A mouse model to study Rio Negro virus pathogenesis: a neglected member of the Venezuelan equine encephalitis virus complex

Abstract

Rio Negro virus (RNV) is an enzootic alphavirus and a member of the Venezuelan equine encephalitis virus (VEEV) complex. Despite its wide circulation in South America, RNV remains a neglected pathogen with no established wild-type animal model to study its pathogenesis. In this study, we developed a lethal mouse model using 18-day-old wild-type C57BL/6 mice to characterize the systemic and neurological features of RNV infection. Following subcutaneous inoculation, RNV exhibited rapid systemic dissemination with a brief and low-titer viremic phase and high viral loads in lymphoid tissues, pancreas, brains, and lungs. Notably, infected mice developed progressive neurological signs, including ataxia and hindlimb paralysis, culminating in 100% lethality. Histopathological analysis revealed significant damage, highlighted by a striking collapse of the splenic architecture, inflammatory and remodeling changes in the lungs, and prominent inflammatory infiltrates with neurodegenerative changes in the brain. The splenic disruption was further evaluated by immunofluorescence analysis of the spleen, which showed a consistent loss of compartmentalization, characterized by an atypical infiltration of CD8+ T cells into B-cell follicles. The terminal stage of disease was characterized by extensive neuroinflammation and neurodegeneration. Histological examination of the brain revealed meningoencephalitis, robust astrogliosis, and widespread somatodendritic and terminal degeneration, particularly clustered around blood vessels. These findings were supported by cytokine analysis of brain homogenates, which showed a significant upregulation of IFN-{gamma}, IL-6, and MCP-1/CCL2 during symptomatic stages. Collectively, these findings establish a reproducible, non-genetically modified animal model that reveals the pathogenic potential of RNV in the context of immune immaturity characteristic of early life. By identifying these specific pathological and neuroinflammatory markers, our study provides a foundational experimental framework to investigate the mechanisms underlying RNV emergence and host-pathogen interactions within the VEEV complex. Author summaryMany viruses circulate silently in nature, hidden within animal populations, until environmental or social changes bring them into contact with humans. The Rio Negro virus is one such example. Despite being closely related to the better-known Venezuelan equine encephalitis virus and showing evidence of circulation in South America, RNV has remained largely overlooked by the scientific community and public health authorities. In this study, we established a new experimental model using infant mice that allowed us to observe how the virus spreads and causes damage. We found that the virus rapidly reaches the brain and other vital organs, causing severe inflammation in the brain, inflammatory changes in the lungs, and a breakdown of the immune systems organization in the spleen. By using a genetically unmodified model, we were able to observe the infection in a host with an intact but naturally immature immune system. This approach avoids the artificial conditions of genetic engineering while providing a more realistic window into how host age and developmental stages can influence the outcome of neglected viral infections. We believe our work is a first step toward understanding how this overlooked virus emerges and highlights the need to monitor and prepare for "silent" pathogens that could pose a risk to public health in an era of intensifying human activity and ecological pressure.