Elucidating the role of dsRNA sensing and Toll6 in antiviral responses of Culex quinquefasciatus cells.

The first step of any immune response is the recognition of foreign molecular structures inside the host organism. An important molecule that is generally foreign to eukaryotic cells is long double-stranded RNA (dsRNA), which can be generated during virus replication. The mechanisms of sensing viral dsRNA are well-studied in mammalian systems but are only poorly understood in insects, including disease vectors such as Culex quinquefasciatus mosquitoes. These mosquitoes are vectors for important arboviruses, such as West Nile virus, and Culex species mosquitoes are distributed across the globe in many temperate and tropical regions. The major antiviral response triggered by dsRNA in mosquitoes is RNA interference - a sequence-specific response which targets complementary viral RNA for degradation. However, here, we aimed to identify whether sequence-independent dsRNA sensing, mimicked by poly(I:C), can elicit an antiviral response. We observed a significant reduction in replication of La Crosse virus (LACV) in Cx. quinquefasciatus mosquito cells following poly(I:C) priming. We identified a number of antimicrobial peptides and Toll receptors that were upregulated at the transcript level by poly(I:C) stimulation. Notably, Toll6 was upregulated and we determined that a knockdown of Toll6 expression resulted also in increased LACV replication. Future efforts require genetic tools to validate whether the observed Toll6 antiviral activity is indeed linked to dsRNA sensing. However, large-scale functional genomic and proteomic approaches are also required to determine which downstream responses are part of the poly(I:C) elicited antiviral response.

Recognition of Arboviruses by the Mosquito Immune System.

Arthropod-borne viruses (arboviruses) pose a significant threat to both human and animal health worldwide. These viruses are transmitted through the bites of mosquitoes, ticks, sandflies, or biting midges to humans or animals. In humans, arbovirus infection often results in mild flu-like symptoms, but severe disease and death also occur. There are few vaccines available, so control efforts focus on the mosquito population and virus transmission control. One area of research that may enable the development of new strategies to control arbovirus transmission is the field of vector immunology. Arthropod vectors, such as mosquitoes, have coevolved with arboviruses, resulting in a balance of virus replication and vector immune responses. If this balance were disrupted, virus transmission would likely be reduced, either through reduced replication, or even through enhanced replication, resulting in mosquito mortality. The first step in mounting any immune response is to recognize the presence of an invading pathogen. Recent research advances have been made to tease apart the mechanisms of arbovirus detection by mosquitoes. Here, we summarize what is known about arbovirus recognition by the mosquito immune system, try to generate a comprehensive picture, and highlight where there are still gaps in our current understanding.

Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector, Culex quinquefasciatus.

Culex quinquefasciatus mosquitoes are a globally widespread vector of multiple human and animal pathogens, including West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. Since the introduction of West Nile virus to the United States in 1999, a cumulative 52,532 cases have been reported to the CDC, including 25,849 (49.2%) neuroinvasive cases and 2456 (5%) deaths. Viral infections elicit immune responses in their mosquito vectors, including the RNA interference (RNAi) pathway considered to be the cornerstone antiviral response in insects. To investigate mosquito host genes involved in pathogen interactions, CRISPR/Cas9-mediated gene-editing can be used for functional studies of mosquito-derived cell lines. Yet, the tools available for the study of Cx. quinquefasciatus-derived (Hsu) cell lines remain largely underdeveloped compared to other mosquito species. In this study, we constructed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Hsu cell cultures. By comparing it to the original Drosophila melanogaster CRISPR/Cas9 plasmid, we showed that the Culex-optimized plasmid demonstrated highly efficient editing of the genomic loci of the RNAi proteins Dicer-2 and PIWI4 in Hsu cells. These new tools support our ability to investigate gene targets involved in mosquito antiviral response, and thus the future development of gene-based vector control strategies.

Investigating Transfusion-related Sepsis Using Culture-Independent Metagenomic Sequencing.

BACKGROUND: Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration. METHODS: In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events. RESULTS: mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations. CONCLUSIONS: Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.