New class of hantaan virus inhibitors based on conjugation of the isoindole fragment to (+)-camphor or (-)-fenchone hydrazonesv.

This work presents the design and synthesis of camphor, fenchone, and norcamphor N-acylhydrazone derivatives as a new class of inhibitors of the Hantaan virus, which causes haemorrhagic fever with renal syndrome (HFRS). A cytopathic model was developed for testing chemotherapeutics against the Hantaan virus, strain 76-118. In addition, a study of the antiviral activity was carried out using a pseudoviral system. It was found that the hit compound possesses significant activity (IC50 = 7.6 ± 2 µM) along with low toxicity (CC50 > 1000 µM). Using molecular docking procedures, the binding with Hantavirus nucleoprotein was evaluated and the correlation between the structure of the synthesised compounds and the antiviral activity was established.

T-cell immunoglobulin and mucin (TIM) contributes to the infection of human airway epithelial cells by pseudotype viruses containing Hantaan virus glycoproteins.

Hantaviruses are rodent-borne hemorrhagic fever viruses leading to serious diseases. Viral attachment and entry represent the first steps in virus transmission and are promising targets for antiviral therapeutic intervention. Here we investigated receptor use in human airway epithelium of the Old and New World hantaviruses Hantaan virus (HTNV) and Andes virus (ANDV). Using a biocontained recombinant vesicular stomatitis virus pseudotype platform, we provide first evidence for a role of the cellular phosphatidylserine (PS) receptors of the T-cell immunoglobulin and mucin (TIM) protein family in HTNV and ANDV infection. In line with previous studies, HTNV, but not ANDV, was able to use glycosaminoglycan heparan sulfate and αvß3 integrin as co-receptors. In sum, our studies demonstrate for the first time that hantaviruses make use of apoptotic mimicry for infection of human airway epithelium, which may explain why these viruses can easily break the species barrier.

IFN-λs inhibit Hantaan virus infection through the JAK-STAT pathway and expression of Mx2 protein.

Hantaan virus (HTNV), member of the newly defined Hantaviridae family, within the order Bunyavirales, can cause a hemorrhagic fever with renal syndrome with high fatality rates in humans. However, no specific antiviral agents are currently available for HTNV infection approved by the US Food and Drug Administration. Although interferon lambdas (IFN-λs) have been shown to induce an antiviral state against HTNV, the molecular mechanisms remain to be determined. In this study, we found that IFN-λs exerted its anti-HTNV effect by activating Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway-mediated antiviral immunity in A549 cells. Simultaneously, IFN-λs downregulated suppressor of cytokine signaling proteins, which are the known negative feedback regulators of the JAK-STAT signaling pathway. Additionally, we demonstrated the role of IFN-λs-induced myxovirus resistance 2 (Mx2, also known as MxB) protein as a potential inhibitor for HTNV infection. These findings indicate that IFN-λs play an important role in cellular defenses against HTNV infection at an early stage and that human Mx2 may represent a potential therapeutic target for HTNV infection.

Determining the Virus Life-Cycle Stage Blocked by an Antiviral.

Among the members of the Arenaviridae family, Junín virus and Lassa virus represent important human health threats generating annual outbreaks of severe human hemorrhagic fever (HF) in endemic areas of Argentina and Western Africa, respectively. Given the lack of a specific and safe chemotherapy, the search for effective antiviral compounds is a continuous demanding effort. During the last two decades, academic research studies originated important results identifying novel molecules to be considered for further in vivo characterization. This chapter summarizes experimental in vitro approaches used to determine the possible mechanism of action of these antiviral agents.

In-Cell Western Assays to Evaluate Hantaan Virus Replication as a Novel Approach to Screen Antiviral Molecules and Detect Neutralizing Antibody Titers.

Hantaviruses encompass rodent-borne zoonotic pathogens that cause severe hemorrhagic fever disease with high mortality rates in humans. Detection of infectious virus titer lays a solid foundation for virology and immunology researches. Canonical methods to assess viral titers rely on visible cytopathic effects (CPE), but Hantaan virus (HTNV, the prototype hantavirus) maintains a relatively sluggish life cycle and does not produce CPE in cell culture. Here, an in-cell Western (ICW) assay was utilized to rapidly measure the expression of viral proteins in infected cells and to establish a novel approach to detect viral titers. Compared with classical approaches, the ICW assay is accurate and time- and cost-effective. Furthermore, the ICW assay provided a high-throughput platform to screen and identify antiviral molecules. Potential antiviral roles of several DExD/H box helicase family members were investigated using the ICW assay, and the results indicated that DDX21 and DDX60 reinforced IFN responses and exerted anti-hantaviral effects, whereas DDX50 probably promoted HTNV replication. Additionally, the ICW assay was also applied to assess NAb titers in patients and vaccine recipients. Patients with prompt production of NAbs tended to have favorable disease outcomes. Modest NAb titers were found in vaccinees, indicating that current vaccines still require improvements as they cannot prime host humoral immunity with high efficiency. Taken together, our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.

Efficacy of arbidol on lethal hantaan virus infections in suckling mice and in vitro.

AIM: Arbidol is an immunomodulator that was first developed in Russia. In this study, we report the antiviral activity of arbidol against Hantaan virus (HTNV) in vitro and in vivo. METHODS: The antiviral activity of arbidol in vitro was determined by plaque-forming assay, ranging from 0.5 to 8 microg/mL. To investigate whether arbidol has an antiviral effect in vivo, suckling BALB/c mice infected with HTNV were treated with arbidol at 24 h before infection with a 5, 10 or 20 mg.kg(-1).d(-1), once per day, for 10 days. On day 12 and 28 post infection (pi), histopathological changes and viral antigen were detected. On days 4, 8, 12, and 16 pi, the viral load of target organs and serum TNF-alpha levels of arbidol-treated animals were determined. RESULTS: Arbidol was found to have potent inhibitory activity against HTNV when added in vitro before or after viral infection, with a 50% inhibitory concentration (IC(50)) of 0.9 and 1.2 microg/mL, respectively. The 50% lethal dose (LD(50)) of arbidol for suckling mice was 78.42 mg.kg(-1).d(-1). Oral administration of arbidol increased both survival rate and mean time to death (MTD). Treatment with arbidol reduced histopathological changes, decreased viral load and viral antigen levels, and modulated the level of serum TNF-alpha. CONCLUSION: Arbidol has the ability to elicit protective antiviral activity against HTNV in vivo and in vitro.Acta Pharmacologica Sinica (2009) 30: 1015-1024; doi: 10.1038/aps.2009.53; published online 8 June 2009.

Synthesis and anti-Hantaan virus activity of N(1)-3-fluorophenyl-inosine.

As part of an ongoing effort to develop new antiviral nucleoside analogs, our interest was drawn to N(1)-aryl purines as a novel structural class and potential scaffold for drug discovery. Herein, we describe the synthesis of N(1)-3-fluorophenyl-inosine (FPI) and N(1)-3-fluorophenyl-hypoxanthine (FP-Hx) and their antiviral activity against hantaviruses. The EC(50) for FPI and FP-Hx were 94 and 234microM, respectively, against Hantaan virus. FPI was not toxic to mammalian cells at concentrations that exhibited antiviral activity. Analysis of its metabolism revealed a low conversion of FPI in Vero E6 or human cells to a 5'-triphosphate, and it was a poor substrate for human purine nucleoside phosphorylase. Further, the compound did not alter GTP levels indicating FPI does not inhibit inosine monophosphate dehydrogenase. With respect to the virus, FPI did not decrease viral RNA levels or increase the mutation frequency of the viral RNA. This suggests that the antiviral activity of FPI might be solely due to the interaction of FPI or its metabolites with viral or host proteins involved in post-replication events that would affect the levels of infectious virus released. Synthesis of other compounds structurally similar to FPI is warranted to identify more potent agents that selectively abrogate production of infectious virus.

Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea.

Results of a clinical study using intravenous (IV) ribavirin for treating Department of Defense personnel with hemorrhagic fever with renal syndrome (HFRS) acquired in Korea from 1987 to 2005 were reviewed to determine the clinical course of HFRS treated with IV ribavirin. A total of 38 individuals enrolled in the study had subsequent serological confirmation of HFRS. Four of the 38 individuals received three or fewer doses of ribavirin and were excluded from treatment analysis. Of the remaining 34 individuals, oliguria was present in one individual at treatment initiation; none of the remaining 33 subjects developed oliguria or required dialysis. The mean peak serum creatinine was 3.46 mg/dl and occurred on day 2 of ribavirin therapy. Both the peak serum creatinine and the onset of polyuria occurred on mean day 6.8 of illness. Reversible hemolytic anemia was the main adverse event of ribavirin, with a >or=25% decrease in hematocrit observed in 26/34 (76.5%) individuals. While inability to adjust for all baseline variables prevents comparison to historical cohorts in Korea where oliguria has been reported in 39-69% cases and dialysis required in approximately 40% HFRS cases caused by Hantaan virus, the occurrence of 3% oliguria and 0% dialysis requirement in the treatment cohort is supportive of a previous placebo-controlled HFRS trial in China where IV ribavirin given early resulted in decreased occurrence of oliguria and decreased severity of renal insufficiency.

[Simulation of the disease caused by Hantaan virus in white mice by a complex of virological, biochemical, and hematological parameters].

The essence of studies was that the disease is simulated in 12-day albino mice subcutaneously infected with Hantaan virus, strain 76-118 in a dose of 10 LD50. As an efficiency index, the study of drugs uses major (death protection coefficient, mean animal lifetime) and auxiliary (virological: pathogen accumulation in the brain tissues of deceased animals) parameters, biochemical (the levels of creatinine, urea, alanine aminotransferase, aspartate aminotransferase, malonic dialdehyde), hematological (count of leukocytes, leukogram) ones; as well as interferon status (the levels of circulatory interferon, leukocytic production of alpha- and gamma-interferons). The procedure for simulating the disease caused by Hantaan virus on an experimental animal is used to choose effective drugs against the pathogen of hemorrhagic nephrosonephritis.

[Inhibiting effects of fucoidans on Hantaan virus adsorption on a model of peritoneal macrophages in vitro].

A model of peritoneal macrophages was used to study the effect of fucoidans from brown seaweeds on the adsorption of Hantaan virus. Fucoidans were found to have antiviral activity, but to differ in the inhibition of hantavirus adsorption, which was associated with their structural features. The mechanism of their action is assumed to be shown via ligand-receptor interaction with certain cell membrane receptors and via blockage of hantavirus glycoproteins (G1 and G2), resulting in adsorption inhibition and preventing viral penetration into the macrophages.

Real-time RT-PCR of Hantaan virus RNA used for the detection of virus response to antiviral drugs.

Hantaan virus (HTN) is an important cause of hemorrhagic fever with renal syndrome (HFRS) in Korea. HTN RNA can be detected with the RT-PCR and the quantity of HTN RNA in infected cells can be measured by competitive RT-PCR. The current study used the real-time RT-PCR for the detection of viral RNA S gene in a more detailed fashion than in the previous study (Nam et al., Virus Genes 26, 31-38, 2003). A standard curve was generated with serial 10-fold dilutions of the HTN RNA. The sensitivity of RNA detection was approximately 10 PFU of HTN. The cells infected with HTN were treated with the antiviral drugs ribavirin, zidovudine, and amantadine. 24 hrs after infection, real-time RT-PCR was used to detect the HTN RNA synthesized in the infected cells. No viral RNA was detected in the HTN-infected cells treated with antiviral drugs, but HTN RNA was detected in untreated HTN-infected cells. This finding suggested that real-time RTPCR should be used for the detection of antiviral activity against HTN.

[The in vitro antiviral activity against the causative agent of hemorrhagic fever with renal syndrome].

It is shown that primary screening of drugs against the pathogens of hemorrhagic fever with renal syndrome (HFV) may be performed, by using two approaches in estimating the suppression of plague formation and that of viral reproduction in the cultured cells. It is expedient to make a primary screening of interferon and its inductors to estimate the suppression of viral reproduction in the cultured Vero E6, PSEV, and CL-17 cells, the infection multiplicity should be hundredth parts of BOE/cell. Among the test agents, there are virazole and realdiron that are the most active drugs against Hantaan virus, 76-118 strain, which virually completely suppress the reproduction of the study causative agent, when used even at concentrations of 1-5 microg/ml and 100 U/ml, respectively.

Ribavirin reveals a lethal threshold of allowable mutation frequency for Hantaan virus.

The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5'-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.

[Screening of biopolymers from sea hydrocoles affecting the adsorption of Hantaan virus].

The biopolymers extracted from sea hydrocoles were screened and a group of sulfated polysaccharides--fucoidans having a pronounced inhibitory effect on the adsorption of Hantaan virus in the cultured Vero E6 cells was identified. The mechanism of action of sulfated polysaccharides was realized through competitive carbohydrate-specific binding to cell membrane receptors and through ligand-receptor interaction and blocking of Hantaan virus glycoproteins (G1 and G2).

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Activity of ribavirin against Hantaan virus correlates with production of ribavirin-5'-triphosphate, not with inhibition of IMP dehydrogenase.

Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5'-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5'-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 mug/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 mug/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.

[Hemorrhagic fever with the renal syndrome: modern aspects of epidemiology and prophylaxis].

Hemorrhagic fever with the renal syndrome (HFRS) is a typical natural-and-focal virus infection having military-and-epidemiological importance for Armed Forces of most countries. Severe forms of this infection are typical for the Far East of Russia, countries of South-East Asia and especially for the American continent. The lethality from HFRS amounts to 15-50%. The main natural carriers and keepers of HFRS stimulus are various mouse-type rodents. In Russia besides well-studied natural focuses on the Far East, Siberia, Ural, Povolge other active HFRS focuses were determined not long ago (Moscow, Orel, Lipetsk, Tula and Voronezh regions, the territory of Big Sochi). A man is infected by an aerosol, forming during drying up of rodents' urine, feces, saliva containing HFRS virus. Vaccinal prevention of HFRS in Russia hasn't yet been worked out. Ribavirin is the most effective in the treatment for HFRS. The means of prevention of population and servicemen must exclude their contact with rodents both in natural conditions and in populated areas.

Effect of cordycepin on Hantaan virus 76-118 infection of primary human embryonic pulmonary fibroblasts--characterization of apoptotic effects.

The cDNA microarray technique was used to study gene epression in human embryonic pulmonary fibroblasts (HEPF) infected with Hantaan virus (HTNV) under the influence of cordycepin (Cor), an inhibitor of post-transcriptional pre-mRNA polyadenylation. Four apoptotic genes, the insulin-like growth factor binding protein 1, NFkB inhibitor alpha, caspase-3 and NFkB1 were up-regulated in both infected and uninfected Cor-treated cells and two cell cycle-associated genes, CDC-like kinase and beta-induced transforming growth factor were up-regulated in Cor-untreated cells but down-regulated in Cor-treated cells. Cell morphology examination, quantitative RT-PCR, and immunofluorescence (IF) test suggested that following the Cor treatment the HTNV infection took place, but late viral gene expression was slightly reduced. Three parameters, namely caspase-3 activity, annexin V binding, and cell cycle were used to detect apoptosis. The results suggested that the induction of apoptosis in HEPF by HTNV started at 6 hrs post infection (p.i.). Following the Cor treatment, however, the caspase-3 activity began to increase at 24 hrs p.i. Thus it is suggested that inhibition of de novo late viral protein synthesis by Cor changes the apoptosis pathway and cell cycle by delaying caspase-3 gene expression and by up/down-regulating of expression of other apoptotic and cell cycle-associated genes. This implicates that HTNV can induce apoptosis in HEPF even without de novo viral protein synthesis and with a reduced and slowed viral maturation.

Inactivation of Hantaan virus-containing samples for subsequent investigations outside biosafety level 3 facilities.

OBJECTIVES: The potential risk of accidental infection by hantaviruses in a clinical or research laboratory necessitates special precautionary measures. A biosafety program must address handling and disposal of infectious materials as well as appropriate virus inactivation or depletion procedures to permit necessary further processing of specimens outside the biosafety level 3 laboratory. METHODS: To study the elimination of hantavirus infectivity, the effects of different chemical and physical inactivation and depletion procedures were investigated on Hantaan virus-containing materials. An infectivity assay for hantaviruses was utilised to verify these procedures which are commonly preceding investigations such as ELISA, flow cytometry analysis, Western blot or immunofluorescence assay. RESULTS: Chemical inactivation with methanol, paraformaldehyde, acetone/methanol and detergent-containing lysis buffer as well as physical forces such as UV irradiation and filtration efficiently reduced viral infectivity in infected cells and their supernatants below the detection limit. CONCLUSION: The virus inactivation and depletion methods described herein are suitable to prepare non-infectious samples for further use in immunological, virological and cell-biological assays.

Peptide antagonists that inhibit Sin Nombre virus and hantaan virus entry through the beta3-integrin receptor.

Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the alpha(v)beta(3) integrin, and transfection of human beta(3) integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-beta(3) antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified alpha(v)beta(3), we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 mug/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use beta(3) integrins and PHV uses a different receptor, beta1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via beta(3) integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.