SUMMARY The current widespread outbreak of Zika virus (ZIKV) infection has been linked to severe clinical birth defects, particularly microcephaly, warranting urgent study of the molecular mechanisms underlying ZIKV pathogenesis. Akt-mTOR signaling is one of the key cellular pathways essential for brain development and autophagy regulation. Here, we show that ZIKV infection of human fetal neural stem cells (fNSCs) causes inhibition of the Akt-mTOR pathway, leading to defective neurogenesis and aberrant activation of autophagy. By screening the three structural proteins and seven nonstructural proteins present in ZIKV, we found that two, NS4A and NS4B, cooperatively suppress the Akt-mTOR pathway and lead to cellular dysregulation. Corresponding proteins from the closely related dengue virus do not have the same effect on neurogenesis. Thus, our study highlights ZIKV NS4A and NS4B as candidate determinants of viral pathogenesis and identifies a mechanism of action for their effects, suggesting potential targets for anti-ZIKV therapeutic intervention.

Summary: Sensitization of the humoral immune response to invading viruses and production of antiviral antibodies forms part of the host antiviral repertoire. Paradoxically, for a number of viral pathogens, under certain conditions, antibodies provide an attractive means of enhanced virus entry and replication in a number of cell types. Known as antibody-dependent enhancement (ADE) of infection, the phenomenon occurs when virus-antibody immunocomplexes interact with cells bearing complement or Fc receptors, promoting internalization of the virus and increasing infection. Frequently associated with exacerbation of viral disease, ADE of infection presents a major obstacle to the prevention of viral disease by vaccination and is thought to be partly responsible for the adverse effects of novel antiviral therapeutics such as intravenous immunoglobulins. There is a growing body of work examining the intracellular signaling pathways and epitopes responsible for mediating ADE, with a view to aiding rational design of antiviral strategies. With in vitro studies also confirming ADE as a feature of infection for a growing number of viruses, challenges remain in understanding the multilayered molecular mechanisms of ADE and its effect on viral pathogenesis.

Blood CD14+ monocytes are the frontline immunomodulators categorized into classical, intermediate or non-classical subsets, subsequently differentiating into M1 pro- or M2 anti-inflammatory macrophages upon stimulation. While Zika virus (ZIKV) rapidly establishes viremia, the target cells and immune responses, particularly during pregnancy, remain elusive. Furthermore, it is unknown whether African- and Asian-lineage ZIKV have different phenotypic impacts on host immune responses. Using human blood infection, we identified CD14+ monocytes as the primary target for African- or Asian-lineage ZIKV infection. When immunoprofiles of human blood infected with ZIKV were compared, a classical/intermediate monocyte-mediated M1-skewed inflammation by African-lineage ZIKV infection was observed, in contrast to a non-classical monocyte-mediated M2-skewed immunosuppression by Asian-lineage ZIKV infection. Importantly, infection of pregnant women’s blood revealed enhanced susceptibility to ZIKV infection. Specifically, Asian-lineage ZIKV infection of pregnant women’s blood led to an exacerbated M2-skewed immunosuppression of non-classical monocytes in conjunction with global suppression of type I interferon-signaling pathway and an aberrant expression of host genes associated with pregnancy complications. 30 ZIKV+ sera from symptomatic pregnant patients also showed elevated levels of M2-skewed immunosuppressive cytokines and pregnancy complication-associated fibronectin-1. This study demonstrates the differential immunomodulatory responses of blood monocytes, particularly during pregnancy, upon infection with different lineages of ZIKV.

Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (μCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.Interleukin-6 | Ross River virus disease | viral arthritis | osteoclastogenesis A rthritogenic alphaviruses including Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), o'nyongnyong virus (ONNV), and Barmah Forest virus (BFV) are classified under the genus Alphavirus ("Old World" alphaviruses) of the Togaviridae family (1). RRV is a small, enveloped, positivesense single-stranded RNA virus transmitted by mosquitoes (2, 3). RRV disease (RRVD) in humans commonly affects the ankles, knees, and peripheral joints. The hallmarks of RRVD include incapacitating joint pain and polyarthralgias, with a level of disability comparable to rheumatoid arthritis (RA) (4, 5). Similar to RA, the onset of RRVD can be sudden and debilitating, and the prolonged manifestations of RRVD in some patients have been proposed to be due to the actions of proinflammatory mediators including interleukin-6 (IL-6), interleukin-1 (IL-1), and chemokine (C-C motif) ligand 2; monocyte chemotactic protein-1 (CCL2; MCP-1) (6-8).Recently, bone lesions in joints of CHIKV-infected patients have been reported (9), providing evidence that alphavirusinduced disease can result in bone pathologies (10, 11). In physiological conditions, osteoblasts (OBs) form bone, and this cell lineage also expresses both receptor activator of nuclear factorkappaB ligand (RANKL) and its soluble decoy receptor, osteoprotegerin (OPG). The expression of RANKL by the OB lineage is stimulated by IL-6 and IL-1β among other proinflammatory cytokines (12, 13), whereas CCL2 is thought to be an important chemoattractant for monocytic precursors during inflammatory processes (14,15). Together with an elevation in RANKL/OPG...

The recent global resurgence of arthritogenic alphaviruses, in particular chikungunya virus (CHIKV), highlights an urgent need for the development of therapeutic intervention strategies. While there has been significant progress in defining the pathophysiology of alphaviral disease, relatively little is known about the mechanisms involved in CHIKV-induced arthritis or potential therapeutic options to treat the severe arthritic symptoms associated with infection. Here, we used microcomputed tomographic (CT) and histomorphometric analyses to provide previously undescribed evidence of reduced bone volume in the proximal tibial epiphysis of CHIKV-infected mice compared to the results for mock controls. This was associated with a significant increase in the receptor activator of nuclear factor-B ligand/osteoprotegerin (RANKL/OPG) ratio in infected murine joints and in the serum of CHIKV patients. The expression levels of the monocyte chemoattractant proteins (MCPs), including MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, were also highly elevated in joints of CHIKV-infected mice, accompanied by increased cellularity within the bone marrow in tibial epiphysis and ankle joints. Both this effect and CHIKV-induced bone loss were significantly reduced by treatment with the MCP inhibitor bindarit. Collectively, these findings demonstrate a unique role for MCPs in promoting CHIKV-induced osteoclastogenesis and bone loss during disease and suggest that inhibition of MCPs with bindarit may be an effective therapy for patients affected with alphavirus-induced bone loss. IMPORTANCE Arthritogenic alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), cause worldwide outbreaks of polyarthritis, which can persist in patients for months following infection. Previous studies have shown that host proinflammatory soluble factors are associated with CHIKV disease severity. Furthermore, it is established that chemokine (C-C motif) ligand 2 (CCL2/MCP-1) is important in cellular recruitment and inducing bone-resorbing osteoclast (OC) formation. Here, we show that CHIKV replicates in bone and triggers bone loss by increasing the RANKL/OPG ratio. CHIKV infection results in MCP-induced cellular infiltration in the inflamed joints, and bone loss can be ameliorated by treatment with an MCP-inhibiting drug, bindarit. Taken together, our data reveal a previously undescribed role for MCPs in CHIKV-induced bone loss: one of recruiting monocytes/OC precursors to joint sites and thereby favoring a pro-osteoclastic microenvironment. This suggests that bindarit may be an effective treatment for alphavirus-induced bone loss and arthritis in humans.