The Role of Interferon Antagonist, Non-Structural Proteins in the Pathogenesis and Emergence of Arboviruses

A myriad of factors favor the emergence and re-emergence of arthropod-borne viruses (arboviruses), including migration, climate change, intensified livestock production, an increasing volume of international trade and transportation, and changes to ecosystems (e.g., deforestation and loss of biodiversity). Consequently, arboviruses are distributed worldwide and represent over 30% of all emerging infectious diseases identified in the past decade. Although some arboviral infections go undetected or are associated with mild, flu-like symptoms, many are important human and veterinary pathogens causing serious illnesses such as arthritis, gastroenteritis, encephalitis and hemorrhagic fever and devastating economic loss as a consequence of lost productivity and high mortality rates among livestock. One of the most consistent molecular features of emerging arboviruses, in addition to their near exclusive use of RNA genomes, is the inclusion of viral, non-structural proteins that act as interferon antagonists. In this review, we describe these interferon antagonists and common strategies that arboviruses use to counter the host innate immune response. In addition, we discuss the complex interplay between host factors and viral determinants that are associated with virus emergence and re-emergence, and identify potential targets for vaccine and anti-viral therapies

Defining early events in La Crosse virus infection: Virus entry and fusion in the neuropathogenesis of La Crosse virus encephalitis and as potential targets for therapeutic intervention

La Crosse virus (LACV) is a major cause of pediatric encephalitis and aseptic meningitis in the Midwestern and Southern United States where it is an emerging pathogen. No specific therapies or vaccines are available for LACV or most other bunyaviruses. Inhibition of the earliest events in LACV infection, including entry and fusion, presents a target for therapeutic intervention. Here, we determined that clathrin-mediated endocytosis (CME) is the primary mechanism of LACV entry and identified key cellular factors in this process. We confirmed the functional requirement of CME for LACV entry using independent assays and, importantly, extended these finding to primary neuronal cultures. Moreover, we determined that LACV entry is dependent upon trafficking into early endosomes. Importantly, these findings will aid in the development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system (CNS). The LACV glycoproteins (Gn and Gc) are the viral attachment proteins and the primary determinants of neuroinvasion. We recently identified and experimentally confirmed the location of the LACV fusion peptide within Gc. Using a novel reverse-genetics system, we generated a panel of recombinant LACVs (rLACVs) containing mutations in the fusion peptide. These rLACVs retained their ability to cause neuronal loss in vitro despite decreased replication and fusion phenotypes, suggesting that the LACV fusion peptide is associated with properties of neuroinvasion, but not with neurovirulence. Subsequently, we used these rLACVs in our age-dependent murine model of LACV encephalitis to determine the neuroinvasive and/or neuropathogenic properties of the fusion peptide. When inoculated into the CNS of adult mice, rLACV fusion peptide mutants were as neurovirulent as wild-type rLACV. In contrast, the fusion peptide mutant rLACVs were less neuroinvasive when suckling or weanling mice were inoculated peripherally demonstrating that the LACV fusion peptide is associated with neuroinvasion, but not neurovirulence. Furthermore, we found that fusion peptide mutant rLACVs elicit a robust immune response in weanling mice that protects from subsequent WT-LACV challenge. Importantly, the high degree of conservation of the fusion peptide makes these findings applicable to other bunyaviruses, especially those that cause neurologic disease

The Role of Interferon Antagonist, Non-Structural Proteins in the Pathogenesis and Emergence of Arboviruses

A myriad of factors favor the emergence and re-emergence of arthropod-borne viruses (arboviruses), including migration, climate change, intensified livestock production, an increasing volume of international trade and transportation, and changes to ecosystems (e.g., deforestation and loss of biodiversity). Consequently, arboviruses are distributed worldwide and represent over 30% of all emerging infectious diseases identified in the past decade. Although some arboviral infections go undetected or are associated with mild, flu-like symptoms, many are important human and veterinary pathogens causing serious illnesses such as arthritis, gastroenteritis, encephalitis and hemorrhagic fever and devastating economic loss as a consequence of lost productivity and high mortality rates among livestock. One of the most consistent molecular features of emerging arboviruses, in addition to their near exclusive use of RNA genomes, is the inclusion of viral, non-structural proteins that act as interferon antagonists. In this review, we describe these interferon antagonists and common strategies that arboviruses use to counter the host innate immune response. In addition, we discuss the complex interplay between host factors and viral determinants that are associated with virus emergence and re-emergence, and identify potential targets for vaccine and anti-viral therapies

Defining early events in La Crosse virus infection: Virus entry and fusion in the neuropathogenesis of La Crosse virus encephalitis and as potential targets for therapeutic intervention

La Crosse virus (LACV) is a major cause of pediatric encephalitis and aseptic meningitis in the Midwestern and Southern United States where it is an emerging pathogen. No specific therapies or vaccines are available for LACV or most other bunyaviruses. Inhibition of the earliest events in LACV infection, including entry and fusion, presents a target for therapeutic intervention. Here, we determined that clathrin-mediated endocytosis (CME) is the primary mechanism of LACV entry and identified key cellular factors in this process. We confirmed the functional requirement of CME for LACV entry using independent assays and, importantly, extended these finding to primary neuronal cultures. Moreover, we determined that LACV entry is dependent upon trafficking into early endosomes. Importantly, these findings will aid in the development of antivirals and therapeutics that may be useful in the treatment of LACV and, more broadly, arboviral infections of the central nervous system (CNS). The LACV glycoproteins (Gn and Gc) are the viral attachment proteins and the primary determinants of neuroinvasion. We recently identified and experimentally confirmed the location of the LACV fusion peptide within Gc. Using a novel reverse-genetics system, we generated a panel of recombinant LACVs (rLACVs) containing mutations in the fusion peptide. These rLACVs retained their ability to cause neuronal loss in vitro despite decreased replication and fusion phenotypes, suggesting that the LACV fusion peptide is associated with properties of neuroinvasion, but not with neurovirulence. Subsequently, we used these rLACVs in our age-dependent murine model of LACV encephalitis to determine the neuroinvasive and/or neuropathogenic properties of the fusion peptide. When inoculated into the CNS of adult mice, rLACV fusion peptide mutants were as neurovirulent as wild-type rLACV. In contrast, the fusion peptide mutant rLACVs were less neuroinvasive when suckling or weanling mice were inoculated peripherally demonstrating that the LACV fusion peptide is associated with neuroinvasion, but not neurovirulence. Furthermore, we found that fusion peptide mutant rLACVs elicit a robust immune response in weanling mice that protects from subsequent WT-LACV challenge. Importantly, the high degree of conservation of the fusion peptide makes these findings applicable to other bunyaviruses, especially those that cause neurologic disease

Characterization of Brain Inflammation, Apoptosis, Hypoxia, Blood-Brain Barrier Integrity and Metabolism in Venezuelan Equine Encephalitis Virus (VEEV TC-83) Exposed Mice by In Vivo Positron Emission Tomography Imaging

Traditional pathogenesis studies of alphaviruses involves monitoring survival, viremia, and pathogen dissemination via serial necropsies; however, molecular imaging shifts this paradigm and provides a dynamic assessment of pathogen infection. Positron emission tomography (PET) with PET tracers targeted to study neuroinflammation (N,N-diethyl-2-[4-phenyl]-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide, [18F]DPA-714), apoptosis (caspase-3 substrate, [18F]CP-18), hypoxia (fluormisonidazole, [18F]FMISO), blood–brain barrier (BBB) integrity ([18F]albumin), and metabolism (fluorodeoxyglucose, [18F]FDG) was performed on C3H/HeN mice infected intranasally with 7000 plaque-forming units (PFU) of Venezuelan equine encephalitis virus (VEEV) TC-83. The main findings are as follows: (1) whole-brain [18F]DPA-714 and [18F]CP-18 uptake increased three-fold demonstrating, neuroinflammation and apoptosis, respectively; (2) [18F]albumin uptake increased by 25% across the brain demonstrating an altered BBB; (3) [18F]FMISO uptake increased by 50% across the whole brain indicating hypoxic regions; (4) whole-brain [18F]FDG uptake was unaffected; (5) [18F]DPA-714 uptake in (a) cortex, thalamus, striatum, hypothalamus, and hippocampus increased through day seven and decreased by day 10 post exposure, (b) olfactory bulb increased at day three, peaked day seven, and decreased day 10, and (c) brain stem and cerebellum increased through day 10. In conclusion, intranasal exposure of C3H/HeN mice to VEEV TC-83 results in both time-dependent and regional increases in brain inflammation, apoptosis, and hypoxia, as well as modest decreases in BBB integrity; however, it has no effect on brain glucose metabolism

Human angiotensin-converting enzyme 2 transgenic mice infected with SARS-CoV-2 develop severe and fatal respiratory disease

The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs