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

Rhesus Macaque Rhadinovirus Encodes a Viral Interferon Regulatory Factor To Disrupt Promyelocytic Leukemia Nuclear Bodies and Antagonize Type I Interferon Signaling

Interferon (IFN) production and the subsequent induction of IFN-stimulated genes (ISGs) are highly effective innate strategies utilized by cells to protect against invading pathogens, including viruses. Critical components involved in this innate process are promyelocytic leukemia nuclear bodies (PML-NBs), which are subnuclear structures required for the development of a robust IFN response. As such, PML-NBs serve as an important hurdle for viruses to overcome to successfully establish an infection. Both Kaposi\u27s sarcoma-associated herpesvirus (KSHV) and the closely related rhesus macaque rhadinovirus (RRV) are unique for encoding viral homologs of IFN regulatory factors (termed vIRFs) that can manipulate the host immune response by multiple mechanisms. All four KSHV vIRFs inhibit the induction of IFN, while vIRF1 and vIRF2 can inhibit ISG induction downstream of the IFN receptor. Less is known about the RRV vIRFs. RRV vIRF R6 can inhibit the induction of IFN by IRF3; however, it is not known whether any RRV vIRFs inhibit ISG induction following IFN receptor signaling. In our present study, we demonstrate that the RRV vIRF R12 aids viral replication in the presence of the type I IFN response. This is achieved in part through the disruption of PML-NBs and the inhibition of robust ISG transcription

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

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

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

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

Aged Rhesus macaques have a delayed and lower B cell proliferative response following CHIKV infection.

<p>Measurement of B-cell proliferative burst following viral infection. PBMC were stained with antibodies directed against CD20, CD27, IgD and Ki67 (as shown in panel A) and subdivided into (B & C) Marginal Zone (MZ)-like B cells and (D & E) memory B cells. Fold increase in number of Ki67+ B cells was calculated at each time point. N = 6 for adult and N = 2 for aged animals.</p