Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection.

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV.

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13.

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host­virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13­treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.