The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme-induced peroxidase.

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme–induced peroxidase

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

Infection Pattern of Mayaro Virus in Aedes aegypti (Diptera: Culicidae) and Transmission Potential of the Virus in Mixed Infections With Chikungunya Virus.

Mayaro virus (MAYV; Togaviridae; Alphavirus) has drawn increasing attention as an arthropod-borne virus with potential to cause outbreaks among the human populations of the Western Hemisphere. In the tropical regions of Central and South America, the virus exists in sylvatic cycles between mosquitoes and primate reservoirs such as marmosets. Although forest-dwelling mosquitoes are regarded as important vectors for MAYV, it has been shown previously that the virus can infect and potentially be transmitted by the mosquitoes, Aedes aegypti and Aedes albopictus (Diptera: Culicidae). Here, we compare the infection and transmission efficiencies of two MAYV strains, IQT 4235 from Iquitos, Peru ('IQT') and the type strain of MAYV from Trinidad, TRVL 4675 ('TRVL') in two laboratory-adapted Ae. aegypti strains, Higgs White Eye and Orlando. The TRVL strain was less efficiently transmitted by both mosquito strains than MAYV IQT. Based on the full-length nucleotide sequences of the two viral genomes, we show that the TRVL prototype strain of MAYV is phylogenetically ancestral and more distantly related to the IQT strain. The TRVL strain efficiently infected wild-type Ae. albopictus from Missouri and readily disseminated in those. Considering scenarios in which natural MAYV transmission cycles may overlap with those of chikungunya virus (CHIKV; Togaviridae; Alphavirus), we assessed the effects of mixed infections of the two viruses in mosquitoes based on coinfection or superinfection. Although coinfection had no measurable effect on the transmission potential of either virus, we observed superinfection exclusion for CHIKV in MAYV-infected mosquitoes but not for MAYV in CHIKV-infected mosquitoes.

Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegypti.

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24⁻32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

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

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.

Infection pattern and transmission potential of chikungunya virus in two New World laboratory-adapted Aedes aegypti strains.

Chikungunya virus (CHIKV) is an emerging mosquito-borne virus belonging to the Togaviridae, which is transmitted to humans by Aedes aegypti and Ae. albopictus. We describe the infection pattern of CHIKV in two New World Ae. aegypti strains, HWE and ORL. Both mosquito strains were susceptible to the virus but showed different infection patterns in midguts and salivary glands. Even though acquisition of a bloodmeal showed moderate levels of apoptosis in midgut tissue, there was no obvious additional CHIKV-induced apoptosis detectable during midgut infection. Analysis of expression of apoptosis-related genes suggested that CHIKV infection dampens rather than promotes apoptosis in the mosquito midgut. In both mosquito strains, the virus was present in saliva within two days post-oral infection. HWE and ORL mosquitoes exhibited no salivary gland infection barrier; however, only 60% (HWE) to 65% (ORL) of the females had released the virus in their saliva at one week post-oral acquisition, suggesting a salivary gland escape barrier. CHIKV induced an apoptotic response in salivary glands of HWE and ORL mosquitoes, demonstrating that the virus caused pathology in its natural vector.

Tissue Barriers to Arbovirus Infection in Mosquitoes.

Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts.