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Extraction of Hemocytes from <em>Drosophila melanogaster</em> Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

Washington State University

Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-β secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection.

Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors.

Innate immunity refers to the body's initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, autoimmune disease may result. The innate immune response during pathogenic infection is initiated through the activation of receptors recognizing conserved molecular patterns, such as nucleic acids from a virus' genome or replicative cycle. Additionally, the host's own nucleic acids are capable of activating an immune response. Therefore, it follows that the nucleic acid-sensing pathways must be tightly controlled to avoid an autoimmune response from recognition of self, yet still be unimpeded to respond to viral infections. In this review, we will describe the nucleic acid sensing pathways and how they respond to virus infection. Moreover, we will discuss autoimmune diseases that develop when these pathways fail to signal properly and identify knowledge gaps that are prime for interrogation.

Nucleic acid sensing and innate immunity: signaling pathways controlling viral pathogenesis and autoimmunity.

 is the causative agent of Q fever, a zoonotic disease that threatens both human and animal health. Due to the paucity of experimental animal models, little is known about how host factors interface with bacterial components and affect pathogenesis. Here, we used , in conjunction with the biosafety level 2 (BSL2) Nine Mile phase II (NMII) clone 4 strain of , as a model to investigate host and bacterial components implicated in infection. We demonstrate that adult  flies are susceptible to  NMII infection and that this bacterial strain, which activates the immune deficiency (IMD) pathway, is able to replicate and cause mortality in the animals. We show that in the absence of Eiger, the only known tumor necrosis factor (TNF) superfamily homolog in , -infected flies exhibit reduced mortality from infection. We also demonstrate that the  type 4 secretion system (T4SS) is critical for the formation of the -containing vacuole and establishment of infection in  Altogether, our data reveal that the  TNF homolog Eiger and the  T4SS are implicated in the pathogenesis of  in flies. The /NMII model mimics relevant aspects of the infection in mammals, such as a critical role of host TNF and the bacterial T4SS in pathogenesis. Our work also demonstrates the usefulness of this BSL2 model to investigate both host and  components implicated in infection.

Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection.

Vector-borne diseases, including arboviruses, pose a serious threat to public health worldwide. Arboviruses of the flavivirus genus, such as Zika virus (ZIKV), dengue virus, yellow fever virus (YFV), and West Nile virus (WNV), are transmitted to humans from insect vectors and can cause serious disease. In 2017, over 2,000 reported cases of WNV virus infection occurred in the United States, with two-thirds of cases classified as neuroinvasive. WNV transmission cycles through two different animal populations: birds and mosquitoes. Mammals, particularly humans and horses, can become infected through mosquito bites and represent dead-end hosts of WNV infection. Because WNV can infect diverse species, research on this arbovirus has investigated the host response in mosquitoes, birds, humans, and horses. With the growing geographical range of the WNV mosquito vector and increased human exposure, improved surveillance and treatment of the infection will enhance public health in areas where WNV is endemic. In this review, we survey the bionomics of mosquito species involved in Nearctic WNV transmission. Subsequently, we describe the known immune response pathways that counter WNV infection in insects, birds, and mammals, as well as the mechanisms known to curb viral infection. Moreover, we discuss the bacterium  and its involvement in reducing flavivirus titer in insects. Finally, we highlight the similarities of the known immune pathways and identify potential targets for future studies aimed at improving antiviral therapeutic and vaccination design.

Alan G. Goodman

School of Molecular Biosciences,
College of Veterinary Medicine,
School of Molecular Biosciences, College of Veterinary Medicine

Washington State University

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Alan G. Goodman

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Washington State University

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

School of Molecular Biosciences,
College of Veterinary Medicine,
School of Molecular Biosciences, College of Veterinary Medicine