Antiviral Activity Of Arbidol Against Hantavirus In Vitro And In Vivo

Hemorrhagic fever with renal syndrome (HFRS) is rodent viral zoonosis that caused by viruses of the genus Hantavirus, family Bunyaviridae. More than 50,000 cases of HFRS are reported annually in Asia and Europe. Most are reported in China. Vaccination is the preferred choice for controlling HFRS and reducing the impact of incidence. Several inactivated Hantaan virus (HTNV) and Seoul virus vaccines for HFRS have been used, but are not yet completely effective or protect against all strains. Additionally, not all people at risk can receive vaccination, especially in those regions having only sporadic outbreaks. During the last few years extensive efforts have been made in the search for effective anti-HTNV agents. Some nucleoside analogs have been found highly inhibitory in animal models, ribavirin is the only antiviral agent known to have been successfully evaluated against HTNV infections in clinical trails. However, the utilization of ribavirin is limited due to its temporal myelosuppression and toxicity. No antiviral agents are approved by the Food and Drug Administration (FDA) to treat HFRS. Therefore, an effort to seek alternative reagents is strongly desirable. Arbidol is an immunomodulator developed in Russia. It has been shown to have broad antiviral effects against such viruses as influenza virus, coxsackievirus, respiratory syncytial virus, and hepatitis C virus. The aim of the present study was to evaluate the anti-HTNV activity of arbidol in vivo and in vitro.ObjectiveTo detect and quantitate HTNV (76-118 strain) after amplification in Vero E6 cell culture, a fluorescence-activated cell sorter (FACS) flow cytometry based method has been developed. To study the anti-HTNV activity of arbidol hydrochloride on Vero E6 cells, cells infected with virus were detected by assay flow cytometry and indirect immunofluorescence (IFA). To further study the action mode of arbidol in vitro, the effect of 5μg/mL arbidol added at -24,-12,0, 2h post inoculation was determined. To investigate whether arbidol has an antiviral effect in vivo, suckling mouse were treated with arbidol at 24 hour before infection at 5,10 or 20mg/kg/d, once per day, for 10 days. To investigate the anti-hantaviral mechanism of arbidol in vivo, four animals were sacrificed for each experimental point (days 4,8,12,16 post infection (pi)), and survivors were euthanized on day 28 pi. Brains, lungs, and kidneys were aseptically dissected from the animals and divided into three parts:one-third was frozen for antigen detection by IF A, one-third was placed in formalin for histological examination, and the rest was stored at -80℃for virological parameters determination.Methods1. A flow cytometry based method has been developed to detect and quantitate HTNV (76-118 strain) after amplification in Vero E6 cell culture. According to the assay we have been developed, two methods of detecting HTNV antigen including flow cytometry and IFA were compared. The kinetics of the virus infection process was determined.2. The antiviral activity of arbidol hydrochloride was evaluated with flow cytometry and IFA. After treated with 5μg/mL arbidol at the -24,-12,0,2 hour post infection (hpi), the positive cells of HTNV infected cells was determined by flow cytometry, and viral mRNA levels was detected by semi-quantited reverse transcriptase polymerase chain reaction.3. Arbidol was evaluated for the dose considered lethally toxic to 2 day old mice. Suckling mice (3 days old) were inoculated ic with 100LD50 of virus stock. The mice were randomly divided into five groups (35 mice per group) and arbidol was tested with the following dosage regimens:5,10, or 20mg/kg/d once a day for 10 days. Treatment was initiated at 24hr before infection, by the same investigator, and with the dose corrected for weight change. Placebo controls (inoculated with 100LD50 of HTNV) and normal controls (inoculated with 2%MEM) received 0.5% methylcellulose solution instead of arbidol. Animals were observed two times and weighed daily. Mice were observed daily for mortality for 28 days after infection. Death occurring 24hr after inoculation of virus was considered traumatic, and such animals were handled as withdrawals. The effect was estimated by the reduction of the rate of mortality and prolongation of mean time to death (MTD). Four mice were sacrificed for each experimental point (on days 4,8, 12,16 pi), and survivors were euthanized on day 28 pi. Histopathological changes in brains, lungs, and kidneys were aseptically dissected from the animals and divided into three parts:one-third was frozen for antigen detection by IFA, one-third was placed in formalin for histological examination, and the rest was stored at -80℃for virological parameters determination. TNF-a and IFN-βwere detected in serum by QuantiKine quantitative sandwich EIAs according to the manufacturer's instruction.Results1. The FACS procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as the 2nd after inoculation with virus,2.6% of infected cells were detected in cells culture, while IFA can detect viral antigen on the 3rd day. The low levels of both methods can detect is 50PFU.2. Compared with untreated control, the percentage of positive cells of pre-treated or treated with arbidol was decreased in a dose-dependent manner. The positive rate of cells infected with HTNV treated with 5μg/mL arbidol at the -24,-12,0,2hpi decreased in a time-dependent manner (p<0.05), the viral mRNA levels of HTNV treated with 5μg/mL arbidol on the -24,-12 hpi demonstrated a significant reduction in comparison to the untreated control (p<0.05)3. Compared with the placebo-treated animals, arbidol-treated animals died at a later time and appearance of clinical signs was reduced in a dose-dependent manner. Weight recovered and symptoms disappeared by day 28 in the survivors. All placebo-treated mice died, with a MTD to 17.17 days, treatment with arbidol increased both survival rate and MTD. Arbidol administered at 10mg/kg/d increased survival rate to 33.3% and MTD to 21.5 days (p<0.05),20mg/kg/d increased survival rate to 60% and MTD to 23.8 days (p<0.05). Orally administered arbidol can reduce histopathological changes, decrease expression of viral antigen and virus loads, and modulate the TNF responses.Conclusions1. Flow cytometry was a rapid, sensitive and accurate method to detecting HTNV antigen in Vero E6 cells culture.2. Arbidol hydrochloride was efficacious against HTNV when added before or after viral infection, no directly virucidal effect of arbidol was found at concentrations lower than 8μg/mL in this experiment.3. Arbidol has the ability to elicit protective antiviral activity against HTNV in vivo.