Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya, eastern- (EEEV), and Venezuelan- (VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology.

Ly6C+ monocytes in the skin promote systemic alphavirus dissemination.

Alphaviruses are mosquito-transmitted pathogens that induce high levels of viremia, which facilitates dissemination and vector transmission. One prevailing paradigm is that, after skin inoculation, alphavirus-infected resident dendritic cells migrate to the draining lymph node (DLN), facilitating further rounds of infection and dissemination. Here, we assess the contribution of infiltrating myeloid cells to alphavirus spread. We observe two phases of virus transport to the DLN, one that occurs starting at 1 h post infection and precedes viral replication, and a second that requires replication in the skin, enabling transit to the bloodstream. Depletion of Ly6C+ monocytes reduces local chikungunya (CHIKV) or Ross River virus (RRV) infection in the skin, diminishes the second phase of virus transport to the DLN, and delays spread to distal sites. Our data suggest that infiltrating monocytes facilitate alphavirus infection at the initial infection site, which promotes more rapid spread into circulation.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response - including recruitment of myeloid cells to the LN - was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Chikungunya virus infection disrupts lymph node lymphatic endothelial cell composition and function via MARCO.

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell-T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we find that during the first 24 h of infection, CHIKV RNA accumulates in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response, including recruitment of myeloid cells to the LN, was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, we find that antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Phagocyte-expressed glycosaminoglycans promote capture of alphaviruses from the blood circulation in a host species-specific manner.

The magnitude and duration of vertebrate viremia are critical determinants of arbovirus transmission, geographic spread, and disease severity-yet, mechanisms determining arbovirus viremia levels are poorly defined. Previous studies have drawn associations between in vitro virion-glycosaminoglycan (GAG) interactions and in vivo clearance kinetics of virions from blood circulation. From these observations, it is commonly hypothesized that GAG-binding virions are rapidly removed from circulation due to ubiquitous expression of GAGs by vascular endothelial cells, thereby limiting viremia. Using an in vivo model for viremia, we compared the vascular clearance of low and enhanced GAG-binding viral variants of chikungunya (CHIKV), eastern-(EEEV), and Venezuelan-(VEEV) equine encephalitis viruses. We find GAG-binding virions are more quickly removed from circulation than their non-GAG-binding variant; however individual clearance kinetics vary between GAG-binding viruses, from swift (VEEV) to slow removal from circulation (EEEV). Remarkably, we find phagocytes are required for efficient vascular clearance of some enhanced GAG-binding virions. Moreover, transient depletion of vascular heparan sulfate (HS) impedes vascular clearance of only some GAG-binding viral variants and in a phagocyte-dependent manner, implying phagocytes can mediate vascular GAG-virion interactions. Finally, in direct contrast to mice, we find enhanced GAG-binding EEEV is resistant to vascular clearance in avian hosts, suggesting the existence of species-specificity in virion-GAG interactions. In summary, these data support a role for GAG-mediated clearance of some viral particles from the blood circulation, illuminate the potential of blood-contacting phagocytes as a site for GAG-virion binding, and suggest a role for species-specific GAG structures in arbovirus ecology. Significance Statement: Previously, evidence of arbovirus-GAG interactions in vivo has been limited to associations between viral residues shown to promote enhanced GAG-binding phenotypes in vitro and in vivo phenotypes of viral dissemination and pathogenesis. By directly manipulating host GAG expression, we identified virion-GAG interactions in vivo and discovered a role for phagocyte-expressed GAGs in viral vascular clearance. Moreover, we observe species-specific differences in viral vascular clearance of enhanced GAG-binding virions between murine and avian hosts. These data suggest species-specific variation in GAG structure is a mechanism to distinguish amplifying from dead-end hosts for arbovirus transmission.

Species-specific MARCO-alphavirus interactions dictate chikungunya virus viremia.

Arboviruses are public health threats that cause explosive outbreaks. Major determinants of arbovirus transmission, geographic spread, and pathogenesis are the magnitude and duration of viremia in vertebrate hosts. Previously, we determined that multiple alphaviruses are cleared efficiently from murine circulation by the scavenger receptor MARCO (Macrophage receptor with collagenous structure). Here, we define biochemical features on chikungunya (CHIKV), o'nyong 'nyong (ONNV), and Ross River (RRV) viruses required for MARCO-dependent clearance in vivo. In vitro, MARCO expression promotes binding and internalization of CHIKV, ONNV, and RRV via the scavenger receptor cysteine-rich (SRCR) domain. Furthermore, we observe species-specific effects of the MARCO SRCR domain on CHIKV internalization, where those from known amplification hosts fail to promote CHIKV internalization. Consistent with this observation, CHIKV is inefficiently cleared from the circulation of rhesus macaques in contrast with mice. These findings suggest a role for MARCO in determining whether a vertebrate serves as an amplification or dead-end host following CHIKV infection.

Animal models of alphavirus infection and human disease.

Alphaviruses are a large group (>30 species) of enveloped, positive-strand RNA viruses. The re-emergence of mosquito-transmitted alphaviruses associated with human diseases ranging from severe and potentially fatal neurological disease to chronic arthritic disease highlights the need to understand the biology and pathogenesis of alphaviruses. Here, we review the development and use of animal models of alphavirus transmission and human disease, and discuss areas for continued refinement of these models including possible avenues for future investigation.

Two Conserved Phenylalanine Residues in the E1 Fusion Loop of Alphaviruses Are Essential for Viral Infectivity.

Alphaviruses infect cells by a low pH-dependent fusion reaction between viral and host cell membranes that is mediated by the viral E1 glycoprotein. Most reported alphavirus E1 sequences include two phenylalanines (F87 and F95) in the fusion loop, yet the role of these residues in viral infectivity remains to be defined. Following introduction of wild type (WT), E1-F87A, and E1-F95A chikungunya virus (CHIKV) RNA genomes into cells, viral particle production was similar in magnitude. However, CHIKV E1-F87A and E1-F95A virions displayed impaired infectivity compared with WT CHIKV particles. Although WT, E1-F87A, and E1-F95A particles bound cells with similar efficiencies, E1-F87A and E1-F95A particles were unable to undergo fusion and entry into cells. Introduction of an F95A mutation in the E1 fusion loop of Mayaro virus or Venezuelan equine encephalitis virus also resulted in poorly infectious virions. We further tested whether an E1-F87A or E1-F95A mutation could be incorporated into a live-attenuated vaccine strain, CHIKV 181/25, to enhance vaccine safety. Infection of immunocompromised Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice with 181/25E1-F87A or 181/25E1-F95A resulted in 0% mortality, compared with 100% mortality following 181/25 infection. Despite this enhanced attenuation, surviving Ifnar1-/- and Irf3-/-Irf5-/-Irf7-/- mice were protected against virulent virus re-challenge. Moreover, single-dose immunization of WT mice with either 181/25, 181/25E1-F87A, or 181/25E1-F95A elicited CHIKV-specific antibody responses and protected against pathogenic CHIKV challenge. These studies define a critical function for residues E1-F87 and E1-F95 in alphavirus fusion and entry into target cells and suggest that incorporation of these mutations could enhance the safety of live-attenuated alphavirus vaccine candidates. IMPORTANCE Alphaviruses are human pathogens that cause both debilitating acute and chronic musculoskeletal disease and potentially fatal encephalitis. In this study, we determined that two highly conserved phenylalanine residues in the alphavirus E1 glycoprotein are required for fusion of viral and host cell membranes and viral entry into target cells. We further demonstrated that mutation of these phenylalanines results in a substantial loss of viral virulence but not immunogenicity. These data enhance an understanding of the viral determinants of alphavirus entry into host cells and could contribute to the development of new antivirals targeting these conserved phenylalanines or new live-attenuated alphavirus vaccines.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.