool to study AMPV replication and/or antiviral therapy.
Keywords: Avian metapneumovirus; RNA interference; Short interfering RNA; Inhibition; Real time PCR