Enterovirus D68 infection in cotton rats results in systemic inflammation with detectable viremia associated with extracellular vesicle and neurologic disease

Enterovirus D68 (EV-D68) is a non-polio enterovirus that causes respiratory illness and is linked to acute flaccid myelitis (AFM) in infants and children. Recent demonstration of association of EV-D68 with extracellular vesicles (EVs) released from infected cells in vitro suggests a role for these vesicles in non-lytic dissemination of virus beyond the respiratory tract. We previously reported the permissiveness of cotton rat (Sigmodon hispidus) to infection with different EV-D68 strains of clades A and B, but did not investigate the virus association with EVs. We present a model of acute respiratory infection with a clinical isolate of EV-D68 of clade B3 in immunocompetent cotton rats featuring systemic dissemination of the virus. EV-D68 was detected in circulation and organs outside of the respiratory tract with the inflammatory response accompanying dissemination. Further analysis demonstrated that the virus was associated with extracellular vesicles purified from plasma. We also present a model of intraperitoneal infection with EV-D68 in young cotton rats featuring dissemination of the virus to spinal cord and brain with associated clinical signs of neurologic disease. EV-D68-associated with EVs produced in cotton rat cells and injected intraperitoneally into young cotton rats also resulted in detection of virus in the CNS. Our results provide the first in vivo experimental support for the notion that respiratory infection with EV-D68 generates virus associated with extracellular vesicles that disseminate outside the respiratory tract. These models of infection could be used to investigate the role of EVs-associated EV-D68 in the pathogenesis of EV-D68 infection and to assess therapeutic interventions.