A Mouse Model for Studying Viscerotropic Disease Caused by Yellow Fever Virus Infection

Mosquito-borne yellow fever virus (YFV) causes highly lethal, viscerotropic disease in humans and non-human primates. Despite the availability of efficacious live-attenuated vaccine strains, 17D-204 and 17DD, derived by serial passage of pathogenic YFV strain Asibi, YFV continues to pose a significant threat to human health. Neither the disease caused by wild-type YFV, nor the molecular determinants of vaccine attenuation and immunogenicity, have been well characterized, in large part due to the lack of a small animal model for viscerotropic YFV infection. Here, we describe a small animal model for wild-type YFV that manifests clinical disease representative of that seen in primates without adaptation of the virus to the host, which was required for the current hamster YF model. Investigation of the role of type I interferon (IFN-α/β) in protection of mice from viscerotropic YFV infection revealed that mice deficient in the IFN-α/β receptor (A129) or the STAT1 signaling molecule (STAT129) were highly susceptible to infection and disease, succumbing within 6–7 days. Importantly, these animals developed viscerotropic disease reminiscent of human YF, instead of the encephalitic signs typically observed in mice. Rapid viremic dissemination and extensive replication in visceral organs, spleen and liver, was associated with severe pathologies in these tissues and dramatically elevated MCP-1 and IL-6 levels, suggestive of a cytokine storm. In striking contrast, infection of A129 and STAT129 mice with the 17D-204 vaccine virus was subclinical, similar to immunization in humans. Although, like wild-type YFV, 17D-204 virus amplified within regional lymph nodes and seeded a serum viremia in A129 mice, infection of visceral organs was rarely established and rapidly cleared, possibly by type II IFN-dependent mechanisms. The ability to establish systemic infection and cause viscerotropic disease in A129 mice correlated with infectivity for A129-derived, but not WT129-derived, macrophages and dendritic cells in vitro, suggesting a role for these cells in YFV pathogenesis. We conclude that the ability of wild-type YFV to evade and/or disable components of the IFN-α/β response may be primate-specific such that infection of mice with a functional IFN-α/β antiviral response is attenuated. Consequently, subcutaneous YFV infection of A129 mice represents a biologically relevant model for studying viscerotropic infection and disease development following wild-type virus inoculation, as well as mechanisms of 17D-204 vaccine attenuation, without a requirement for adaptation of the virus

YFV strains Asibi and 17D exhibit differential infectivity for murine DCs and macrophages <i>in vitro</i>.

<p>Cultures of primary bone marrow-derived DCs (A) and macrophages (B) generated from WT129 (circle) or A129 (triangle) mice were infected with wild-type Asibi virus (closed) or live-attenuated 17D-204 virus (open) at MOI = 0.1 PFU/cell. Viral replication was measured by plaque assay titration of progeny virions in culture supernatants. Data are expressed as log₁₀ PFU/mL where datum points are <i>n = 3±SD</i>. These data were reproducible in three separate experiments.</p

Pathologic changes were evident in liver and spleen of Asibi virus-infected A129 mice.

<p>A129 mice were mock-infected or inoculated with 10⁴ PFU of Asibi or 17D-204 viruses in each rear footpad. H&E-stained sections of liver and spleen are presented. Liver (A) and spleen (B) sections from mock-infected A129 animals were indistinguishable from 17D-204-infected liver (C) and spleen (D) sections, 4 d p.i. (E and F) Liver sections from Asibi virus-infected A129 mice 4 d p.i., showing diffuse inflammatory infiltrates, well-developed fatty acid steatosis involving the majority of the lobular area. Note prominent Küpffer cells (black arrows) and some accumulation of inflammatory cells in hepatic sinusoids (arrow heads). <i>Original magnification: (E) 200×; (F) 400×</i>. (G) Asibi virus-infected liver 6 d p.i., showing and a focus of spotty necrosis (center of field) surrounded by mononuclear inflammatory cells and higher magnification (H) showing more severe inflammatory cell infiltration surrounding portal triad and hepatocytes with intranuclear viral inclusion bodies (black arrows). <i>Original magnification: (G) 200×; (H) 400×</i>. (I and J) Spleen sections from Asibi virus-infected A129 mice 4 d p.i. and (K and L) 6 d p.i., showing diminishing marginal zone (MZ) and white pulp lymphoid follicles (WP), increasing number of splenic macrophages (arrow heads) and widely-distributed cells with pyknotic nuclei, morphologically consistent with apoptotic death. <i>Original magnification: (I, K, L) 200×; (J) 400×</i>.</p

Virulence of wild-type YFV in mice is IFN-α/β-dependent while attenuation of live-attenuated 17D-204 virus is not.

<p>Adult WT129 (solid line; open circle), G129 (solid line; open square), A129 (dotted line; closed circle), AG129 (dashed line; closed square) and STAT129 (dot-dash line; closed triangle) mice were subcutaneously inoculated in each rear footpad with 10⁴ PFU of wild-type YFV strains, Asibi (A and D) or Angola73 (B and E) or with 17D-204 vaccine virus (C and F). Changes in weight from mock-infected counterparts were calculated daily for (A) Asibi virus-infected, (B) Angola73 virus-infected or (C) 17D-204 virus-infected mice as an indicator of morbidity. Percent survivals of (D) Asibi virus-infected, (E) Angola73 virus-infected and (F) 17D-204 virus-infected mice were calculated each day and are presented as Kaplan-Meier survival curves. Datum points are n≥4 and data are representative of at least two separate experiments.</p

Cytokine levels are elevated in Asibi virus-infected A129 mice.

<p>Serum levels of (A) IL-6 and (B) MCP-1 were measured in serum collected from mock-infected WT129 (open diamond) and A129 (closed diamond) mice, Asibi virus-infected WT129 (open circle) and A129 (closed circle) mice and 17D-204 virus-infected A129 mice (closed square) by cytokine bead array analysis (BioRad BioPlex Assay). Data are expressed as concentration of cytokine in pg/mL serum where datum points are <i>n = 3±SD</i>. Mock values were generated from pooled data over the course of infection.</p

Susceptibility of mice to subcutaneously inoculated Asibi and Angola73 wild-type or 17D-204 vaccine strains of yellow fever virus.

1<p>AST±SD (d): average survival time±standard deviation of the mean (days).</p>2<p>- : not applicable.</p>3<p>ND: Not done.</p

Wild-type Asibi virus exhibits viscerotropism in type I IFN-deficient mice, whereas 17D-204 virus does not.

<p>WT129 (data not shown) and A129 mice were subcutaneously inoculated with 10⁴ PFU of Asibi (black bar) or 17D-204 (white bar) virus in each rear footpad. Viral titers in individual, perfused tissues are expressed as log₁₀ PFU/ml of serum, PFU/draining lymph node (DLN) or PFU/g of other tissues: (A) DLN; (B) serum; (C) spleen; (D) liver; (E) bone marrow; and (F) brain. Dotted lines represent the lower limit of detection for the plaque assay. Datum points are <i>n = 3±SD</i> for all data sets.</p

Alpha/Beta Interferon Inhibits Cap-Dependent Translation of Viral but Not Cellular mRNA by a PKR-Independent Mechanism▿

The alpha/beta interferon (IFN-α/β) response is critical for host protection against disseminated replication of many viruses, primarily due to the transcriptional upregulation of genes encoding antiviral proteins. Previously, we determined that infection of mice with Sindbis virus (SB) could be converted from asymptomatic to rapidly fatal by elimination of this response (K. D. Ryman et al., J. Virol. 74:3366-3378, 2000). Probing of the specific antiviral proteins important for IFN-mediated control of virus replication indicated that the double-stranded RNA-dependent protein kinase, PKR, exerted some early antiviral effects prior to IFN-α/β signaling; however, the ability of IFN-α/β to inhibit SB and protect mice from clinical disease was essentially undiminished in the absence of PKR, RNase L, and Mx proteins (K. D. Ryman et al., Viral Immunol. 15:53-76, 2002). One characteristic of the PKR/RNase L/Mx-independent antiviral effect was a blockage of viral protein accumulation early after infection (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). We show here that IFN-α/β priming induces a PKR-independent activity that inhibits m7G cap-dependent translation at a step after association of cap-binding factors and the small ribosome subunit but before formation of the 80S ribosome. Furthermore, the activity targets mRNAs that enter across the cytoplasmic membrane, but nucleus-transcribed RNAs are relatively unaffected. Therefore, this IFN-α/β-induced antiviral activity represents a mechanism through which IFN-α/β-exposed cells are defended against viruses that enter the cytoplasm, while preserving essential host activities, including the expression of antiviral and stress-responsive genes

Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury.

Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS

Silencing Bruton's tyrosine kinase in alveolar neutrophils protects mice from LPS/immune complex-induced acute lung injury.

Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS