Chikungunya virus infection results in higher and persistent viral replication in aged Rhesus macaques due to defects in anti-viral immunity

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne Alphavirus that causes a clinical disease involving fever, myalgia, nausea and rash. The distinguishing feature of CHIKV infection is the severe debilitating poly-arthralgia that may persist for several months after viral clearance. Since its re-emergence in 2004, CHIKV has spread from the Indian Ocean region to new locations including metropolitan Europe, Japan, and even the United States. The risk of importing CHIKV to new areas of the world is increasing due to high levels of viremia in infected individuals as well as the recent adaptation of the virus to the mosquito species Aedes albopictus. CHIKV re-emergence is also associated with new clinical complications including severe morbidity and, for the first time, mortality. In this study, we characterized disease progression and host immune responses in adult and aged Rhesus macaques infected with either the recent CHIKV outbreak strain La Reunion (LR) or the West African strain 37997. Our results indicate that following intravenous infection and regardless of the virus used, Rhesus macaques become viremic between days 1-5 post infection. While adult animals are able to control viral infection, aged animals show persistent virus in the spleen. Virus-specific T cell responses in the aged animals were reduced compared to adult animals and the B cell responses were also delayed and reduced in aged animals. Interestingly, regardless of age, T cell and antibody responses were more robust in animals infected with LR compared to 37997 CHIKV strain. Taken together these data suggest that the reduced immune responses in the aged animals promotes long-term virus persistence in CHIKV-LR infected Rhesus monkeys

Src family kinase inhibitors block translation of alphavirus subgenomic mRNAs

Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)–phosphatidylinositol 3-kinase (PI3K)–AKT–mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O’nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigm-shifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development

Src Family Kinase Inhibitors Block Translation of Alphavirus Subgenomic mRNAs

Alphaviruses are arthropod-transmitted RNA viruses that can cause arthralgia, myalgia, and encephalitis in humans. Since the role of cellular kinases in alphavirus replication is unknown, we profiled kinetic changes in host kinase abundance and phosphorylation following chikungunya virus (CHIKV) infection of fibroblasts. Based upon the results of this study, we treated CHIKV-infected cells with kinase inhibitors targeting the Src family kinase (SFK)–phosphatidylinositol 3-kinase (PI3K)–AKT–mTORC signaling pathways. Treatment of cells with SFK inhibitors blocked the replication of CHIKV as well as multiple other alphaviruses, including Mayaro virus, O’nyong-nyong virus, Ross River virus, and Venezuelan equine encephalitis virus. Dissecting the effect of SFK inhibition on alphavirus replication, we found that viral structural protein levels were significantly reduced, but synthesis of viral genomic and subgenomic RNAs was unaffected. By measuring the association of viral RNA with polyribosomes, we found that the SFK inhibitor dasatinib blocks alphavirus subgenomic RNA translation. Our results demonstrate a role for SFK signaling in alphavirus subgenomic RNA translation and replication. Targeting host factors involved in alphavirus replication represents an innovative, perhaps paradigmshifting, strategy for exploring the replication of CHIKV and other alphaviruses while promoting antiviral therapeutic development

Macrophage and dendritic cell population changes following CHIKV infection observed in adult animals are reduced in aged Rhesus macaques.

<p>Measurement of dendritic cell and monocyte/macrophage populations following CHIKV infection. PBMC were stained with antibodies directed against CD3, CD11c, CD14, CD20, CD123 and HLA-DR (shown in panel A) and subdivided into plasmacytoid DCs (B); myeloid DCs (C); non-P/non-M DC's (D); monocyte/macrophages (E). Fold increase in numbers of cells was calculated. N = 6 for adult and N = 2 for aged animals.</p

Animal groupings.

<p>Animal groupings.</p

Magnitude and breadth of anti-CHIKV T cell responses is reduced in aged Rhesus macaques.

<p>IFN-γ ELISPOT assays were used to quantify anti-CHIKV T cell responses in peripheral blood lymphocytes at 35 dpi in adult and aged animals (n = 4). In triplicate wells, PBMC (2×10⁵) were incubated overnight with overlapping peptides that corresponded to each of the 9 CHIKV proteins (NSP-1-4, Core, E1-3 and 6k). PMA/ionomycin stimulation was used as a positive control and medium plus DMSO was used as a negative control. A) Representative wells from IFN-γ ELISPOT assay performed with PBMC from adult and aged animals. B) Average cumulative anti-CHIKV responses to all viral proteins were compared for adult vs. aged Rhesus macaques (n = 4). C) Average response to individual CHIKV proteins (n = 4).</p

CHIKV-LR persists in spleen of aged Rhesus macaques.

<p>CHIKV load was quantified in spleen tissue at necropsy (35 dpi) by virus-specific qRT-PCR from total RNA samples prepared using Trizol.</p

Aged Rhesus macaques have lower T cell responses following CHIKV infection.

<p>T-cell proliferative burst was measured following CHIKV infection. PBMC were stained with antibodies directed against CD4, CD8, CD28, CD95 and Ki67 (as shown in panel A). Fold increase in number of Ki67+ cells was calculated for each time point. Cell populations were subdivided: B & C) CD4+ Central memory (CM) T-cells; D & E) CD4+ Effector memory (EM) T-cells; F & G) CD8+ CM; H & I) CD8+ EM for animals infected with CHIKV-LR (panels B, D, F, H) or CHIKV-37997 (panels C, E, G, I). N = 6 for adult and N = 2 for aged animals.</p

CHIKV viremia in blood peaks 1 to 2 days post infection of Rhesus macaques.

<p>CHIKV load in plasma and PBMC was quantified by qRT-PCR using virus specific primers and probes. A and C) Virus was detected in RNA prepared from 10 µl of plasma using ZR Viral RNA extraction kit (Zymol). B and D) Virus was detected in 0.1 µg of total RNA prepared with Trizol from 1×10⁶ PBMC.</p

Aged Rhesus macaques have reduced anti-virion antibody titers compared to adult animals following CHIKV infection.

<p>The production of anti-CHIKV antibodies in aged Rhesus macaques was compared to adult animals. CHIKV-specific endpoint IgG titers were measured using standard ELISA detecting either CHIKV purified virions (A & C) or CHIKV infected cell lysates (B & D) as the antigenic source. N = 6 for adult and N = 2 for aged animals. Aged animals have lower antibody titers to whole virions compared to adult animals, whereas the production of IgG responses to antigen present in CHIKV-infected cellular lysates were not age dependent.</p