Age dependent susceptibility and immune responses to La Crosse virus infection in non-human primates

Abstract

La Crosse virus (LACV) is a primary cause of pediatric viral encephalitis in the United States but rarely causes disease in adults. We tested whether cynomolgus macaques displayed a similar age-dependent susceptibility to LACV. Immune responses from naïve or LACV infected weanling (9-15 months), juvenile (19-23 months) or adult (> 6 years) animals were measured and infected animals were monitored for disease. Naïve weanling animals had fewer dendritic cells in their blood and weaker induction of IFN-stimulated genes (ISG) and chemokines when PBMCs were stimulated in vitro. While no infected animals developed disease, the weaker innate response in naive weanlings correlated with increased viral RNA in plasma from 2 of 3 infected weanlings out to 7 days post infection (dpi). Activated CD8⁺ T cells and neutralizing antibody proportions were similar amongst all ages. However, CD4⁺ T cells proportions were increased in young animals relative to adults. This suggests the CD4⁺ adaptive response in young animals may be bolstering an initially weak innate response to clear virus. Finally, because macaques were resistant to disease, we infected 3 common marmosets intranasally with LACV. Marmoset were selected due to their susceptibility to viral encephalitis. Although no animals showed disease signs, one animal had evidence of infection in the nasal mucosa out to 23 days with associated vacuolization, edema and immune cell infiltration.

Keywords: Age-related susceptibility; Immune response; Interferon stimulated gene; La Crosse virus; Non-human primate; T cell.

Fig. 1

Dendritic cells proportions of are lower in naïve weanling cynomolgus macaques relative to older animals. Flow cytometric analysis of PBMCs from naïve weanling, juvenile and adult cynomolgus macaques was performed to identify CD11b⁺ CD14⁺ CD11c- monocytes (A-C), CD11b⁺ CD16⁺ CD11c- monocytes (A-C) and CD11c⁺ dendritic cells (A,B). Quantifications of the proportions of each cell type as a proportion of total PBMCs is in shown in (D-F).

Fig. 2

Naïve weanling cynomolgus macaques have a weaker early innate response to virus and synthetic viral RNA mimics. PBMCs isolated from naïve weanling, juvenile and adult cynomolgus macaques were cultured for 5 h with mock inoculum, LACV (MOI 0.01) or the viral double-stranded RNA mimic poly I: C (1 µg/ml). RNA was isolated from cells, cDNA synthesized and qRT PCR assays for Isg15 (A), Isg20 (B), Oas1 (C), Il6 (D), Ifit1 (E), Ifit2 (F) and Il-1b (G) transcripts were performed. Target transcript expression is plotted as a percent of the house-keeping transcript Gapdh. A two-way ANOVA with a Dunnett’s multiple comparisons test was used to compare the indicated transcript expression between each of the three age groups. *p < 0.05.

Fig. 3

There are minimal age-dependent differences in the dendritic cell response to LACV. 3 dpi flow cytometric analysis of PBMCs from LACV-infected weanling, juvenile and adult cynomolgus macaques was performed to identify CD11c⁺ dendritic cells (A,B), CD11c⁺ CD123⁺ plasmacytoid dendritic cells and CD11C⁺ CD123⁺ HLADR^hi activated plasmacytoid dendritic cells (C). Quantifications of the proportions of each cell type as percent of total cells is in shown in (D). A Wilcoxon matched-pairs signed rank test was used to compare the proportions of cells in each age group.

Fig. 4

ISG responses in PBMCs from mock and LACV infected cynomolgus macaques were similar regardless of age. ISG transcript expression from PBMCs at 3 dpi was compared between LACV infected weanling, juvenile and adults NHPs and a mock infected juvenile and adult NHP. Data are presented as target transcript expression as a percent of the house-keeping gene Gapdh.

Fig. 5

Activated CD4⁺ CD44⁺ T cells are proportionally increased in LACV-infected young NHP. PBMCs isolated from mock and LACV-infected weanling, juvenile and adult cynomolgus macaques were analyzed by flow cytometry for expression of T cell markers (A,B) including CD44 which is a marker of T cell activation (C,D). The proportion of CD4⁺ T cells was determined in LACV-infected weanling (E), and mock and LACV-infected juvenile (F) and adult (G) animals. Activated CD4⁺ CD44⁺ T cells were determined in the same animals (H-J). All data are presented as the proportion of T cells within total PBMCs.

Fig. 6

Intranasally inoculation of LACV induces pathology in the nasal epithelium of the common marmoset. Nasal turbinate tissue from marmoset MA250 was stained with H&E (A,C,D) and fluorescently immunolabled with a polyclonal antibody raised against LACV (B). Areas of infection were infrequent and delimited with most tissue being normal in appearance (A). In areas of infection pathology (B-D), LACV protein was evident by immunolabeling (B, yellow arrows) which was at times coincident with vacuoles (lower arrow in B and C,D) or adjacent to vacuoles (upper arrow in B). Hypertrophic goblet cells were also present in areas of vacuolization (C,D, orange arrowheads) indicating increased mucus production in the area. Vacuoles were commonly found near degenerating cells (C,D, red arrowheads) and infiltrating immune cells (C,D, green arrowheads). Edema was commonly observed in the basal cell layer near areas of infection (D, *). Images of H&E staining (A,C,D) were all taken at 400x. The scale bar in (B) = 20 μm.