Mammarenaviruses are endemic in rodent populations worldwide and their zoonotic transmission can lead to a severe life-threatening hemorrhagic fever (HF) syndrome. In the Americas, five New World mammarenaviruses (NWAs) are known to cause HF. In the absence of FDA-licensed vaccines or antiviral therapies, these viruses pose a significant public health risk and a threat to national security. Research to understand NWA pathogenesis and develop effective countermeasures is severely limited by the lack of small-animal models that faithfully mirror human disease. We recently demonstrated that transgenic expression of human transferrin receptor 1 (huTfR1), the known cellular receptor used by the pathogenic NWAs, confers susceptibility in mice to lethal disease following challenge with the Junin arenavirus (JUNV), the agent of Argentine HF. However, the mice do not manifest signs of hemorrhagic disease that are prominent features of severe cases of NWA HF in humans. We and others have shown that golden Syrian hamsters infected with related nonpathogenic NWAs that do not use huTfR1 develop a severe HF-like syndrome with many of the cardinal features of hemorrhagic disease, including coagulopathy, extensive petechia, bleeding from the oronasal mucosal region and vascular leak. Moreover, we recently showed that expression of huTfR1 in hamster cell lines significantly augments JUNV infection. Thus, we hypothesize that hamsters engineered to express huTfR1 will be susceptible to JUNV infection, resulting in a HF-like syndrome more representative of human disease. To explore this hypothesis, we will pursue the following Specific Aims:
Aim 1. Develop a huTfR1 knock-in (KI) golden Syrian hamster line. We will design and test single guide (sg)RNAs targeting the 3? end of the hamster TfR1 gene for insertion of the huTfR1 open reading frame. By appending the huTfR1 cDNA via a T2A peptide linker immediately before the stop codon of the hamster TfR1 gene, we will ensure expression of huTfR1 at physiological levels and with normal tissue distribution. The huTfR1 hamster line will be generated by a well-established pronuclear injection procedure with the Cas9/sgRNA ribonucleoprotein complex and the huTfR1 cDNA donor construct.
Aim 2. Evaluate the susceptibility of the huTfR1 hamster to JUNV infection. Wild-type and huTfR1 KI hamsters will be challenged with JUNV to assess differences in viral replication and development of disease. We anticipate that expression of huTfR1 will boost viral replication leading to a HF-like syndrome in the hamsters. Disease parameters evaluated daily will include body weight, temperature and clinical disease signs including petechia and mucosal bleeding. Viral loads will be determined from blood samples taken one week after JUNV challenge, a point at which huTfR1 hamsters are expected to develop signs of disease and have measurable viremia. The ultimate goal of this project is to produce a novel hamster model of NWA HF to support development of host-directed therapies that would complement direct-acting antivirals to improve outcomes in patients suffering from severe disease caused by pathogenic NWAs.
Five New World mammarenaviruses (NWAs) cause a severe life-threatening hemorrhagic fever syndrome and pose a significant public health risk and a threat to national security. Research to understand NWA pathogenesis and develop effective countermeasures is severely limited by the lack of small-animal models that faithfully mirror human disease. The goal of this project is to produce a novel hamster model of NWA hemorrhagic fever to support the development of promising host-directed therapies that would complement direct-acting antivirals to improve outcomes in patients suffering from severe disease caused by pathogenic NWAs.
