Distinct cellular and molecular responses to infection in three target cell types from horses, a species naturally susceptible to Ross River virus.

Overview

• This study investigates how different cell types from horses, a natural host of Ross River virus (RRV), respond at the cellular and molecular levels to RRV infection.
• The research reveals distinctive antiviral and inflammatory responses in these cells and highlights impaired mitochondrial function despite strong immune activation.

Background and Objective

• Ross River virus is a mosquito-borne alphavirus causing disease in humans and horses.
• Most previous knowledge about RRV pathogenesis comes from mouse models that do not fully replicate the clinical symptoms seen in natural hosts.
• The study aimed to create more relevant cell-based models from horses to better understand host-virus interactions during RRV infection.

Methodology

• Three cell types from horses were used:
  • Peripheral blood mononuclear cells (PBMCs) obtained ex vivo
  • Primary synovial fibroblast cultures (cells from joint connective tissue)
  • Primary epidermal keratinocyte cultures (skin cells)
• Transcriptomic (gene expression) analysis was performed to detect how gene expression changed upon RRV infection.
• Virus growth kinetics were measured to assess how well RRV replicated in the different cell types.
• Cellular metabolic flux assays evaluated mitochondrial function and cellular metabolism in infected cells.
• PBMCs from seronegative (no prior exposure) and seropositive (prior exposure) horses were compared to understand immune status effects.

Key Findings – Gene Expression and Antiviral Responses

• The transmembrane protein matrix remodeling associated 8 (mxra8), known as the receptor facilitating RRV entry into cells, was downregulated in all infected cell types compared to controls, indicating possible cellular defense or viral manipulation mechanisms.
• Both synovial fibroblasts and epidermal keratinocytes strongly upregulated antiviral and inflammatory genes when infected:
  • Many genes induced by double-stranded RNA were increased, signaling activation of antiviral pathways triggered by viral replication intermediates.
  • Toll-like receptor 3 (tlr-3), which senses viral double-stranded RNA, was upregulated, but other TLRs that sense single-stranded RNA (tlr-7, -8, and -9) were not, suggesting limited viral replication in these cells.
• This gene expression profile suggests an abortive viral replication cycle, where the virus enters cells but struggles to produce new infectious particles efficiently in synovial fibroblasts and keratinocytes.

Virus Replication Efficiency

• Virus growth kinetic studies supported the transcriptomic findings by showing inefficient replication of RRV in synovial fibroblasts and epidermal keratinocytes.
• Limited viral production in these cell types indicates these cells mount effective antiviral responses that restrict viral spread.

Mitochondrial Function and Metabolic Impact

• Metabolic flux studies showed RRV infection reduced mitochondrial function in both PBMCs and synovial fibroblasts.
• Reduced mitochondrial capacity can impair cellular energy production and may influence immune responses.
• This suggests that despite antiviral defenses, viral infection affects cell metabolism and mitochondrial health, which could contribute to disease pathology.

Differences Between Seronegative and Seropositive Horses

• PBMCs from seropositive horses (previously exposed and likely immune) displayed an enhanced antiviral state compared to seronegative horses.
• Seropositive PBMCs exhibited reduced expression of inflammation-related genes upon infection, indicating a more controlled immune response.
• These differences highlight the importance of immune memory in modulating responses to RRV infection.

Conclusions and Implications

• The study provides novel insights into how three relevant horse cell types respond distinctly to RRV infection.
• Potent interferon-mediated antiviral and inflammatory responses limit viral replication in synovial fibroblasts and keratinocytes.
• However, mitochondrial function is detrimentally affected by infection, which may influence disease progression.
• Differences in immune response between seronegative and seropositive horses reveal the impact of prior exposure on controlling infection and inflammation.
• These findings improve understanding of RRV pathogenesis in a natural host and may guide development of better models and interventions for Ross River virus infection.