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.

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o’nyong ‘nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes debilitating musculoskeletal pain and inflammation and can persist for months to years after acute infection. Although studies in humans and experimentally-infected animals suggest that CHIKV infection persists in musculoskeletal tissues, the mechanisms for this remain poorly understood. To evaluate this further, we isolated CHIKV from the serum of persistently infected mice at day 28. When inoculated into naïve WT mice, this persistently circulating CHIKV strain displayed a capacity for earlier dissemination and greater pathogenicity compared with the parental virus. Sequence analysis revealed a nonsynonymous mutation in the E2 glycoprotein (E2 K200R) and a deletion within the 3' untranslated region (3' -UTR). Introduction of these changes into the parental virus conferred enhanced virulence in mice although a primary tropism for musculoskeletal tissues was maintained. The E2 K200R mutation was largely responsible for enhanced viral dissemination and pathogenicity, although these effects were augmented by the 3' -UTR deletion. Finally, studies in and mice suggest that the E2 K200R mutation enhances viral dissemination from the site of inoculation independently of IRF3, IRF7, and IFNAR1 mediated responses. As our findings reveal viral determinants of CHIKV dissemination and pathogenicity, their further study should help to elucidate host-virus interactions that determine acute and chronic CHIKV infection. CHIKV is a globally-spreading, mosquito-transmitted virus that causes debilitating acute and chronic musculoskeletal disease in humans. The viral genetic determinants that dictate acute and chronic disease severity are not understood. To improve our understanding of CHIKV pathogenesis, we evaluated a CHIKV strain isolated from the serum of chronically-infected immunocompromised mice. Sequence analysis of this persistent CHIKV strain identified two mutations, an amino acid change in the E2 viral attachment protein and a deletion within the 3' -UTR of the viral genome. We identified roles for these mutations in enhancement of viral dissemination from the inoculation site and in disease severity. These data improve our understanding of the viral determinants of CHIKV pathogenesis and adaptive changes that occur during viral persistence.

During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α.