Yohichi Kumaki

A new mouse-adapted strain of SARS-CoV as a lethal model for evaluating antiviral agents in vitro and in vivo

Abstract Severe acute respiratory syndrome (SARS) is a highly lethal emerging disease caused by coronavirus SARS-CoV. New lethal animal models for SARS were needed to facilitate antiviral research. We adapted and characterized a new strain of SARS-CoV (strain v2163) that was highly lethal in 5- to 6-week-old BALB/c mice. It had nine mutations affecting 10 amino acid residues. Strain v2163 increased IL-1α, IL-6, MIP-1α, MCP-1, and RANTES in mice, and high IL-6 expression correlated with mortality. The infection largely mimicked human disease, but lung pathology lacked hyaline membrane formation. In vitro efficacy against v2163 was shown with known inhibitors of SARS-CoV replication. In v2163-infected mice, Ampligen™ was fully protective, stinging nettle lectin (UDA) was partially protective, ribavirin was disputable and possibly exacerbated disease, and EP128533 was inactive. Ribavirin, UDA, and Ampligen™ decreased IL-6 expression. Strain v2163 provided a valuable model for anti-SARS research.

Favipiravir (T-705) protects against peracute Rift Valley fever virus infection and reduces delayed-onset neurologic disease observed with ribavirin treatment.

Rift Valley fever is a zoonotic, arthropod-borne disease that affects livestock and humans. The etiologic agent, Rift Valley fever virus (RVFV; Bunyaviridae, Phlebovirus) is primarily transmitted through mosquito bites, but can also be transmitted by exposure to infectious aerosols. There are presently no licensed vaccines or therapeutics to prevent or treat severe RVFV infection in humans. We have previously reported on the activity of favipiravir (T-705) against the MP-12 vaccine strain of RVFV and other bunyaviruses in cell culture. In addition, efficacy has also been documented in mouse and hamster models of infection with the related Punta Toro virus. Here, hamsters challenged with the highly pathogenic ZH501 strain of RVFV were used to evaluate the activity of favipiravir against lethal infection. Subcutaneous RVFV challenge resulted in substantial serum and tissue viral loads and caused severe disease and mortality within 2-3 days of infection. Oral favipiravir (200 mg/kg/day) prevented mortality in 60% or greater of hamsters challenged with RVFV when administered within 1 or 6h post-exposure and reduced RVFV titers in serum and tissues relative to the time of treatment initiation. In contrast, although ribavirin (75 mg/kg/day) was effective at protecting animals from the peracute RVFV disease, most ultimately succumbed from a delayed-onset neurologic disease associated with high RVFV burden observed in the brain in moribund animals. When combined, T-705 and ribavirin treatment started 24 h post-infection significantly improved survival outcome and reduced serum and tissue virus titers compared to monotherapy. Our findings demonstrate significant post-RVFV exposure efficacy with favipiravir against both peracute disease and delayed-onset neuroinvasion, and suggest added benefit when combined with ribavirin.

Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model.

Abstract Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p <0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 106 to 108 PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12h post infection (p <0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.

Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin

Abstract Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1±0.4μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575PFU) of virus for 4h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0mg/kg/day for 4 days. Treatment with UDA at 5mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p <0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p <0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells.

Inhibition of novel reassortant avian influenza H7N9 virus infection in vitro with three antiviral drugs, oseltamivir, peramivir and favipiravir.

Background: A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from three patients and was identified as H7N9 in China. Antiviral agents are required to treat patients with avian influenza H7N9 virus infection. Methods: In this study, we assessed the antiviral potential of oseltamivir, peramivir, favipiravir (T-705), amantadine and rimantadine against novel reassortant avian-origin influenza H7N9 virus in vitro. Results: All three avian influenza H7N9 virus strains were sensitive to oseltamivir, peramivir and favipiravir (T-705), but resistant to amantadine and rimantadine. Conclusions: Our data show a pattern of antiviral sensitivity for this novel H7N9 strain of influenza that suggests the compounds oseltamivir, peramivir and favipiravir should be useful for therapy.

Interferon alfacon 1 inhibits SARS-CoV infection in human bronchial epithelial Calu-3 cells

Abstract The primary targets for SARS-CoV infection are the epithelial cells in the respiratory and intestinal tract. The angiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor for SARS-CoV. ACE-2 has been shown to be expressed at the apical domain of polarized Calu-3 cells. In this report, interferon alfacon 1 was examined for inhibitory activities against SARS-CoV on human lung carcinoma epithelial Calu-3 cell line and the other three African green monkey kidney epithelial cell lines. Interferon alfacon 1 demonstrated significant antiviral activity in neutral red uptake assay and virus yield reduction assay. The data might provide an important insight into the mechanism of pathogenesis of SARS-CoV allowing further development of antiviral therapies for treating SARS infections.

Induction of Interferon-γ-Inducible Protein 10 by SARS-CoV Infection, Interferon Alfacon 1 and Interferon Inducer in Human Bronchial Epithelial Calu-3 Cells and BALB/c Mice

Background: The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is poorly understood. Several mechanisms involving both direct effects on target cells and indirect effects via the immune system might exist. SARS-CoV has been shown in vitro to induce changes of cytokines and chemokines in various human and animal cells. We previously reported that interferon (IFN) alfacon-1 was more active against SARS-CoV infection in human bronchial epithelial Calu-3 cells than in African green monkey kidney epithelial cells on day 3 post-infection. Methods: In the current study, we first evaluated the efficacy of IFN-alfacon 1 in Calu-3 cells during the first 7 days of virus infection. We then used the two-antibody sandwich ELISA method to detect IFN-γ-inducible protein 10 (IP-10). We further evaluated the efficacy of antivirals directed against SARS-CoV infection in BALB/c mice. Results: A potent, prolonged inhibition of SARS-CoV replication in Calu-3 cells with IFN-alfacon 1 was observed. Furthermore, IP-10, an IFN-inducible leukocyte chemoattractant, was detected in Calu-3 cells after SARS-CoV infection. Interestingly, IP-10 expression was shown to be significantly increased when SARS-CoV-infected Calu-3 cells were treated with IFN alfacon-1. IP-10 expression was detected in the lungs of SARS-CoV-infected BALB/c mice. Significantly high levels of mouse IP-10 in BALB/c mice was also detected when SARS-CoV-infected mice were treated with the interferon inducer, polyriboinosinic-polyribocytidylic acid stabilized with poly-L-lysine and carboxymethyl cellulose (poly IC:LC). Treatment with poly IC:LC by intranasal route were effective in protecting mice against a lethal infection with mouse-adapted SARS-CoV and reduced the viral lung titres. Conclusion: Our data might provide an important insight into the mechanism of pathogenesis of SARS-CoV and these properties might be therapeutically advantageous.

Inhibition of adenovirus serotype 14 infection by octadecyloxyethyl esters of (S)-[(3-hydroxy-2-phosphonomethoxy)propyl]- nucleosides in vitro

Abstract On September 22, 2008, a physician on Prince of Wales Island, Alaska, notified the Alaska Department of Health and Social Services (ADHSS) of an unusually high number of adult patients with recently diagnosed pneumonia (n = 10), including three persons who required hospitalization and one who died. ADHSS and CDC conducted an investigation to determine the cause and distribution of the outbreak, identify risk factors for hospitalization, and implement control measures. This report summarizes the results of that investigation, which found that the outbreak was caused by adenovirus 14 (Ad14), an emerging adenovirus serotype in the United States that is associated with a higher rate of severe illness compared with other adenoviruses. Among the 46 cases identified in the outbreak from September 1 through October 27, 2008, the most frequently observed characteristics included the following: male (70%), Alaska Native (61%), underlying pulmonary disease (44%), aged > or = 65 years (26%), and current smoker (48%). Patients aged > or = 65 years had a fivefold increased risk for hospitalization. The most commonly reported symptoms were cough (100%), shortness of breath (87%), and fever (74%). Of the 11 hospitalized patients, three required intensive care, and one required mechanical ventilation. One death was reported. Ad14 isolates obtained during the outbreak were identical genetically to those in recent community‐acquired outbreaks in the United States which suggests the emergence of a new, and possibly more virulent Ad14 variant. Clinicians should consider Ad14 infection in the differential diagnosis for patients with community‐acquired pneumonia, particularly when unexplained clusters of severe respiratory infections are detected. HighlightsAdenovirus type 14 had been reported to cause severe and fatal pneumonia in rare cases in people of all ages.No antiviral compounds have yet been approved for the treatment of such adenovirus infections.Four nucleoside analogue compounds were evaluated against adenovirus type 14 and some other adenoviruses in vitro.All the ODE‐nucleoside analogues demonstrated antiviral activities in neutral red uptake and virus yield reduction assays.

Prophylactic and therapeutic intranasal administration with an immunomodulator, Hiltonol® (Poly IC:LC), in a lethal SARS-CoV-infected BALB/c mouse model

&NA; Hiltonol®, (Poly IC:LC), a potent immunomodulator, is a synthetic, double‐stranded polyriboinosinic‐polyribocytidylic acid (poly IC) stabilized with Poly‐L‐lysine and carboxymethyl cellulose (LC). Hiltonol® was tested for efficacy in a lethal SARS‐CoV‐infected BALB/c mouse model. Hiltonol® at 5, 1, 0.5 or 0.25 mg/kg/day by intranasal (i.n.) route resulted in significant survival benefit when administered at selected times 24 h prior to challenge with a lethal dose of mouse‐adapted severe acute respiratory syndrome coronavirus (SARS‐CoV). The infected BALB/c mice receiving the Hiltonol® treatments were also significantly effective in protecting mice against weight loss due to infection (p < 0.001). Groups of 20 mice were dosed with Hiltonol® at 2.5 or 0.75 mg/kg by intranasal instillation 7, 14, and 21 days before virus exposure and a second dose was given 24 h later, prophylactic Hiltonol® treatments (2.5 mg/kg/day) were completely protective in preventing death, and in causing significant reduction in lung hemorrhage scores, lung weights and lung virus titers. Hiltonol® was also effective as a therapeutic when give up to 8 h post virus exposure; 100% of the‐infected mice were protected against death when Hiltonol® was administered at 5 mg/kg/day 8 h after infection. Our data suggest that Hiltonol® treatment of SARS‐CoV infection in mice leads to substantial prophylactic and therapeutic effects and could be used for treatment of other virus disease such as those caused by MERS‐CoV a related coronavirus. These properties might be therapeutically advantageous if Hiltonol® is considered for possible clinical use. HighlightsWe evaluated the use of Hiltonol® for treating lethal SARS‐CoV‐infected BALB/c mice.Hiltonol® treatment leads to prophylactic and therapeutic effects.These properties might be therapeutically advantageous.