Japanese encephalitis virus (JEV), an enveloped Flavivirus with a positive-sense 
RNA genome, causes acute encephalitis with high mortality in humans. We used a 
virulent (RP-9) and an attenuated (RP-2ms) JEV strain to assess the role of 
autophagy in JEV infection. By monitoring the levels of lipidated LC3, we found 
that autophagy was induced in human NT-2 cells infected with RP-2ms, especially 
at the late stage, and to a lesser extent with RP-9. The induction of autophagy 
by rapamycin increased viral production, whereas the inhibition of autophagy by 
3-methyladenine reduced viral yields for both RP-9 and RP-2ms. The viral 
replication of RP-9 and RP-2ms was also reduced in cells with downregulated ATG5 
or Beclin 1 expression, suggesting a proviral role of autophagy in JEV 
replication. To determine the step of JEV life cycle affected by autophagy, we 
used an mCherry-LC3 fusion protein as the autophagosome marker. Little of no 
colocalization of LC3 puncta with dsRNA was noted, whereas the input JEV 
particles were targeted to autophagosomes stained positive for early endosome 
marker. Overall, we show for the first time that the cellular autophagy process 
is involved in JEV infection and the inoculated viral particles traffic to 
autophagosomes for subsequent steps of viral infection.