An Immunocompetent Mouse Model of Zika Virus Infection

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1-/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned

The IRE1α-XBP1 arm of the unfolded protein response is a host factor activated in SARS-CoV-2 infection

SARS-CoV-2 infection can cause severe pneumonia, wherein exacerbated inflammation plays a major role. This is reminiscent of the process commonly termed cytokine storm, a condition dependent on a disproportionated production of cytokines. This state involves the activation of the innate immune response by viral patterns and coincides with the biosynthesis of the biomass required for viral replication, which may overwhelm the capacity of the endoplasmic reticulum and drive the unfolded protein response (UPR). The UPR is a signal transduction pathway composed of three branches that is initiated by a set of sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor 6 (ATF6). These sensors control adaptive processes, including the transcriptional regulation of proinflammatory cytokines. Based on this background, the role of the UPR in SARS-CoV-2 replication and the ensuing inflammatory response was investigated using in vivo and in vitro models of infection. Mice and Syrian hamsters infected with SARS-CoV-2 showed a sole activation of the Ire1α-Xbp1 arm of the UPR associated with a robust production of proinflammatory cytokines. Human lung epithelial cells showed the dependence of viral replication on the expression of UPR-target proteins branching on the IRE1α-XBP1 arm and to a lower extent on the PERK route. Likewise, activation of the IRE1α-XBP1 branch by Spike (S) proteins from different variants of concern was a uniform finding. These results show that the IRE1α-XBP1 system enhances viral replication and cytokine expression and may represent a potential therapeutic target in SARS-CoV-2 severe pneumonia.This work was supported by Junta de Castilla y León/Fondo Social Europeo Grants CSI035P17 (M.S.C.) and VA175P20 (N.F.). Fondo COVID-19 del Instituto de Salud Carlos III/Junta de Castilla y León. European Commission-NextGenerationEU, (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). Plan Nacional de Salud y Farmacia Grant SAF2017-83079-R and Grant PID2020-113751RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 (J.J.F., M.S.C., N.F.). Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA) (P.M., R.K.G.). U.S. Food and Drug Administration Medical Countermeasures Initiative contract (75F40120C00085) (J.A.H.). MRC (MR/W005611/1) G2P-UK: A national virology consortium to address phenotypic consequences of SARS-CoV-2 genomic variation (co-Is JPS and JAH). National Institutes of Health (NIH) grant: R21AI147172 (N.I.), NIH/NIAID R01AI160706, NIH/NIAID R21AI176069, and NIH/NIDDK R01DK130425 (M.S.). This work was also partly supported by NIAID grant U19AI135972, and by CRIPT (Center for Research on Influenza Pathogenesis and Transmission), a NIAID funded Center of Excellence for Influenza Research and Response (CEIRR, contract # 75N93021C00014) (A.G.S.). This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences.Peer reviewe

ISRE-Reporter Mouse Reveals High Basal and Induced Type I IFN Responses in Inflammatory Monocytes

Summary: Type I and type III interferons (IFNs) are critical for controlling viral infections. However, the precise dynamics of the IFN response have been difficult to define in vivo. Signaling through type I IFN receptors leads to interferon-stimulated response element (ISRE)-dependent gene expression and an antiviral state. As an alternative to tracking IFN, we used an ISRE-dependent reporter mouse to define the cell types, localization, and kinetics of IFN responding cells during influenza virus infection. We find that measurable IFN responses are largely limited to hematopoietic cells, which show a high sensitivity to IFN. Inflammatory monocytes display high basal IFN responses, which are enhanced upon infection and correlate with infection of these cells. We find that inflammatory monocyte development is independent of IFN signaling; however, IFN is critical for chemokine production and recruitment following infection. The data reveal a role for inflammatory monocytes in both basal IFN responses and responses to infection. : Uccellini and García-Sastre create an ISRE reporter mouse and track interferon (IFN) responses in vivo in response to pathogen-associated molecular pattern (PAMP) stimulation and influenza infection. They find that IFN responses are highest in hematopoietic cells during infection. Specifically, Ly6Chi inflammatory monocytes have high basal IFN responses that are further enhanced upon infection. Keywords: influenza, interferon, monocytes, innate immunit

Development of a Macrophage-Based ADCC Assay

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies

STAT2 Limits Host Species Specificity of Human Metapneumovirus

The host tropism of viral infection is determined by a variety of factors, from cell surface receptors to innate immune signaling. Many viruses encode proteins that interfere with host innate immune recognition in order to promote infection. STAT2 is divergent between species and therefore has a role in species restriction of some viruses. To understand the role of STAT2 in human metapneumovirus (HMPV) infection of human and murine tissues, we first infected STAT2−/− mice and found that HMPV could be serially passaged in STAT2−/−, but not WT, mice. We then used in vitro methods to show that HMPV inhibits expression of both STAT1 and STAT2 in human and primate cells, but not in mouse cells. Transfection of the murine form of STAT2 into STAT2-deficient human cells conferred resistance to STAT2 inhibition. Finally, we sought to understand the in vivo role of STAT2 by infecting hSTAT2 knock-in mice with HMPV, and found that mice had increased weight loss, inhibition of type I interferon signaling, and a Th2-polarized cytokine profile compared to WT mice. These results indicate that STAT2 is a target of HMPV in human infection, while the murine version of STAT2 restricts tropism of HMPV for murine cells and tissue

RIG-I detects mRNA of intracellular Salmonella enterica serovar Typhimurium during bacterial infection

The cytoplasmic helicase RIG-I is an established sensor for viral 5'-triphosphorylated RNA species. Recently, RIG-I was also implicated in the detection of intracellular bacteria. However, little is known about the host cell specificity of this process and the bacterial pathogen-associated molecular pattern (PAMP) that activates RIG-I. Here we show that RNA of Salmonella enterica serovar Typhimurium activates production of beta interferon in a RIG-I-dependent fashion only in nonphagocytic cells. In phagocytic cells, RIG-I is obsolete for detection of Salmonella infection. We further demonstrate that Salmonella mRNA reaches the cytoplasm during infection and is thus accessible for RIG-I. The results from next-generation sequencing analysis of RIG-I-associated RNA suggest that coding bacterial mRNAs represent the activating PAMP. IMPORTANCE S. Typhimurium is a major food-borne pathogen. After fecal-oral transmission, it can infect epithelial cells in the gut as well as immune cells (mainly macrophages, dendritic cells, and M cells). The innate host immune system relies on a growing number of sensors that detect pathogen-associated molecular patterns (PAMPs) to launch a first broad-spectrum response to invading pathogens. Successful detection of a given pathogen depends on colocalization of host sensors and PAMPs as well as potential countermeasures of the pathogen during infection. RIG-I-like helicases were mainly associated with detection of RNA viruses. Our work shows that S. Typhimurium is detected by RIG-I during infection specifically in nonimmune cells