The strategies adopted by a densovirus to cross the insect midgut barrier

Densoviruses (DNVs) are parvoviruses highly pathogenic for arthropods, mostly insect at larval stages, including agronomical pest and insect vector borne disease. They have limited host range and are not pathogenic to vertebrates, characteristic that make them particularly interesting as potential pest control agents alternative to chemical pesticides. As a model, we studied the Junonia coenia Densovirus (JcDNV) infection of the Lepidopteran pest Spodoptera frugiperda. In natural condition JcDNV infection is initiated after ingestion of viral particles. The virus does not replicate in intestinal cells, so the midgut epithelium represents a barrier that the virus has to overcome to reach the internal target tissue. To understand the early step of infection, we first analyzed the JcDNV entry in midgut cells in culture. We showed that JcDNV enters specifically columnar cells, but not stem and goblet cells, the three cells types that form the midgut epithelium. Mounting isolated midgut in a Ussing chamber in the presence of JcDNV in the luminal compartment, we showed that after 10 min of incubation viral particles are detectable in the cytoplasm of midgut cells and after 30 min or more of exposition the virus appears in the intercellular spaces. We also demonstrated that JcDNV internalization induces a raise of cytosolic Ca2+ concentration in columnar cells, an intracellular mediator that causes an increase of the epithelium permeability through the paracellular pathway. We effectively verified, in Ussing chamber, that JcDNV causes a significant decrease of the paracellular electrical resistance, i.e an increase of the septate junction (SJ) permeability to ions. Using the same apparatus we also observed that after 10 min of incubation the amount of virus able to cross the epithelium is lower compared to that measured after 30 min. Therefore our results indicate that the internalization of JcDNV into midgut cells causes the raise of intracellular calcium concentration and, consequently, the increase of the permeability of the paracellular route, the pathway used by the virus to cross the midgut epithelium to reach in a massive amount the internal target tissues. To characterized the mechanism involved in JcDNV internalization into midgut cells, isolated midguts were mounted in a Ussing chamber and incubated with the virus in the absence or in the presence of drugs able to inhibit specific endocytic pathway. Our results show that clathrin-coated vesicle formation and lipid rafts-dependent endocytosis are essential for JcDNV entry into columnar cells. Using specific molecular markers for vesicle trafficking we also demonstrated that early and late endosomes are involved in JcDNV intracellular trafficking.