Overexpression of wild-type HRAS drives non-alcoholic steatohepatitis to hepatocellular carcinoma in mice

Hepatocellular carcinoma(HCC),a prevalent solid carcinoma of significant concern,is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes.The etiology and pathological progression of non-alcoholic steatohepatitis(NASH)-related HCC is multifactorial and multistage.However,no single animal model can accurately mimic the full NASH-related HCC pathological progression,posing considerable challenges to transition and mechanistic studies.Herein,a novel conditional inducible wild-type human HRAS overexpressed mouse model(HRAS-HCC)was established,demonstrating 100%morbidity and mortality within approximately one month under normal dietary and lifestyle conditions.Advanced symptoms of HCC such as ascites,thrombus,internal hemorrhage,jaundice,and lung metastasis were successfully replicated in mice.In-depth pathological features of NASH-related HCC were demonstrated by pathological staining,biochemical analyses,and typical marker gene detections.Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival,further confirming the accuracy and reliability of the model.Based on protein-protein interaction(PPI)network and RNA sequencing analyses,we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis,with subsequent progression to HCC.Collectively,our study successfully duplicated natural sequential progression in a single murine model over a very short period,providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.

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),antibodydependent 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.

Histones released by NETosis enhance the infectivity of SARS-CoV-2 by bridging the spike protein subunit 2 and sialic acid on host cells

Neutrophil extracellular traps(NETs)can capture and kill viruses,such as influenza viruses,human immunodeficiency virus(HIV),and respiratory syncytial virus(RSV),thus contributing to host defense.Contrary to our expectation,we show here that the histones released by NETosis enhance the infectivity of SARS-CoV-2,as found by using live SARS-CoV-2 and two pseudovirus systems as well as a mouse model.The histone H3 or H4 selectively binds to subunit 2 of the spike(S)protein,as shown by a biochemical binding assay,surface plasmon resonance and binding energy calculation as well as the construction of a mutant S protein by replacing four acidic amino acids.Sialic acid on the host cell surface is the key molecule to which histones bridge subunit 2 of the S protein.Moreover,histones enhance cell-cell fusion.Finally,treatment with an inhibitor of NETosis,histone H3 or H4,or sialic acid notably affected the levels of sgRNA copies and the number of apoptotic cells in a mouse model.These findings suggest that SARS-CoV-2 could hijack histones from neutrophil NETosis to promote its host cell attachment and entry process and may be important in exploring pathogenesis and possible strategies to develop new effective therapies for COVID-19.

The Infection and Pathogenicity of SARS-CoV-2 Variant B.1.351 in hACE2 Mice

Dear Editor,Since the outbreak of COVID-19 at the end of 2019,its causative agent SARS-Co V-2 has been spreading around the world for one and half a year.During the long global circulation of SARS-Co V-2,mutations in the viral genome gradually emerged and accumulated。

A mouse model for SARS-CoV-2-induced acute respiratory distress syndrome

Dear Editor,The COVID-19 pandemic has covered more than 200 countries and regions around the world since its outbreak in January 2020.To date,the SARS-CoV-2 virus has caused>1.2 million deaths.The mortality rate of COVID-19 is closely concerned with the clinical symptoms of the patients from mild-to-severe disease.Notably,in its most severe form,COVID-19 leads to life-threatening pneumonia and acute respiratory distress syndrome(ARDS),which is mostly accom-panied by a hyperactive immune response called"cytokine storm"and has high death rates from 40 to 50%.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

To discover new drugs to combat COVID-19,an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed.Here,for the first time,we report the crucial role of cathepsin L(CTSL)in patients with COVID-19.The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity.Correspondingly,SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo,while CTSL overexpression,in turn,enhanced pseudovirus infection in human cells.CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry,as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo.Furthermore,amantadine,a licensed anti-influenza drug,significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo.Therefore,CTSL is a promising target for new anti-COVID-19 drug development.

Enhanced protective immunity against SARS-CoV-2 elicited by a VSV vector expressing a chimeric spike protein

SARS-CoV-2 and SARS-CoV are genetically related coronavirus and share the same cellular receptor ACE2.By replacing the VSV glycoprotein with the spikes(S)of SARS-CoV-2 and SARS-CoV,we generated two replication-competent recombinant viruses,rVSVSARS-CoV-2 and rVSV-SARS-CoV.Using wild-type and human ACE2(hACE2)knock-in mouse models,we found a single dose of rVSV-SARS-CoV could elicit strong humoral immune response via both intranasal(i.n.)and intramuscular(i.m.)routes.Despite the high genetic similarity between SARS-CoV-2 and SARS-CoV,no obvious cross-neutralizing activity was observed in the immunized mice sera.In macaques,neutralizing antibody(NAb)titers induced by one i.n.dose of rVSV-SARS-CoV-2 were eight-fold higher than those by a single i.m.dose.Thus,our data indicates that rVSV-SARS-CoV-2 might be suitable for i.n.administration instead of the traditional i.m.immunization in human.Because rVSV-SARS-CoV elicited significantly stronger NAb responses than rVSV-SARS-CoV2 in a route-independent manner,we generated a chimeric antigen by replacing the receptor binding domain(RBD)of SARS-CoV S with that from the SARS-CoV-2.rVSV expressing the chimera(rVSV-SARS-CoV/2-RBD)induced significantly increased NAbs against SARS-CoV-2 in mice and macaques than rVSV-SARS-CoV-2,with a safe Th1-biased response.Serum immunized with rVSV-SARS-CoV/2-RBD showed no cross-reactivity with SARS-CoV.hACE2 mice receiving a single i.m.dose of either rVSV-SARS-CoV-2 or rVSV-SARSCoV/2-RBD were fully protected against SARS-CoV-2 challenge without obvious lesions in the lungs.Our results suggest that transplantation of SARS-CoV-2 RBD into the S protein of SARS-CoV might be a promising antigen design for COVID-19 vaccines.