A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses.

Zika virus (ZIKV) is a mosquito borne flavivirus, which was a neglected tropical pathogen until it emerged and spread across the Pacific Area and the Americas, causing large human outbreaks associated with fetal abnormalities and neurological disease in adults. The factors that contributed to the emergence, spread and change in pathogenesis of ZIKV are not understood. We previously reported that ZIKV evades cellular antiviral responses by targeting STAT2 for degradation in human cells. In this study, we demonstrate that Stat2-/- mice are highly susceptible to ZIKV infection, recapitulate virus spread to the central nervous system (CNS), gonads and other visceral organs, and display neurological symptoms. Further, we exploit this model to compare ZIKV pathogenesis caused by a panel of ZIKV strains of a range of spatiotemporal history of isolation and representing African and Asian lineages. We observed that African ZIKV strains induce short episodes of severe neurological symptoms followed by lethality. In comparison, Asian strains manifest prolonged signs of neuronal malfunctions, occasionally causing death of the Stat2-/- mice. African ZIKV strains induced higher levels of inflammatory cytokines and markers associated with cellular infiltration in the infected brain in mice, which may explain exacerbated pathogenesis in comparison to those of the Asian lineage. Interestingly, viral RNA levels in different organs did not correlate with the pathogenicity of the different strains. Taken together, we have established a new murine model that supports ZIKV infection and demonstrate its utility in highlighting intrinsic differences in the inflammatory response induced by different ZIKV strains leading to severity of disease. This study paves the way for the future interrogation of strain-specific changes in the ZIKV genome and their contribution to viral pathogenesis.

Chemokine Receptor Ccr7 Restricts Fatal West Nile Virus Encephalitis.

West Nile virus (WNV) is a mosquito-transmitted flavivirus that can cause debilitating encephalitis. To delineate the mechanisms behind this pathology, we studied Ccr7-deficient mice, which afforded us the capacity to study infection in mice with disrupted peripheral cellular trafficking events. The loss of Ccr7 resulted in an immediate pan-leukocytosis that remained elevated throughout the infection. This leukocytosis resulted in a significant enhancement of leukocyte accumulation within the central nervous system (CNS). Despite an excess of virus-specific T cells in the CNS, Ccr7-deficient mice had significantly higher CNS viral loads and mortality rates than wild-type animals. Mechanistically, the elevated trafficking of infected myeloid cells into the brain in Ccr7-deficient mice resulted in increased levels of WNV in the CNS, thereby effectively contributing to neuroinflammation and lowering viral clearance. Combined, our experiments suggest that during WNV infection, Ccr7 is a gatekeeper for nonspecific viral transference to the brain.IMPORTANCE In this study, we show that Ccr7 is required for the sufficient migration of dendritic cells and T cells into the draining lymph node immediately following infection and for the restriction of leukocyte migration into the brain. Further, the severe loss of dendritic cells in the draining lymph node had no impact on viral replication in this organ, suggesting that WNV may migrate from the skin into the lymph node through another mechanism. Most importantly, we found that the loss of Ccr7 results in a significant leukocytosis, leading to hypercellularity within the CNS, where monocytes/macrophages contribute to CNS viremia, neuroinflammation, and increased mortality. Together, our data point to Ccr7 as a critical host defense restriction factor limiting neuroinflammation during acute viral infection.

Dual Function of Ccr5 during Langat Virus Encephalitis: Reduction in Neutrophil-Mediated Central Nervous System Inflammation and Increase in T Cell-Mediated Viral Clearance.

Tick-borne encephalitis virus (TBEV) is a vector-transmitted flavivirus that causes potentially fatal neurologic infection. There are thousands of cases reported annually, and despite the availability of an effective vaccine, the incidence of TBEV is increasing worldwide. Importantly, up to 30% of affected individuals develop long-term neurologic sequelae. We investigated the role of chemokine receptor Ccr5 in a mouse model of TBEV infection using the naturally attenuated tick-borne flavivirus Langat virus (LGTV). Ccr5-deficient mice presented with an increase in viral replication within the CNS and decreased survival during LGTV encephalitis compared with wild-type controls. This enhanced susceptibility was due to the temporal lag in lymphocyte migration into the CNS. Adoptive transfer of wild-type T cells, but not Ccr5-deficient T cells, significantly improved survival outcome in LGTV-infected Ccr5-deficient mice. Concomitantly, a significant increase in neutrophil migration into the CNS in LGTV-infected Ccr5(-/-) mice was documented at the late stage of infection. Ab-mediated depletion of neutrophils in Ccr5(-/-) mice resulted in a significant improvement in mortality, a decrease in viral load, and a decrease in overall tissue damage in the CNS compared with isotype control-treated mice. Ccr5 is crucial in directing T cells toward the LGTV-infected brain, as well as in suppressing neutrophil-mediated inflammation within the CNS.

Differential Roles of Chemokines CCL2 and CCL7 in Monocytosis and Leukocyte Migration during West Nile Virus Infection.

West Nile virus (WNV) is a re-emerging pathogen and the leading cause of epidemic encephalitis in the United States. Inflammatory monocytes are a critical component of the cellular infiltrate found in the CNS during WNV encephalitis, although the molecular cues involved in their migration are not fully understood. In mice, we previously showed that WNV infection induces a CCR2-dependent monocytosis that precedes monocyte migration into the CNS. Currently, the relative contribution of the CCR2 ligands, chemokines CCL2 and CCL7, in directing monocyte mobilization and leukocyte migration into the CNS is unclear. In this study, we demonstrate that, although both CCL2 and CCL7 are required for efficient monocytosis and monocyte accumulation in the CNS, only CCL7 deficiency resulted in increased viral burden in the brain and enhanced mortality. The enhanced susceptibility in the absence of CCL7 was associated with the delayed migration of neutrophils and CD8(+) T cells into the CNS compared with WT or Ccl2(-/-) mice. To determine whether CCL7 reconstitution could therapeutically alter the survival outcome of WNV infection, we administered exogenous CCL7 i.v. to WNV-infected Ccl7(-/-) mice and observed a significant increase in monocytes and neutrophils, but not CD8(+) T cells, within the CNS, as well as an enhancement in survival compared with Ccl7(-/-) mice treated with a linear CCL7 control peptide. Our experiments suggest that CCL7 is an important protective signal involved in leukocyte trafficking during WNV infection, and it may have therapeutic potential for the treatment of acute viral infections of the CNS.

Differences in Early Cytokine Production Are Associated With Development of a Greater Number of Symptoms Following West Nile Virus Infection.

BACKGROUND: West Nile virus (WNV) is an emerging cause of meningitis and encephalitis in the United States. Although severe neuroinvasive disease and death can occur in rare instances, the majority of infected individuals remain asymptomatic or present with a range of clinical manifestations associated with West Nile fever. METHODS: To better understand the interindividual variability associated with the majority of WNV infections, we evaluated the association of cytokine/chemokine production and outcome of infection among 115 WNV-positive US blood donors identified in 2008-2011. All subjects self-reported symptoms as having occurred during the 2 weeks following blood donation, using a standardized questionnaire. RESULTS: We discovered that, prior to seroconversion, an early potent, largely type I interferon-mediated response correlated with development of a greater number of symptoms in WNV-infected individuals. Interestingly, individuals who developed fewer symptoms had not only a more modest type I interferon response initially, but also a protracted cytokine response after seroconversion, marked by the production of monocyte and T-cell-associated chemokines. CONCLUSIONS: Collectively, our data suggest that, although an early type I interferon response appears to be crucial to control WNV infection, successful immunity may require a modest early response that is maintained during the course of infection.

Passenger Mutations Confound Phenotypes of SARM1-Deficient Mice.

The Toll/IL-1R-domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Roles for SARM1 in TLR signaling, viral infection, inflammasome activation, and chemokine and Xaf1 expression have also been described. Much of the evidence for SARM1 function relies on SARM1-deficient mice generated in 129 ESCs and backcrossed to B6. The Sarm1 gene lies in a gene-rich region encompassing Xaf1 and chemokine loci, which remain 129 in sequence. We therefore generated additional knockout strains on the B6 background, confirming the role of SARM1 in axonal degeneration and WNV infection, but not in VSV or LACV infection, or in chemokine or Xaf1 expression. Sequence variation in proapoptotic Xaf1 between B6 and 129 results in coding changes and distinct splice variants, which may account for phenotypes previously attributed to SARM1. Reevaluation of phenotypes in these strains will be critical for understanding the function of SARM1.

CCL7 Is a Negative Regulator of Cutaneous Inflammation Following Leishmania major Infection.

The chemokine CCL7 (MCP3) is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 upregulation has been associated with many inflammatory settings including infection, cardiovascular disease, and the tumor microenvironment. CCL7's pleotropic effects are due in part to its ability to bind numerous chemokine receptors, namely CCR1, CCR2, CCR3, CCR5, and CCR10. CCL7-blockade or CCL7-deficiency is often marked by decreased inflammation and poor pathogen control. In the context of Leishmania major infection, CCL7 is specifically upregulated in the skin one-2 weeks after infection but its role in L. major control is unclear. To determine CCL7's impact on the response to L. major we infected WT and CCL7-/- C57BL/6 mice. L. major infection of CCL7-deficient mice led to an unexpected increase in inflammation in the infected skin 2 weeks post-infection. A broad increase in immune cell subsets was observed but was dominated by enhanced neutrophilic infiltration. Increased neutrophil recruitment was associated with an enhanced IL-17 gene profile in the infected skin. CCL7 was shown to directly antagonize neutrophil migration in vitro and CCL7 add-back in vivo specifically reduced neutrophil influx into the infected skin revealing an unexpected role for CCL7 in limiting neutrophil recruitment during L. major infection. Enhanced neutrophilic infiltration in CCL7-deficient mice changed the balance of L. major infected host cells with an increase in the ratio of infected neutrophils over monocytes/macrophages. To determine the consequence of CCL7 deficiency on L. major control we analyzed parasite load cutaneously at the site of infection and viscerally in the draining LN and spleen. The CCL7-/- mice supported robust cutaneous parasite control similar to their WT C57BL/6 counterparts. In contrast, CCL7-deficiency led to greater parasite dissemination and poor parasite control in the spleen. Our studies reveal a novel role for CCL7 in negatively regulating cutaneous inflammation, specifically neutrophils, early during L. major infection. We propose that CCL7-mediated dampening of the early immune response in the skin may limit the ability of the parasite to disseminate without compromising cutaneous control.

Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity.

Zika virus (ZIKV) is spreading rapidly into regions around the world where other flaviviruses, such as dengue virus (DENV) and West Nile virus (WNV), are endemic. Antibody-dependent enhancement has been implicated in more severe forms of flavivirus disease, but whether this also applies to ZIKV infection is unclear. Using convalescent plasma from DENV- and WNV-infected individuals, we found substantial enhancement of ZIKV infection in vitro that was mediated through immunoglobulin G engagement of Fcγ receptors. Administration of DENV- or WNV-convalescent plasma into ZIKV-susceptible mice resulted in increased morbidity-including fever, viremia, and viral loads in spinal cord and testes-and increased mortality. Antibody-dependent enhancement may explain the severe disease manifestations associated with recent ZIKV outbreaks and highlights the need to exert great caution when designing flavivirus vaccines.

The role of chemokines in the pathogenesis of neurotropic flaviviruses.

Neurotropic flaviviruses are important emerging and reemerging arthropod-borne pathogens that cause significant morbidity and mortality in humans and other vertebrates worldwide. Upon entry and infection of the CNS, these viruses can induce a rapid inflammatory response characterized by the infiltration of leukocytes into the brain parenchyma. Chemokines and their receptors are involved in coordinating complex leukocyte trafficking patterns that regulate viral pathogenesis in vivo. In this review, we will summarize the current literature on the role of chemokines in regulating the pathogenesis of West Nile, Japanese encephalitis, and tick-borne encephalitis virus infections in mouse models and humans. Understanding how viral infections trigger chemokines, the key cellular events that occur during the infection process, as well as the immunopathogenic role of these cells, are critical areas of research that may ultimately guide a much needed effort toward developing specific immunomodulators and/or antiviral therapeutics.