Coagulation factors, fibrinogen and plasminogen activator inhibitor-1, are differentially regulated by yellow fever virus infection of hepatocytes.

Yellow fever virus (YFV) infection poses a great risk to un-vaccinated individuals living or traveling in the endemic regions of Africa and South America. It is estimated that approximately 30,000 people die each year of this disease. The liver is the main target of YFV, where as many as 80% of the hepatocytes may become involved in the infection. The overwhelming infection of the liver is associated with the observed hemorrhagic disease manifestations such as petechiae, ecchymoses, and hematemesis which are all thought to be linked with the observed coagulation abnormalities that include prolonged clotting times, reduction in clotting factors, fibrin-split products (D-dimers) and elevated prothrombin times. Many factors involved in the coagulation pathway are produced by hepatocytes, such as fibrinogen (FBG) and plasminogen activator inhibitor-1 (PAI-1). Both of these proteins have been indicated in another flavivirus related disease, dengue, as having roles related to the bleeding abnormalities observed and overall outcome of infection. In this study we wanted to determine if FBG and PAI-1 expression levels by human hepatocytes was disrupted or altered by infection with either wild-type Asibi or vaccine strain17-D YFVs. Our findings indicate that YFV infection does affect the transcriptional and translational expression of FBG and PAI-1 in human hepatocytes and that these results are further affected by IL-6 during early stages of infection. These results may lead to further understanding of the molecular mechanism associated with bleeding abnormalities observed during late stage YFV infection.

Infection of hepatocytes with 17-D vaccine-strain yellow fever virus induces a strong pro-inflammatory host response.

Yellow fever virus (YFV) causes serious disease in endemic areas of South America and Africa, even though a very well tolerated vaccine is available. YFV primarily targets the liver where as many as 80 % of hepatocytes may be involved during infection. The objective of this project was to compare and contrast the cytokine response from hepatocytes infected with either wild-type (Asibi) or vaccine (17-D-204) strains of YFV, with the goal of identifying responses that might be correlated with disease severity or vaccine efficacy. We report here that PH5CH8 hepatocytes support a productive infection with both wild-type and vaccine-strain YFV. Infection with either virus resulted in elevated expression of several pro- and anti-inflammatory cytokines [interleukin (IL)-1ß, IL-4, IL-6, IL-8, IL-10 and tumour necrosis factor-α) with a corresponding increase in transcription. Hepatocytes infected with vaccine virus had a more profound response than did cells infected with wild-type virus. Pre-stimulation of hepatocytes with IL-6 resulted in reduced viral titres, elevated concentrations of cytokines released from Asibi virus-infected cells and improved cell viability in cells infected with 17-D virus. Data reported here suggest that 17-D virus stimulates an appropriate antiviral inflammatory response in hepatocytes, while Asibi virus can attenuate the host response. These data identify potential mechanisms that are associated with increased virulence in wild-type virus infections and also provide clues towards potential immune-response limitations that may be associated with vaccine-related adverse events.

Differential cytokine responses from primary human Kupffer cells following infection with wild-type or vaccine strain yellow fever virus.

Wild-type yellow fever virus (YFV) infections result in a hepatotropic disease which is often fatal, while vaccination with the live-attenuated 17-D strain results in productive infection yet is well-tolerated with few adverse events. Kupffer cells (KCs) are resident liver macrophages that have a significant role in pathogen detection, clearance and immune signaling. Although KCs appear to be an important component of YF disease, their role has been under-studied. This study examined cytokine responses in KCs following infection with either wild-type or vaccine strains of YFV. Results indicate that KCs support replication of both wild-type and vaccine strains, yet wild-type YFV induced a prominent and prolonged pro-inflammatory cytokine response (IL-8, TNF-α and RANTES/CCL5) with little control by a major anti-inflammatory cytokine (IL-10). This response was significantly reduced in vaccine strain infections. These data suggest that a differentially regulated infection in KCs may play a critical role in development of disease.

Antiviral activities of ISG20 in positive-strand RNA virus infections.

ISG20 is an interferon-inducible 3'-5' exonuclease that inhibits replication of several human and animal RNA viruses. However, the specificities of ISG20's antiviral action remain poorly defined. Here we determine the impact of ectopic expression of ISG20 on replication of several positive-strand RNA viruses from distinct viral families. ISG20 inhibited infections by cell culture-derived hepatitis C virus (HCV) and a pestivirus, bovine viral diarrhea virus and a picornavirus, hepatitis A virus. Moreover, ISG20 demonstrated cell-type specific antiviral activity against yellow fever virus, a classical flavivirus. Overexpression of ISG20, however, did not inhibit propagation of severe acute respiratory syndrome coronavirus, a highly-pathogenic human coronavirus in Huh7.5 cells. The antiviral effects of ISG20 were all dependent on its exonuclease activity. The closely related cellular exonucleases, ISG20L1 and ISG20L2, did not inhibit HCV replication. Together, these data may help better understand the antiviral specificity and action of ISG20.

A broad-spectrum antiviral targeting entry of enveloped viruses.

We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus-cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure-activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus-cell but not cell-cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus-cell fusion.

A mutation in the envelope protein fusion loop attenuates mouse neuroinvasiveness of the NY99 strain of West Nile virus.

Substitutions were engineered individually and in combinations at the fusion loop, receptor-binding domain and a stem-helix structure of the envelope protein of a West Nile virus strain, NY99, and their effects on mouse virulence and presentation of epitopes recognized by monoclonal antibodies (MAbs) were assessed. A single substitution within the fusion loop (L107F) attenuated mouse neuroinvasiveness of NY99. No substitutions attenuated NY99 neurovirulence. The L107F mutation also abolished binding of a non-neutralizing MAb, 3D9, whose epitope had not been previously identified. MAb 3D9 was subsequently shown to be broadly cross-reactive with other flaviviruses, consistent with binding near the highly conserved fusion loop.