Chemotactic and Inflammatory Responses in the Liver and Brain Are Associated with Pathogenesis of Rift Valley Fever Virus Infection in the Mouse

Rift Valley fever virus (RVFV) is a major human and animal pathogen associated with severe disease including hemorrhagic fever or encephalitis. RVFV is endemic to parts of Africa and the Arabian Peninsula, but there is significant concern regarding its introduction into non-endemic regions and the potentially devastating effect to livestock populations with concurrent infections of humans. To date, there is little detailed data directly comparing the host response to infection with wild-type or vaccine strains of RVFV and correlation with viral pathogenesis. Here we characterized clinical and systemic immune responses to infection with wild-type strain ZH501 or IND vaccine strain MP-12 in the C57BL/6 mouse. Animals infected with live-attenuated MP-12 survived productive viral infection with little evidence of clinical disease and minimal cytokine response in evaluated tissues. In contrast, ZH501 infection was lethal, caused depletion of lymphocytes and platelets and elicited a strong, systemic cytokine response which correlated with high virus titers and significant tissue pathology. Lymphopenia and platelet depletion were indicators of disease onset with indications of lymphocyte recovery correlating with increases in G-CSF production. RVFV is hepatotropic and in these studies significant clinical and histological data supported these findings; however, significant evidence of a pro-inflammatory response in the liver was not apparent. Rather, viral infection resulted in a chemokine response indicating infiltration of immunoreactive cells, such as neutrophils, which was supported by histological data. In brains of ZH501 infected mice, a significant chemokine and pro-inflammatory cytokine response was evident, but with little pathology indicating meningoencephalitis. These data suggest that RVFV pathogenesis in mice is associated with a loss of liver function due to liver necrosis and hepatitis yet the long-term course of disease for those that might survive the initial hepatitis is neurologic in nature which is supported by observations of human disease and the BALB/c mouse model

Rescue from Cloned cDNAs and In Vivo Characterization of Recombinant Pathogenic Romero and Live-Attenuated Candid #1 Strains of Junin Virus, the Causative Agent of Argentine Hemorrhagic Fever Disease ▿

The New World arenavirus Junin virus (JUNV) is the causative agent of Argentine hemorrhagic fever (AHF), which is associated with high morbidity and significant mortality. Several pathogenic strains of JUNV have been documented, and a highly attenuated vaccine strain (Candid #1) was generated and used to vaccinate the human population at risk. The identification and functional characterization of viral genetic determinants associated with AHF and Candid #1 attenuation would contribute to the elucidation of the mechanisms contributing to AHF and the development of better vaccines and therapeutics. To this end, we used reverse genetics to rescue the pathogenic Romero and the attenuated Candid #1 strains of JUNV from cloned cDNAs. Both recombinant Candid #1 (rCandid #1) and Romero (rRomero) had the same growth properties and phenotypic features in cultured cells and in vivo as their corresponding parental viruses. Infection with rRomero caused 100% lethality in guinea pigs, whereas rCandid #1 infection was asymptomatic and provided protection against a lethal challenge with Romero. Notably, Romero and Candid #1 trans-acting proteins, L and NP, required for virus RNA replication and gene expression were exchangeable in a minigenome rescue assay. These findings support the feasibility of studies aimed at determining the contribution of each viral gene to JUNV pathogenesis and attenuation. In addition, we rescued Candid #1 viruses with three segments that efficiently expressed foreign genes introduced into their genomes. This finding opens the way for the development of a safe multivalent arenavirus vaccine

Mouse daily weight and temperature.

<p>Daily weight (g) and temperature (°C) of mock-infected C57BL/6 mice and mice infected with either MP-12 or ZH501.</p

Viral Titers.

<p>Viral titers in the serum, liver, spleen, and brain after infection with (A) MP-12 or (B) ZH501. Each point with the exception of 96 hpi in the ZH501 graph represents the mean virus titer of five mice. The 96 hpi points in the ZH501 graph represent only the 3 mice that survived until that point. Error bars for the standard deviation were removed for clarity. (C) Immunohistochemical staining for RVFV antigen in liver from a ZH501 infected mouse at 60 hpi. Intracytoplasmic viral antigen is depicted by brown staining, 10×. (D) Immunohistochemical stain of liver from a ZH501 infected mouse at 84 hpi. Intracytoplasmic viral antigen is depicted by brown staining, 20×. (E) Immunohistochemical stain of spleen for RVFV antigen from a ZH501 infected mouse at 84 hpi. The red pulp sinusoids contain numerous cells with cytoplasmic brown, granular material depicting viral antigen. There are no viral antigen positive cells in the lymphoid follicle. Note the increased lymphocytolysis depicted by pyknotic nuclei and cellular fragments, 20×. (F) Brain from a ZH501 infected animal at 84 hpi with no pathologic changes, H&E 2.5×.</p

Liver function enzymes.

<p>Alanine aminotransferase (ALT), glucose, and total bilirubin concentrations in the serum of mock (A), MP-12 (B) and ZH501 (C) infected mice. The first row provides serum ALT concentrations, the second row glucose concentrations and the third total bilirubin concentrations. Each symbol represents an individual mouse. There were five mice in each group, except at 96 hpi where only three animals had survived until this point of the study. The horizontal bar represents the mean of the mice for that group. Please note that the maximum for the Y-axis in (C)-ALT is 2000 U/L rather than 150 U/L as in (A) and (B) ALT graphs.</p

IFN-β concentration following infection.

<p>IFN-β concentration in the (A) liver, (B) spleen, and (C) brain in mice after infection. The first row represents mock infected mice, the second row represents MP-12 infected mice, and the third is ZH501 infected mice. Each symbol represents an individual mouse. There were five mice in each group with the exception of the 96 time-point for ZH501 when only three animals were surviving. The horizontal bar represents the mean of the mice for each group. Brackets indicate that the average value for ZH501 infected animals is significantly different from both mock and MP-12 infected animals.</p

Liver and spleen pathology.

<p>(A) Liver from a mock-infected mouse at 84 hpi. The liver is essentially normal H&E 20×. (B) Liver from an MP-12 infected mouse at 84 hpi. The random, small microgranulomas composed of individually-necrotic hepatocytes surrounded by small numbers of neutrophils and mononuclear cells are unrelated to the study, H&E 20×. (C) Liver from a ZH501 infected mouse at 84 hpi, showing piecemeal hepatocyte necrosis (white arrowhead), hepatocellular intranuclear eosinophilic inclusions (white arrow). Note the presence of neutrophils (black arrow) within foci of necrosis, H&E stain 40×. (D) Spleen from a mock-infected mouse at 84 hpi. The spleen is essentially normal, H&E 20×. (E) Spleen from an MP-12 infected mouse at 84 hpi. The spleen is essentially normal, H&E 20×. (F) Spleen from a ZH501 infected mouse at 84 hpi with moderate amounts of necrotic cellular debris and macrophages containing hemosiderin (brown pigment) within red pulp sinusoids, H&E 10×.</p

Mice Lacking Alpha/Beta and Gamma Interferon Receptors Are Susceptible to Junin Virus Infection▿

Junin virus (JUNV) causes a highly lethal human disease, Argentine hemorrhagic fever. Previous work has demonstrated the requirement for human transferrin receptor 1 for virus entry, and the absence of the receptor was proposed to be a major cause for the resistance of laboratory mice to JUNV infection. In this study, we present for the first time in vivo evidence that the disruption of interferon signaling is sufficient to generate a disease-susceptible mouse model for JUNV infection. After peripheral inoculation with virulent JUNV, adult mice lacking alpha/beta and gamma interferon receptors developed disseminated infection and severe disease

Junín virus infection activates the type I interferon pathway in a RIG-I-dependent manner.

Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment

RIG-I enhanced interferon independent apoptosis upon Junin virus infection.

Junin virus (JUNV) is the etiological agent of Argentine hemorrhagic fever (AHF), a human disease with a high case-fatality rate. It is widely accepted that arenaviral infections, including JUNV infections, are generally non-cytopathic. In contrast, here we demonstrated apoptosis induction in human lung epithelial carcinoma (A549), human hepatocarcinoma and Vero cells upon infection with the attenuated Candid#1 strain of, JUNV as determined by phosphatidylserine (PS) translocation, Caspase 3 (CASP3) activation, Poly (ADP-ribose) polymerase (PARP) cleavage and/or chromosomal DNA fragmentation. Moreover, as determined by DNA fragmentation, we found that the pathogenic Romero strain of JUNV was less cytopathic than Candid#1 in human hepatocarcinoma and Vero, but more apoptotic in A549 and Vero E6 cells. Additionally, we found that JUNV-induced apoptosis was enhanced by RIG-I signaling. Consistent with the previously reported role of RIG-I like helicase (RLH) signaling in initiating programmed cell death, we showed that cell death or DNA fragmentation of Candid#1-infected A549 cells was decreased upon siRNA or shRNA silencing of components of RIG-I pathway in spite of increased virus production. Similarly, we observed decreased DNA fragmentation in JUNV-infected human hepatocarcinoma cells deficient for RIG-I when compared with that of RIG-I-competent cells. In addition, DNA fragmentation detected upon Candid#1 infection of type I interferon (IFN)-deficient Vero cells suggested a type I IFN-independent mechanism of apoptosis induction in response to JUNV. Our work demonstrated for the first time apoptosis induction in various cells of mammalian origin in response to JUNV infection and partial mechanism of this cell death