Animal models for COVID-19: advances, gaps and perspectives.

COVID-19, caused by SARS-CoV-2, is the most consequential pandemic of this century. Since the outbreak in late 2019, animal models have been playing crucial roles in aiding the rapid development of vaccines/drugs for prevention and therapy, as well as understanding the pathogenesis of SARS-CoV-2 infection and immune responses of hosts. However, the current animal models have some deficits and there is an urgent need for novel models to evaluate the virulence of variants of concerns (VOC), antibody-dependent enhancement (ADE), and various comorbidities of COVID-19. This review summarizes the clinical features of COVID-19 in different populations, and the characteristics of the major animal models of SARS-CoV-2, including those naturally susceptible animals, such as non-human primates, Syrian hamster, ferret, minks, poultry, livestock, and mouse models sensitized by genetically modified, AAV/adenoviral transduced, mouse-adapted strain of SARS-CoV-2, and by engraftment of human tissues or cells. Since understanding the host receptors and proteases is essential for designing advanced genetically modified animal models, successful studies on receptors and proteases are also reviewed. Several improved alternatives for future mouse models are proposed, including the reselection of alternative receptor genes or multiple gene combinations, the use of transgenic or knock-in method, and different strains for establishing the next generation of genetically modified mice.

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures (preprint)

Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek M). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

Clinical characteristics of COVID-19 and establishment of a disease risk prediction model (preprint)

Background: COVID-19 is spreading worldwide. No specific medicine has been used for the treatment of coronavirus infections. The aim of this study is to establish a new risk predictive model to screen potential critical patients for early intervention.Methods: In this study, Clinical characteristics were collected and analyzed from 317 confirmed cases of COVID-19. A total of 175 of the 317 cases with detailed examination results were included to establish models for predicting the risk of disease progression. Major independent risk factors were incorporated into MuLBSTA model to establish new models for predicting critical risk. We further tracked 25 mild or moderate patients with COVID-19 to research dynamic changes of the major independent risk factors in COVID-19 progression.Results: The average age of all of the 317 patients was 47.76 (SD 17.22). A total of 48 (15.14%) were diagnosed with mild disease with a median age of 34(39.29±13.04), 116(36.59%) were diagnosed with moderate disease with a median age of 34(38.78±12.32), 38(11.99%) were diagnosed as severe with a median age of 56(58.24±15.12), and 115(36.28) were diagnosed as critical with a median age of 59(56.89+17.09). The most common symptom at onset of illness were fever(211[66.56%] patients). Age>50, CK>64, CD4≤461, and CD8≤241 were predicted to be major independent risk factors that could promote COVID-19 progression. Compared with the MuLBSTA model, the predictive ability of the CD4-CD8-MuLBSTA model and the CD4-MuLBSTA model were improved by 11.87% and 11.79%, respectively. In the prospective study, CK value began to show significant differences from day13. The average CD4 in Severe Group began to decline significantly on the fourth day, and the CD8 maintained a relatively low level in the Severe Group after day13.Conclusions: Severe COVID-19 patients were significantly older than non-severe patients. Immune systems of severe COVID-19 patients were significantly suppressed, and advanced age(>50 years), low levels of CD4(≤461) or CD8(≤241) was important clinical manifestations of rapid deterioration. CK values in severe COVID-19 patients were significantly higher than in no severe patients. CD4 and CD8 were incorporated into the MuLBSTA to establish a new model, which is an ideal risk prediction model for COVID-19 patients.

Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2 (preprint)

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of coronavirus SARS-CoV-2. Existing direct-acting antiviral (DAA) drugs cannot be applied immediately to new viruses because of virus-specificity, and the development of new DAA drugs from the beginning is not timely for outbreaks. Thus, host-targeting antiviral (HTA) drugs have many advantages to fight against a broad spectrum of viruses, by blocking the viral replication and overcoming the potential viral mutagenesis simultaneously. Herein, we identified two potent inhibitors of DHODH, S312 and S416, with favorable drug-like and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus (H1N1, H3N2, H9N2), Zika virus, Ebola virus, and particularly against the recent novel coronavirus SARS-CoV-2. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knocking-out cells. We also proposed the drug combination of DAA and HTA was a promising strategy for anti-virus treatment and proved that S312 showed more advantageous than Oseltamivir to treat advanced influenza diseases in severely infected animals. Notably, S416 is reported to be the most potent inhibitor with an EC50 of 17nM and SI value >5882 in SARS-CoV-2-infected cells so far. This work demonstrates that both our self-designed candidates and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-repression may have clinical potentials not only to influenza but also to COVID-19 circulating worldwide, no matter such viruses mutate or not.