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 metalloproteinases (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.

Malaria parasite immune evasion and adaptation to its mosquito host is influenced by the acquisition of multiple blood meals

“…To examine if an additional blood- or protein-meal could influence the integrity of the basal lamina, we utilized a collagen hybridizing peptide (CHP) that specifically binds unfolded collagen chains, to serve as an indicator of tissue damage (Hwang et al, 2017). Collagen IV serves as a primary component of the midgut basal lamina (Arrighi and Hurd, 2002; Arrighi et al, 2005; Dong et al, 2017) that becomes degraded following blood-feeding (Dong et al, 2017). We demonstrate that CHP stains dissected midguts shortly after blood-or protein-feeding (Figure 2A), with the intensity of CHP staining reaching peak levels ∼18hr after blood- (Figure 2B) or protein-feeding (Figure 2C), before being repaired shortly thereafter.…”

“…These results suggest that the effects of P. berghei killing are independent of the host blood meal, and more likely caused by the distention of the mosquito midgut following feeding. Ingestion of a blood- or protein-meal results in dramatic changes to midgut epithelium cell morphology, causing a flattening of the columnar cells, the loss of microvilli, and substantial degradation of the basal lamina (Dong et al, 2017; Sodja et al, 2007). This agrees with our CHP assays demonstrating the presence of degraded collagen shortly after taking a blood- or protein-meal, suggesting that the integrity of the basal lamina is compromised during distention, enabling the exposure of developing oocysts to components of the mosquito hemolymph.…”

Successive Bloodmeals Enhance Virus Dissemination Within Mosquitoes And Increase Transmission Potential

“…http://dx.doi.org/10.1101/246306 doi: bioRxiv preprint first posted online Jan. 10, 2018; 4 midgut, apoptosis and regeneration of midgut epithelial cells, and altered permeability of the basal lamina layer-that could enhance or accelerate virus dissemination out of the midgut 20,21 .…”

“…The basal lamina layer surrounding the midgut epithelium represents a physical barrier that is impermeable to virus particles 26 , yet viruses are still able to escape from the gut. While a second non-infectious bloodmeal is not required for escape, a recent study showed that the integrity of the basal lamina was compromised 24-32 hours post-bloodmeal coincident with increased collagenase activity 21 . Future studies are needed to identify mechanisms mediating arbovirus escape from the midgut and how multiple bloodmeals enhance this event.…”

Molecular Basis for Arbovirus Transmission by <b><i>Aedes aegypti</i></b> Mosquitoes

“…During the extrinsic incubation period (EIP), a virus that enters the mosquito midgut and is able to pass into the hemolymph may infect fat body cells, trachea, hemocytes, ovaries, nerve tissue, and reach salivary glands [2, 18, 32, 33]. For an arbovirus to be transmitted to a new vertebrate host in a subsequent blood meal, it must first be secreted with the saliva by the acinar (epithelial) cells to the luminal side of the salivary glands.…”