Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion

Abstract

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24–32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metallo proteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.

Author summary

The biological nature of the midgut escape barrier in insects for arthropod-borne viruses has been a mystery for decades. Here we show that the basal lamina (BL) surrounding the mosquito midgut acts as a barrier for chikungunya virus, an alphavirus, which has emerged in the New World hemisphere around three years ago. The barrier became permissive for the virus during digestion of a viremic bloodmeal inside the midgut lumen. Concurrent with BL permissiveness, we observed that collagen IV, a major component of the BL became temporally degraded while the BL was visibly damaged. Based on previous findings, we hypothesized that matrix metalloproteinases such as Ae. aegypti (Ae)MMP1 may be involved in BL degradation. We confirmed that recombinant AeMMP1 exhibited strong gelatinase activity, which was profoundly reduced when recombinant AeMMP1 interacted in vitro with the recombinant Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP). When transgenically overexpressing AeTIMP in an attempt to temporally inhibit general MMP activity in the mosquito midgut, we observed that the dissemination efficiency of chikungunya virus became significantly increased, while its midgut infection was not affected. It is possible that AeTIMP overexpression affected BL degradation/restoration permitting increased quantities of virus to escape from the midgut.