Zoonotic barriers: defining species-specific porcine restriction factors against coronaviruses

Abstract

The pandemic nature of coronaviruses (CoVs) is underpinned by their ability to mutate and overcome barriers caused by evolutionary differences between species and establish disease in new hosts. One such barrier is the interferon (IFN) response. Systematic analysis using arrayed human IFN-stimulated gene (ISG) expression libraries has been effective at identifying antiviral factors. By comparing antiviral activity against viruses adapted to different species, species-specific restriction factors have been identified that act as barriers against zoonosis. However, most of these studies were focused on humans and libraries for other species are lacking.

Pigs are important livestock species for public health as they are hosts of influenza virus and at least six CoVs. We have generated an arrayed lentiviral-based porcine Type-I ISG expression library comprising 432 ISGs to enable cross-species screening. We hypothesized that a pig endemic virus would be more adept at evading the IFN response of the natural host, compared to a virus, such as SARS-CoV-2, that has not adapted to pigs. Indeed, our results indicate that porcine respiratory coronavirus (PRCV), a highly adapted endemic CoV in pigs that causes pneumonia resembling SARS in humans, was less sensitive than SARS-CoV-2 to porcine IFN. This suggests species-specific restriction factors have greater antiviral effects against non-adapted viruses. To identify such factors, we have applied the library to PRCV and SARS-CoV-2. We have developed and optimised resources to carry out such screening experiments in porcine cell lines, as existing protocols for human cell lines were not applicable. Using an RT-qPCR-based read-out, we have identified and validated two novel porcine ISGs, ZCWPW1 and PHACTR1, which potently restrict PRCV and SARS-CoV-2 replication. We also showed that the ISG, USF1, restricts SARS-CoV-2 to a greater extent than PRCV.

In addition to the IFN response, receptor compatibility could be a major determinant in the lack of susceptibility of pigs to SARS-CoV-2 in vivo. Human ACE2 is the entry receptor for SARS-CoV-2. We have developed a human ACE2 transgenic pig model highly susceptible to SARS-CoV-2, displaying clinical signs, disease progression, and lung inflammation that faithfully replicates COVID-19 in humans. This large animal model could serve as a unique tool in addition to non-human primates and rodents for testing mechanisms of disease and developing improved vaccines and therapeutics against COVID-19.