Biology subjects

Barkhymer, A.

Publications and source records attributed to Barkhymer, A..

3 recordsLinked to original sources

bioRxiv2026

A chicken interferon stimulated gene screen reveals limited genes restricting influenza replication

Avian influenza A virus (IAV) is a major global health threat. Not only does it cause significant economic losses to the agricultural industry, but it also presents a risk of spillover into the human population. However, several obstacles must be overcome for a successful cross-species transmission event to occur. One of these obstacles is the innate immunity of the host. While the innate immune response to IAV in humans has been well-studied, the innate immune response to IAV in birds is not as well understood. Here, we explore the interferon-induced immune response by identifying interferon-stimulated genes (ISGs) in chicken, quail, and duck cells, and find that they are largely species-specific. Using this data, we performed a CRISPR knockout ISG screen against two strains of IAV, the mouse-adapted PR8, and the low pathogenicity avian IAV isolate WF10. We find that for PR8, IFITM3 is the dominant restrictor of viral replication with several additional hits with modest effects, consistent with previous work in mammals. For avian WF10, IFITM3 was also a hit however there were two additional hits absent for PR8, the HSP70 co-chaperone protein BAG5 and deubiquitinase OTUD4. Overall, these findings confirm the divergence of interferon-mediated immunity between species and establish a screening system that can be used to identify ISGs important for viral restriction in avian species. IMPORTANCEWild birds, especially waterfowl and shore birds, are the primary natural reservoir for influenza A viruses. These birds can transmit IAV to domestic poultry, where the spillover risk to humans increases. Chickens are the most common species of domestic poultry world-wide and are frequently infected with avian influenza viruses. Despite this, relatively little is known about how their immune system responds to influenza infection. This lack of knowledge creates a barrier to finding ways of preventing or mitigating IAV infection in chicken flocks. In this study, we characterized the interferon-induced immune response to IAV in chickens and found that only a small subset of ISGs were responsible for restricting IAV in chicken cells.

microbiology↗

bioRxiv2022

Primary infection with Zika virus provides one-way heterologous protection against Spondweni virus infection in rhesus macaques

Spondweni virus (SPONV) is the closest known relative of Zika virus (ZIKV). SPONV pathogenesis resembles that of ZIKV in pregnant mice, and both viruses are transmitted by Aedes aegypti mosquitoes. We aimed to develop a translational model to further understand SPONV transmission and pathogenesis. We found that cynomolgus macaques (Macaca fascicularis) inoculated with ZIKV or SPONV were susceptible to ZIKV, but resistant to SPONV infection. In contrast, rhesus macaques (Macaca mulatta) supported productive infection with both ZIKV and SPONV and developed robust neutralizing antibody responses. Crossover serial challenge in rhesus macaques revealed that SPONV immunity did not protect against ZIKV infection, whereas ZIKV immunity was fully protective against SPONV infection. These findings establish a viable model for future investigation into SPONV pathogenesis, and suggest the risk of SPONV emergence is low in areas with high ZIKV seroprevalence due to one-way cross-protection between ZIKV and SPONV. TeaserIdentification of asymmetric immune interactions between Zika and Spondweni viruses in macaque monkeys.

microbiology↗

bioRxiv2021

Highly efficient SARS-CoV-2 infection of human cardiomyocytes: spike protein-mediated cell fusion and its inhibition

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to a bystander effect: the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) that beat spontaneously. These cardiomyocytes express the angiotensin I converting-enzyme 2 (ACE2) receptor and a subset of the proteases that mediate spike protein cleavage in the lungs, but not transmembrane protease serine 2 (TMPRSS2). Nevertheless, SARS-CoV-2 infection was productive: viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CM, smooth walled exocytic vesicles contained numerous 65-90 nm particles with typical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand the mechanisms of SARS-CoV-2 spread in hiPSC-CM we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm signal from cell to cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin abolished cell fusion. A spike mutant with the single amino acid change R682S that inactivates the furin cleavage site was fusion inactive. Thus, SARS-CoV-2 can replicate efficiently in hiPSC-CM and furin activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform provides an opportunity for target-based drug discovery in cardiac COVID-19. Author SummaryIt is unclear whether the cardiac complications frequently observed in COVID-19 patients are due exclusively to systemic inflammation and thrombosis. Viral replication has occasionally been confirmed in cardiac tissue, but rigorous analyses are restricted to rare autopsy materials. Moreover, there are few animal models to study cardiovascular complications of coronavirus infections. To overcome these limitations, we developed an in vitro model of SARS-CoV-2 spread in induced pluripotent stem cell-derived cardiomyocytes. In these cells, infection is highly productive: viral transcription levels exceed those documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread we expressed a fluorescent version of its spike protein that allowed to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin cleavage site lost cytopathic function. The spike protein of the Middle East Respiratory Syndrome (MERS) coronavirus drove cardiomyocyte fusion with slow kinetics, whereas the spike proteins of SARS-CoV and the respiratory coronavirus 229E were inactive. These fusion activities correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 has the potential to cause cardiac damage by fusing cardiomyocytes.

microbiology↗