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Biochem Biophys Res Commun ; 553: 25-29, 2021 05 14.
Article in English | MEDLINE | ID: covidwho-1147359


The current COVID-19 pandemic is caused by infections with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A sex-bias has been observed, with increased susceptibility and mortality in male compared to female patients. The gene for the SARS-CoV-2 receptor ACE2 is located on the X chromosome. We previously generated TP53 mutant pigs that exhibit a sex-specific patho-phenotype due to altered regulation of numerous X chromosome genes. In this study, we explored the effect of p53 deficiency on ACE2 expression in pigs. First, we identified the p53 binding site in the ACE2 promoter and could show its regulatory effect on ACE2 expression by luciferase assay in porcine primary kidney fibroblast cells. Later, quantitative PCR and western blot showed tissue- and gender-specific expression changes of ACE2 and its truncated isoform in p53-deficient pigs. We believe these findings will broaden the knowledge on ACE2 regulation and COVID-19 susceptibility.

Angiotensin-Converting Enzyme 2/metabolism , Gene Expression Regulation , Organ Specificity , Sex Characteristics , Sus scrofa/metabolism , Tumor Suppressor Protein p53/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Animals , Base Sequence , Binding Sites , COVID-19/metabolism , COVID-19/virology , Disease Models, Animal , Female , Fibroblasts , Gene Deletion , Male , Promoter Regions, Genetic/genetics , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , X Chromosome/genetics
Elife ; 92020 09 02.
Article in English | MEDLINE | ID: covidwho-740561


Porcine reproductive and respiratory syndrome virus (PRRSV) and transmissible gastroenteritis virus (TGEV) are two highly infectious and lethal viruses causing major economic losses to pig production. Here, we report generation of double-gene-knockout (DKO) pigs harboring edited knockout alleles for known receptor proteins CD163 and pAPN and show that DKO pigs are completely resistant to genotype 2 PRRSV and TGEV. We found no differences in meat-production or reproductive-performance traits between wild-type and DKO pigs, but detected increased iron in DKO muscle. Additional infection challenge experiments showed that DKO pigs exhibited decreased susceptibility to porcine deltacoronavirus (PDCoV), thus offering unprecedented in vivo evidence of pAPN as one of PDCoV receptors. Beyond showing that multiple gene edits can be combined in a livestock animal to achieve simultaneous resistance to two major viruses, our study introduces a valuable model for investigating infection mechanisms of porcine pathogenic viruses that exploit pAPN or CD163 for entry.

Pig epidemics are the biggest threat to the pork industry. In 2019 alone, hundreds of billions of dollars worldwide were lost due to various pig diseases, many of them caused by viruses. The porcine reproductive and respiratory virus (PRRS virus for short), for instance, leads to reproductive disorders such as stillbirths and premature labor. Two coronaviruses ­ the transmissible gastroenteritis virus (or TGEV) and the porcine delta coronavirus ­ cause deadly diarrhea and could potentially cross over into humans. Unfortunately, there are still no safe and effective methods to prevent or control these pig illnesses, but growing disease-resistant pigs could reduce both financial and animal losses. Traditionally, breeding pigs to have a particular trait is a slow process that can take many years. But with gene editing technology, it is possible to change or remove specific genes in a single generation of animals. When viruses infect a host, they use certain proteins on the surface of the host's cells to find their inside: the PRRS virus relies a protein called CD163, and TGEV uses pAPN. Xu, Zhou, Mu et al. used gene editing technology to delete the genes that encode the CD163 and pAPN proteins in pigs. When the animals were infected with PRRS virus or TGEV, the non-edited pigs got sick but the gene-edited animals remained healthy. Unexpectedly, pigs without CD163 and pAPN also coped better with porcine delta coronavirus infections, suggesting that CD163 and pAPN may also help this coronavirus infect cells. Finally, the gene-edited pigs reproduced and produced meat as well as the control pigs. These experiments show that gene editing can be a powerful technology for producing animals with desirable traits. The gene-edited pigs also provide new knowledge about how porcine viruses infect pigs, and may offer a starting point to breed disease-resistant animals on a larger scale.

CD13 Antigens/deficiency , Coronavirus Infections/prevention & control , Coronavirus/pathogenicity , Gastroenteritis, Transmissible, of Swine/prevention & control , Porcine Reproductive and Respiratory Syndrome/prevention & control , Porcine respiratory and reproductive syndrome virus/pathogenicity , Receptors, Cell Surface/deficiency , Transmissible gastroenteritis virus/pathogenicity , Animals , Animals, Genetically Modified , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/genetics , Antigens, Differentiation, Myelomonocytic/immunology , Body Composition , CD13 Antigens/genetics , CD13 Antigens/immunology , Coronavirus/immunology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/virology , Disease Susceptibility , Gastroenteritis, Transmissible, of Swine/genetics , Gastroenteritis, Transmissible, of Swine/immunology , Gastroenteritis, Transmissible, of Swine/virology , Gene Knockdown Techniques , Host Microbial Interactions , Meat-Packing Industry , Phenotype , Porcine Reproductive and Respiratory Syndrome/genetics , Porcine Reproductive and Respiratory Syndrome/immunology , Porcine Reproductive and Respiratory Syndrome/virology , Porcine respiratory and reproductive syndrome virus/immunology , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Sus scrofa/genetics , Swine , Transmissible gastroenteritis virus/immunology , Weight Gain
J Virol ; 94(17)2020 08 17.
Article in English | MEDLINE | ID: covidwho-601769


Coronaviruses (CoVs) have repeatedly emerged from wildlife hosts and infected humans and livestock animals to cause epidemics with significant morbidity and mortality. CoVs infect various organs, including respiratory and enteric systems, as exemplified by newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The constellation of viral factors that contribute to developing enteric disease remains elusive. Here, we investigated CoV interferon antagonists for their contribution to enteric pathogenesis. Using an infectious clone of an enteric CoV, porcine epidemic diarrhea virus (icPEDV), we generated viruses with inactive versions of interferon antagonist nonstructural protein 1 (nsp1), nsp15, and nsp16 individually or combined into one virus designated icPEDV-mut4. Interferon-responsive PK1 cells were infected with these viruses and produced higher levels of interferon responses than were seen with wild-type icPEDV infection. icPEDV-mut4 elicited robust interferon responses and was severely impaired for replication in PK1 cells. To evaluate viral pathogenesis, piglets were infected with either icPEDV or icPEDV-mut4. While the icPEDV-infected piglets exhibited clinical disease, the icPEDV-mut4-infected piglets showed no clinical symptoms and exhibited normal intestinal pathology at day 2 postinfection. icPEDV-mut4 replicated in the intestinal tract, as revealed by detection of viral RNA in fecal swabs, with sequence analysis documenting genetic stability of the input strain. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV. These results identify nsp1, nsp15, and nsp16 as virulence factors that contribute to the development of PEDV-induced diarrhea in swine. Inactivation of these CoV interferon antagonists is a rational approach for generating candidate vaccines to prevent disease and spread of enteric CoVs, including SARS-CoV-2.IMPORTANCE Emerging coronaviruses, including SARS-CoV-2 and porcine CoVs, can infect enterocytes, cause diarrhea, and be shed in the feces. New approaches are needed to understand enteric pathogenesis and to develop vaccines and therapeutics to prevent the spread of these viruses. Here, we exploited a reverse genetic system for an enteric CoV, porcine epidemic diarrhea virus (PEDV), and outline an approach of genetically inactivating highly conserved viral factors known to limit the host innate immune response to infection. Our report reveals that generating PEDV with inactive versions of three viral interferon antagonists, nonstructural proteins 1, 15, and 16, results in a highly attenuated virus that does not cause diarrhea in animals and elicits a neutralizing antibody response in virus-infected animals. This strategy may be useful for generating live attenuated vaccine candidates that prevent disease and fecal spread of enteric CoVs, including SARS-CoV-2.

Coronavirus Infections/immunology , Coronavirus/immunology , Interferons/immunology , Porcine epidemic diarrhea virus/immunology , Vaccines, Attenuated/immunology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Betacoronavirus/immunology , COVID-19 , Chlorocebus aethiops , Coronavirus Infections/prevention & control , Diarrhea/pathology , Diarrhea/virology , Disease Models, Animal , Endoribonucleases/antagonists & inhibitors , Feces/virology , Ileum/pathology , Immunity, Innate , Jejunum/pathology , Pandemics , Pneumonia, Viral/immunology , Porcine epidemic diarrhea virus/genetics , RNA, Viral , RNA-Dependent RNA Polymerase , SARS-CoV-2 , Swine , Swine Diseases/virology , Vero Cells , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/immunology