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.

Rescue of a recombinant Machupo virus from cloned cDNAs and in vivo characterization in interferon (αß/γ) receptor double knockout mice.

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.

In vivo imaging systems (IVIS) detection of a neuro-invasive encephalitic virus.

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

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-ß 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.

Natural killer cell mediated pathogenesis determines outcome of central nervous system infection with Venezuelan equine encephalitis virus in C3H/HeN mice.

TC83 is a human vaccine with investigational new drug status and is used as a prototype Venezuelan equine encephalitis virus for pathogenesis and antiviral research. Differing from other experimental models, the virus causes high titer infection in the brain and 90-100% mortality in the C3H/HeN murine model. To better characterize the susceptibility to disease development in C3H/HeN mice, we have analyzed the gene transcriptomes and cytokine production in the brains of infected mice. Our analysis indicated the potential importance of natural killer cells in the encephalitic disease development. This paper describes for the first time a pathogenic role for natural killer cells in VEEV encephalitis.

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.

Functional interferon system is required for clearance of lassa virus.

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever (LF) in humans, a deadly disease endemic to West Africa that results in 5,000 to 10,000 deaths annually. Here we present results demonstrating that functional type I and type II interferon (IFN) signaling is required for efficient control of LASV dissemination and clearance.

Rapid, non-invasive imaging of alphaviral brain infection: reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.

Rapid and accurate identification of disease progression are key factors in testing novel vaccines and antivirals against encephalitic alphaviruses. Typical efficacy studies utilize a large number of animals and severe morbidity or mortality as an endpoint. New technologies provide a means to reduce and refine the animal use as proposed in Hume's 3Rs (replacement, reduction, refinement) described by Russel and Burch. In vivo imaging systems (IVIS) and bioluminescent enzyme technologies accomplish the reduction of animal requirements while shortening the experimental time and improving the accuracy in localizing active virus replication. In the case of murine models of viral encephalitis in which central nervous system (CNS) viral invasion occurs rapidly but the disease development is relatively slow, we visualized the initial brain infection and enhance the data collection process required for efficacy studies on antivirals or vaccines that are aimed at preventing brain infection. Accordingly, we infected mice through intranasal inoculation with the genetically modified pathogen, Venezuelan equine encephalitis, which expresses a luciferase gene. In this study, we were able to identify the invasion of the CNS at least 3 days before any clinical signs of disease, allowing for reduction of animal morbidity providing a humane means of disease and vaccine research while obtaining scientific data accurately and more rapidly. Based on our data from the imaging model, we confirmed the usefulness of this technology in preclinical research by demonstrating the efficacy of Ampligen, a TLR-3 agonist, in preventing CNS invasion.

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.

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.

TC83 replicon vectored vaccine provides protection against Junin virus in guinea pigs.

Junin virus (JUNV) is the etiological agent of the potentially lethal, reemerging human disease, Argentine hemorrhagic fever (AHF). The mechanism of the disease development is not well understood and no antiviral therapy is available. Candid 1, a live-attenuated vaccine, has been developed by the US Army and is being used in the endemic area to prevent AHF. This vaccine is only approved for use in Argentina. In this study we have used the alphavirus-based approach to engineer a replicon system based on a human (United States Food and Drug Administration Investigational New Drug status) vaccine TC83 that express heterologous viral antigens, such as glycoproteins (GPC) of Junin virus (JUNV). Preclinical studies testing the immunogenicity and efficacy of TC83/GPC were performed in guinea pigs. A single dose of the live-attenuated alphavirus based vaccine expressing only GPC was immunogenic and provided partial protection, while a double dose of the same vaccine provided a complete protection against JUNV. This is the first scientific report to our knowledge that the immune response against GPC alone is sufficient to prevent lethal disease against JUNV in an animal model.

Superior efficacy of a recombinant flagellin:H5N1 HA globular head vaccine is determined by the placement of the globular head within flagellin.

Transmission of highly pathogenic avian influenza (HPAI) between birds and humans is an ongoing threat that holds potential for the emergence of a pandemic influenza strain. A major barrier to an effective vaccine against avian influenza has been the generally poor immunopotency of many of the HPAI strains coupled with the manufacturing constraints employing conventional methodologies. Fusion of flagellin, a toll-like receptor-5 ligand, to vaccine antigens has been shown to enhance the immune response to the fused antigen in preclinical studies. Here, we have evaluated the immunogenicity and efficacy of a panel of flagellin-based hemagglutinin (HA) globular head fusion vaccines in inbred mice. The HA globular head of these vaccines is derived from the A/Vietnam/1203/04 (VN04; H5N1) HA molecule. We find that replacement of domain D3 of flagellin with the VN04 HA globular head creates a highly effective vaccine that elicits protective HAI titers which protect mice against disease and death in a lethal challenge model.

CD4+ T cells provide protection against acute lethal encephalitis caused by Venezuelan equine encephalitis virus.

Studying the mechanisms of host survival resulting from viral encephalitis is critical to the development of vaccines. Here we have shown in several independent studies that high dose treatment with neutralizing antibody prior to intranasal infection with Venezuelan equine encephalitis virus had an antiviral effect in the visceral organs and prolonged survival time of infected mice, even in the absence of alphabeta T cells. Nevertheless, antibody treatment did not prevent the development of lethal encephalitis. On the contrary, the adoptive transfer of primed CD4(+) T cells was necessary to prevent lethal encephalitis in mice lacking alphabeta T cell receptor.

Endoluminal suturing may overcome the limitations of clip closure of a gaping wide colon perforation (with videos).

BACKGROUND: It is unclear whether large gaping perforations of the colon can be closed by the endoluminal route. OBJECTIVE: To evaluate the feasibility and the outcome of closure of large perforations of colon with clips and sutures by using through-the-endoscope novel devices. DESIGN: Prospective animal study. SETTING: University hospital. PATIENTS: Ten pigs. INTERVENTIONS: Closure of a 4-cm full-thickness colon perforation freshly created by an insulated-tip knife with the InScope Multi-Clip Applier (n = 6) and with the tissue approximation device (n = 4). MAIN OUTCOME MEASUREMENTS: (a) Technical feasibility of closure, (b) clinical monitoring for 2 weeks, (c) necropsy (day 14), (d) healing by a dye-leak test and histology. RESULTS: Endoluminal closure of a 4-cm-long colon perforation was successful in 9 of 10 animals. The clips failed to close a gaping wide colon perforation in 1 animal. The sutures were successful in the closure of both nongaping and gaping perforations. Successful closure resulted in a leakproof sealing at 2 weeks and prevented clinical peritonitis in all the animals in the clip-closure group and in 3 of 4 animals in the suture-closure group. Necropsy at 2 weeks revealed mild peritonitis in 2 of the 5 animals in the clip closure group and in 2 of the 4 animals in the suture-closure group; none developed fecal peritonitis. LIMITATIONS: None. CONCLUSIONS: Endoluminal closure of a 4-cm colon perforation with clips was successful in the majority of cases. Sutures were useful in the closure of gaping colon perforations that could not be closed with clips.

Study of full-thickness endoluminal segmental resection of colon in a porcine colon model (with videos).

BACKGROUND: Entrapment injury of the adjacent bowel is frequently encountered during full-thickness endoluminal colon suction-resection. OBJECTIVE: Our purpose was to develop a technique that can create a full-thickness resection of the colon without the risk of entrapment injury to adjacent viscera. DESIGN: Pilot study. SETTING: University medical center. PATIENTS: Five pigs. INTERVENTIONS: Traction-resection of the colon was created by using a grasping forceps to pull the colon into a band ligator loaded on a double-channel endoscope, followed by the application of a band, and subsequent snare resection (n = 14). Suction-resection of the colon was created by using a double-channel endoscope loaded with a band ligator (n = 12) and a single-channel endoscope with a band ligator (n = 6). MAIN OUTCOME MEASUREMENTS: Number of full-thickness colon resections, frequency of the adjacent bowel and mesenteric injury, and the size of the resections were measured. RESULTS: The suction-resection technique resulted in significant injury to adjacent viscera compared with the traction-resection technique (56% vs 0%, P = .0013). The traction-resection method resulted in a significantly larger resection compared with the suction-resection method (mean +/- SEM: 2.91 +/- 0.3 cm vs 2.1 +/- 0.1 cm, P = .024). A double-channel endoscope suction-resection method resulted in a significantly larger resection compared with a single channel suction-resection technique (mean +/- SEM: 2.1 +/- 0.1 cm vs 0.91 +/- 0.2 cm, P = .0022). LIMITATIONS: None. CONCLUSIONS: The traction-resection technique is safer than the suction-resection method in removing larger specimens of the colon. In addition, the traction-resection technique reduces the risk of injury of the mesentery or adjacent small intestine.

Endoluminal clip closure of a circular full-thickness colon resection in a porcine model (with videos).

BACKGROUND: Linear perforations of the colon can be closed by the application of clips through a colonoscope. It is unclear whether circular perforations after full-thickness resection of the colon can be closed with clips. OBJECTIVE: To develop an animal model for circular perforation of the colon and to study different techniques to accomplish a leakproof sealing of the circular perforation of the colon. DESIGN: Pilot study. SETTING: University medical center. ANIMALS: Ten pigs: 2 perforations in the 1st pig and 1 perforation in the 2nd to 9th pigs were closed with clips. In the 10th pig, 5 perforations were created, and the dimensions of the perforation were measured. INTERVENTIONS: Creation of a circular full-thickness resection of the colon with a band-ligation-resection device, followed by longitudinal or transverse endoluminal closure of the perforation by using the first clip opened and applied in the 3- to 9-o'clock or the 6- to 12-o'clock direction in relation to the circular perforation, respectively. MAIN OUTCOME MEASUREMENTS: The mean (standard deviation) size of circular perforation was 1.7 +/- 0.075 cm (range, 1.5-2.0 cm). Necropsy immediately after closure of the perforation was done to examine the closure and to confirm the quality of sealing with the methylene blue dye leak test. RESULTS: The transverse closure was unsuccessful in the closure of 3 perforations, whereas the longitudinal closure resulted in a leakproof sealing in 6 of the 7 closures. LIMITATIONS: Perforation of the adjacent viscera limits it to a nonsurvival study. CONCLUSIONS: Endoluminal application of clips by using the longitudinal closure technique results in a leak proof sealing of circular perforations of the colon.

Controlled trial of immediate endoluminal closure of colon perforations in a porcine model by use of a novel clip device (with videos).

BACKGROUND: Although endoluminal closure of a small perforation of the colon is technically feasible, the outcome of such a closure is unclear. OBJECTIVE: Our purpose was to evaluate the feasibility and the outcome of endoluminal closure of a small perforation of the colon with a novel clip device, the InScope MultiClip Applier (IMCA), and to assess the number of clips required for successful closure. DESIGN: Prospective controlled study. SETTING: University hospital. ANIMALS: 17 pigs. INTERVENTIONS: A 2-cm full-thickness colon perforation was randomized to 3 groups: control, no closure (n = 4), 2-clip closure (n = 7), and 4-clip closure (n = 6). MAIN OUTCOME MEASUREMENTS: (1) Technical feasibility of closure, (2) closure time, (3) clinical monitoring for 2 weeks, (4) necropsy (day 14), and (5) healing by a dye leak test and histologic examination. RESULTS: Endoscopic closure of the colon perforation was technically successful in 12 of 13 animals. A wide gaping hole prevented satisfactory closure in 1 animal. The median time for closure with 2 and 4 clips was 2 and 3 minutes, respectively. Clip closure of perforation prevented clinical sepsis (P = .008) and diminished the risk for fibrinous peritonitis (P = .02 for a single test of hypothesis; however, correction for the multiple testing of data removes this significance) and adhesion formation (P = .008) compared with controls, without any leakage. The outcomes of 2- and 4-clip closure were similar. CONCLUSIONS: Endoluminal closure of a 2-cm colon perforation with clips is successful in preventing peritonitis and adhesions and it can be accomplished quickly with this novel device. Clip closure at 1-cm intervals is sufficient for successful closure of a 2-cm colon perforation.