Enterovirus D68 infection of human inducible pluripotent stem cell-derived skeletal muscles resulted in structural destruction, loss of muscle function and hampered muscle regeneration

Abstract

Enterovirus D68 (EV-D68) is an emerging respiratory virus that commonly causes mild to severe respiratory diseases. EV-D68 infection is also associated with extra-respiratory complications, especially acute flaccid myelitis. However, how the virus invades the central nervous system (CNS) and infects motor neurons is not fully understood. One possible neuroinvasive route is through infection of skeletal muscles, which allows the virus to infect motor neurons via the neuromuscular junction. However, we hypothesise that direct EV-D68 infection of human skeletal muscles can impair muscle function and thus contribute to the development of EV-D68-associated muscle weakness. Here, we inoculated human induced pluripotent stem cell-derived skeletal muscle myotubes grown in 2D with different EV-D68 isolates, which resulted in a productive infection and cell death. We showed, through neuraminidase treatment, that sialic acids facilitate infection of these cells. EV-D68 infection of 3D tissue engineered skeletal muscles led to tissue damage, reduction of contractile force and depletion of muscle and satellite cells. Altogether, we showed that human skeletal muscle can act as an extra-respiratory replication site and infection of skeletal muscles may contribute to EV-D68-associated muscle weakness. Author summaryAfter causing a global outbreak in 2014, enterovirus D68 (EV-D68) - a respiratory virus - has been associated with polio-like paralysis. How the virus spreads from the respiratory system to the central nervous system is poorly understood. One of the possible routes for this spread is by infecting skeletal muscles before spreading to motor neurons. However, it is not known whether the virus can infect human skeletal muscles and whether EV-D68 infection of skeletal muscles can also lead to paralysis. Here, we used 2D and 3D human skeletal muscle models to investigate EV-D68 infection of human skeletal muscles. We found that EV-D68 can replicate in these 2D and 3D models and the infection results in destruction of muscle fibres and loss of muscle function. We also found that the infection results in loss of satellite cells, which are important for muscle regeneration, suggesting that EV-D68 infection hampers skeletal muscle regeneration. Our study provides new insights into the potential role of human skeletal muscles in EV-D68-associated paralysis.