Microbial interactions in the mosquito gut determineSerratiacolonization and blood-feeding propensity

How microbe–microbe interactions dictate microbial complexity in the mosquito gut is unclear. Previously we found that, Serratia, a gut symbiont that alters vector competence and is being considered for vector control, poorly colonized Aedes aegypti yet was abundant in Culex quinquefasciatus reared under identical conditions. To investigate the incompatibility between Serratia and Ae. aegypti, we characterized two distinct strains of Serratia marcescens from Cx. quinquefasciatus and examined their ability to infect Ae. aegypti. Both Serratia strains poorly infected Ae. aegypti, but when microbiome homeostasis was disrupted, the prevalence and titers of Serratia were similar to the infection in its native host. Examination of multiple genetically diverse Ae. aegypti lines found microbial interference to S. marcescens was commonplace, however, one line of Ae. aegypti was susceptible to infection. Microbiome analysis of resistant and susceptible lines indicated an inverse correlation between Enterobacteriaceae bacteria and Serratia, and experimental co-infections in a gnotobiotic system recapitulated the interference phenotype. Furthermore, we observed an effect on host behavior; Serratia exposure to Ae. aegypti disrupted their feeding behavior, and this phenotype was also reliant on interactions with their native microbiota. Our work highlights the complexity of host–microbe interactions and provides evidence that microbial interactions influence mosquito behavior

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Citation: Londono-Renteria, B., Troupin, A., Conway, M. J., Vesely, D., Ledizet, M., Roundy, C. M., . . . Colpitts, T. M. (2015). Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein. Plos Pathogens, 11(10), 23. doi:10.1371/journal.ppat.1005202Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were >= 5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses

Susceptibility of South Texas Aedes aegypti to Pyriproxyfen

An integral part to integrated mosquito management is to ensure chemical products used for area-wide control are effective against a susceptible population of mosquitoes. Prior to conducting an intervention trial using an insect growth regulator, pyriproxyfen, in South Texas to control Aedes aegypti, we conducted a larval bioassay to evaluate baseline levels of susceptibility. We used seven serially-diluted doses ranging from 2.5 ppb to 6.3 × 10−4 ppb. We observed 100% inhibition emergence (IE) at even the lowest dose of 6.3 × 10−4 ppb in our susceptible reference colony of Ae. aegypti Liverpool. In our field strain of Ae. aegypti (F5 colonized from South Texas) we observed 79.8% IE at 6.3 × 10−4 ppb, 17.7% IE at 1.25 × 10−3 ppb, 98.7% IE at 1.25 × 10−2 ppb, and 100% emergence inhibition for the remainder of the doses. Given that commercial pyriproxyfen products are labeled for doses ranging to 50 ppb, we conclude that the field population sampled by this study are susceptible to this insect growth regulator

Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV‑2

Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the novel severe acute respiratory syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and angiotensin-converting enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 μM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 μM. The current study demonstrates that two kinetically controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals

ZIKV Demonstrates Minimal Pathologic Effects and Mosquito Infectivity in Viremic Cynomolgus Macaques

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3⁻4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed

Differential Vector Competency of Aedes albopictus Populations from the Americas for Zika Virus

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-07-27T16:26:27Z No. of bitstreams: 1 Azar SR Differential Vector Competency of Aedes ......pdf: 606491 bytes, checksum: 1e91eba1233227a8a7f24b30b5b0e97c (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-07-27T16:55:28Z (GMT) No. of bitstreams: 1 Azar SR Differential Vector Competency of Aedes ......pdf: 606491 bytes, checksum: 1e91eba1233227a8a7f24b30b5b0e97c (MD5)Made available in DSpace on 2018-07-27T16:55:28Z (GMT). No. of bitstreams: 1 Azar SR Differential Vector Competency of Aedes ......pdf: 606491 bytes, checksum: 1e91eba1233227a8a7f24b30b5b0e97c (MD5) Previous issue date: 2017Institute for Human Infections and Immunity. NIH grants R24AI120942 and R01AI121452 (SCW), 1U01AI115577 (NV) and 1R15AI113628-01 KAH), and grants from Brazilian National Council of Technological and Scientific Development (440891/2016-7 and 400830/2013-2) and the Coordination for the Improvement of Higher Education (440891/2016-7) (GSR).University of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, Texas / University of Texas Medical Branch. Department of Microbiology and Immunology. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, Texas / University of Texas Medical Branch. Department of Microbiology and Immunology. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TexasInstituto Nacional de Salud Pública. Centro Regional de Salud Pública. Tapachula, Chiapas, MéxicoUniversity of Texas Rio Grande Valley. Edinburg, TexasFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Instituto de Saúde Coletiva. Salvador, BA, BrasilHarris County Public Health. Mosquito and Vector Control Division. Houston, TexasHarris County Public Health. Mosquito and Vector Control Division. Houston, TexasHarris County Public Health. Mosquito and Vector Control Division. Houston, TexasHarris County Public Health. Mosquito and Vector Control Division. Houston, TexasEmory University. Graduate Division of Biological and Biomedical Sciences. Department of Environmental Sciences. Population Biology, Ecology, and Evolution Graduate Program. Atlanta, GeorgiaFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Instituto de Saúde Coletiva. Salvador, BA, BrasilNew Mexico State University. Department of Biology. Las Cruces, New MexicoUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, TexasUniversity of Texas Medical Branch. Institute for Human Infections and Immunity. Department of Pathology. Galveston, Texas / University of Texas Medical Branch. Center for Tropical Diseases. Galveston, Texas / University of Texas Medical Branch. Department of Microbiology and Immunology. Galveston, TexasTo evaluate the potential role of Aedes albopictus (Skuse) as a vector of Zika virus (ZIKV), colonized mosquitoes of low generation number (≤ F5) from Brazil, Houston, and the Rio Grande Valley of Texas engorged on viremic mice infected with ZIKV strains originating from Senegal, Cambodia, Mexico, Brazil, or Puerto Rico. Vector competence was established by monitoring infection, dissemination, and transmission potential after 3, 7, and 14 days of extrinsic incubation. Positive saliva samples were assayed for infectious titer. Although all three mosquito populations were susceptible to all ZIKV strains, rates of infection, dissemination, and transmission differed among mosquito and virus strains. Aedes albopictus from Salvador, Brazil, were the least efficient vectors, demonstrating susceptibility to infection to two American strains of ZIKV but failing to shed virus in saliva. Mosquitoes from the Rio Grande Valley were the most efficient vectors and were capable of shedding all three tested ZIKV strains into saliva after 14 days of extrinsic incubation. In particular, ZIKV strain DakAR 41525 (Senegal 1954) was significantly more efficient at dissemination and saliva deposition than the others tested in Rio Grande mosquitoes. Overall, our data indicate that, while Ae. albopictus is capable of transmitting ZIKV, its competence is potentially dependent on geographic origin of both the mosquito population and the viral strain

Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States

Zika virus has recently spread throughout the Americas. Although Aedes aegypti mosquitoes are considered the primary vector, Culex quinquefasciatus and mosquitoes of other species may also be vectors. We tested Cx. quinquefasciatus and Ae. taeniorhynchus mosquitoes from the US Gulf Coast; both were refractory to infection and incapable of transmission

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

Summary: Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV) causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccin

CRVP379 tandem affinity purification assay results.

<p>CRVP379 tandem affinity purification assay results.</p

DENV infection optimally enhances CRVP379 expression.

<p>Aag2 cells were transfected with an insect expression vector encoding CRVP379 (AcCRVP379) or GFP (AcGFP) and A. CRVP379 expression was measured by qRT-PCR at 48 h post-transfection. B. Cells were infected with DENV (MOI of 1.0) at 48 h post-transfection and infection levels were measure by qRT-PCR at 24 hpi.</p