Mingxue Li,Yueying Yng,Yng Liu,Mengzhu Zheng,Dejun Sun ,Hu Li ,,Lixi Chen

a.Wuya College of Innovation,Key Laboratory of Structure-Based Drug Design &Discovery,Ministry of Education,Shenyang Pharmaceutical University,Shenyang 110016,China;b.Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,School of Pharmacy,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China

Abstract Cancer is a serious threat to human life and a big problem in clinical treatment.Some natural active substances extracted from Traditional Chinese Medicine can effectively inhibit the growth of cancer cells,which is a new direction for finding more effective anticancer drugs.Osthole is a natural coumarin compound extracted from Traditional Chinese Medicines such as Cnidium monnieri,Angelica pubescens and Peucedanum praeruptorum Dunn.It has significant inhibitory activity against a variety of cancers.This paper summarizes the anticancer effects and molecular mechanisms of osthole in the treatment of cancers in recent years in order to provide references for further research.

Key words: osthole;anticancer effects;molecular mechanisms

1 Introduction

In 2019,a novel coronavirus broke out and is still spreading,causing widespread concern.On January 12,2020,the World Health Organization(WHO) named the new coronavirus 2019-nCoV.On February 11,2020,the International Committee on Taxonomy of Viruses (ICTV) announced that the new coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARSCoV-2).WHO announced on the same day that the official name of the disease caused by this virus was coronavirus disease pneumonia 2019 (COVID-19).The emergence of SARS-CoV-2 poses a great threat to global health and the economy.As of May 16,2020,more than 4 million people worldwide had been infected with SARS-CoV-2 and more than 300,000 people had died of the infection.

Since the outbreak of the epidemic,many researchers have strengthened their efforts to tackle this problem and carried out many studies around SARS-CoV-2.Researchers carried out studies on the basic characteristics of SARS-CoV-2 pathogen,the symptoms of the infected person,the possible transmission route of the virus,the diagnosis,the occurrence of thrombocytopenia in patients with COVID-19,the latest COVID-19 patients,vaccine development,prognosis and prevention [1-11].Based on the current research progress and results of SARS-CoV-2,this review elaborates on its structure studies,natural and intermediate hosts,virus sources and pathogenesis.

2 Structure study

SARS-CoV-2 is a

β

-type coronavirus (a diameter of 60 to 140 nm) with an envelope and round or oval particles,usually polymorphic [12].The genetic characteristics of SARS-CoV-2 are significantly different from those of severe acute respiratory syndrome coronavirus (SARS-CoV)and Middle East respiratory syndrome coronavirus(MERS-CoV),but all three coronaviruses are positive-sense,single-stranded RNA viruses.Coronavirus genetic material is the largest among all RNA viruses,with a genome full length between 26 and 32 kb.Approximately 2/3 of the 5’ end of the viral genome is composed of overlapping open reading frames (ORFs),ORF1a and ORF1b,which are mainly responsible for encoding non-structural proteins such as enzymes related to virus replication and transcription.The next 1/3 genome encodes major structural proteins such as Spike protein (S),Envelope membrane protein (E),Membrane protein(M),and Nucleocapsid protein (N).Moreover,a series of genes nested in the 3’ end genome encode accessory proteins.Structures of several important proteins of SARS-COV-2 are summarized in Table 1.

The entry of coronavirus into the host cells is mediated by S protein,which contains two functional subunits,S1 and S2.S1 subunit is responsible for binding to host cell receptors and S2 subunit is responsible for viral and cell membrane fusion.McLellan’s team analyzed the structure of SARSCoV-2 S protein using a cryo-electron microscope with the resolution of 3.5 Å.Its base sequence is similar to that of SARS-CoV,but there are significant differences in spatial structure [13].The SARSCoV-2 S protein shares 98% sequence identity with the S protein of bat coronavirus RaTG13,and the difference between the two is that a segment of furin cleavage site is inserted between the SARS-CoV-2 S1/ S2 subunit,which is rare in coronaviruses,and is only arginine in SARS-CoV.Wu et al.has proved that furin is one of the important reasons for the high infectivity of SARS-CoV-2 and a potential therapeutic target of COVID-19 [14].Moreover,the study has also reported that the affinity of SARSCoV-2 S protein for ACE2 receptor is 10-20 times higher than that of SARS-CoV S protein,which provides an explanation for the strong spreading ability of SARS-CoV-2 in human [13].The structure of RBD of SARS-CoV-2 is highly similar to that of SARS-CoV,while the RBD antibody of SARSCoV has no obvious binding to SARS-CoV-2 [13].Therefore,there is no cross-immunity of SARS-CoV antibody to SARS-CoV-2 [13].This study analyzed the crystal structure of SARS-CoV-2 S protein at the near-atomic level,providing an important structural biological basis for vaccine design and the discovery of antiviral drugs.

Veesler’s team has reported that hACE2 can regulate SARS-CoV-2 S protein-mediated cell entry and is a functional receptor for this emerging coronavirus.In addition,the affinity of SARSCoV-2 for hACE2 recognition is similar to that of SARS-CoV.The tight integration with hACE2 can partially explain the efficient transmission of SARS-CoV-2 in humans,as is the case with SARSCoV.They identified a furin cleavage site at the border of the S1/ S2 subunit of SARS-CoV-2 S protein,which was cleaved during the biosynthesis of S protein.This new feature distinguished this coronavirus from SARS-CoV and SARSr-CoV.They have analyzed the structure of SARS-CoV-2 S protein extracellular domain trimer and found that SARS-CoV-2 S extracellular domain is a 160 Å long trimer with a triangular cross section,similar to SARS-CoV S structure.SARS-CoV-2 S trimer exists in many different conformational states due to the Sopening at the top of the trimer.Like other

β

-coronavirus S glycoproteins(including SARS-CoV S),S1 subunit has a V-shaped structure.Each promoter of SARS-CoV-2 S contains 22 N-linked glycosylation sequences,16 of which are resolved in the cryo-EM profile.In SARS-CoV-2 S protein and SARS-CoV S protein,9 out of the 13 glycans of S1 subunit and all 9 glycans of S2 subunit are conserved,and most of the S2 oligosaccharides are conserved among SARSr-COV S glycoproteins [15].The striking structural similarity and sequence conservation between SARS-CoV-2 S protein and SARS-CoV S protein highlight the close relationship between these two coronaviruses that enter target cells by recognizing hACE2.In this study,Veesler et al.found that the polyclonal antibody response elicited by SARS-CoV-2 S protein strongly neutralized S protein-mediated cell entry,further enhancing this similarity.This research provides a blueprint for the design of vaccines and viral invasion inhibitors.

The structure of SARS-CoV-2 S protein has been analyzed by cryo-electron microscopy,but in what state does ACE2 exist on the cell membrane?What are the similarities and differences between SARS-CoV-2 and SARS-CoV in binding to ACE2?Elucidating the full-length protein of ACE2 and its complex structure with the S protein will hopefully help answer these questions.Zhou’s team resolved the cryo-EM structure of the fulllength human ACE2-neutral amino acid transporter BAT1 complex at the resolution of 2.9 Å.Studies have shown that ACE2-BAT1 complex exists as a dimer of a heterodimer with open and closed conformations [16].The conversion of these two conformations is achieved by the rotation of the peptidase domains (PDs) of ACE2,and PDs are the direct binding sites of coronavirus S protein and the entrance of the virus to the human body.In both open and closed states,S protein is located on the lateral surface of ACE2 dimer [15].Dimeric ACE2 may contain two S protein trimers simultaneously.The results suggested the presence of potential aggregation between dimer ACE2 and trimer S proteins,which might be important for membrane entrapment and endocytosis of virus particles,a process similar to other receptor-mediated endocytosis.Researchers also identified seven glycosylated sites on the ACE2-BAT1 complex,three of which were located near the binding sites in the receptor binding domain [16].Glycosylation allows the virus to escape recognition and attack by the body’s immune cells.By comparing the interaction interface of SARS-CoV-2-RBD and SARS-CoV-RBD with ACE2,it was found that some changes might enhance the interaction between SARS-CoV-2-RBD and ACE2,while other changes might reduce the affinity of SARS-CoV-RBD and ACE2 [17].This research is an important progress in understanding how SARS-CoV-2 infects human cells.

By immunostaining and flow cytometry,Yan et al.first identified S1 CTD (SARS-CoV-2-CTD) as the key region of interaction with hACE2 receptor in SARS-CoV-2.Subsequently,the crystal structure of SARS-CoV-2-CTD combined with hACE2 was resolved.Through structural analysis,the receptor binding pattern of SARS-CoV-2-CTD and hACE2 was revealed.Compared with SARS-RBD,SARSCoV-2-CTD and hACE2 formed more atomic interactions and had higher receptor binding affinity.Notably,a group of monoclonal antibodies (mAbs)and mouse polyclonal anti-serum against SARS-S1/RBD cannot bind to SARS-CoV-2 S protein,indicating a significant difference in antigenicity between SARS-CoV and SARS-CoV-2 [18].In other words,the candidate vaccine developed previously based on SARS-RBD is unlikely to have any clinical benefit in the prevention of SARS-CoV-2.Taken together,these findings also shed light on the entry of the virus and its pathogenesis.

It is well known that the S protein on the surface of SARS-CoV-2 plays a key role when it invades human cells.The S protein binds to the receptor ACE2 on human cells through the receptor domain (RBD),which is the first step in initiating infection and an important target for neutralizing antibodies and vaccine development.Based on this key issue,Lan et al.expressed and purified the SARS-CoV-2 S protein RBD and human ACE2 receptor using the insect cell system,which successfully produced complex crystals.X-ray diffraction technology was used to obtain highresolution crystal structure data,which illustrated how SARS-CoV-2 RBD interacted with the receptor at the atomic level.The results showed that the ACE2 binding pattern of SARS-CoV-2 RBD was almost the same as that of SARS-CoV RBD.Through structural analysis,the residues in SARSCoV-2 RBD that are critical for ACE2 binding were identified,most of which were highly conserved or had side chain characteristics similar to those in SARS-CoV RBD.This structural and sequence similarity indicated convergent evolution between SARS-CoV-2 RBD and SARS-CoV RBD,thus enhancing the binding to ACE2,although SARSCoV-2 did not resemble SARS and SARS-related coronavirus in the evolutionary tree.The researchers also analyzed two SARS-CoV antibody epitopes targeting RBD using SARS-CoV-2 RBD,providing insight for further identification of cross-reactive antibodies [19].

Li et al.also determined the crystal structure of the composite of SARS-CoV-2 RBD and hACE2 based on X-ray diffraction data with the resolution of 2.68 Å.Compared with the amino acid sequence of SARS-CoV,two structural features of SARSCoV-2 and hACE2 with stronger binding ability were identified.On the one hand,SARS-CoV-2 shows a more compact conformation near hACE2 and is in closer contact with the receptor.On the other hand,several residue changes in SARS-CoV-2 RBD make the binding of RBD and hACE2 more stable [20].In summary,these structural data indicate that SARS-CoV-2 RBD is more capable of recognizing hACE2 than SARS-CoV RBD.This study provides guidance for the intervention strategy of SARS-CoV-2 targeted receptor recognition.

Mpro is essential for processing polyproteins translated from viral RNA,and inhibiting the activity of this enzyme prevents the virus from replicating.Zhang et al.determined the threedimensional structure of SARS-CoV-2 Mpro at the resolution of 1.75Å,and analyzed the structure of the functional protein as well as the crystal structure of the drug compound and SARS-CoV-2 Mpro complex.The researchers found that the three-dimensional structure of SARS-CoV-2 Mpro was highly similar to that of SARS-CoV Mpro,with a sequence identity of 96%.Among them,chymotrypsin and picornavirus 3C protease-like domains I and II formed a six-stranded antiparallel

β

barrels with a substrate binding site between them.Domain III was a spherical cluster of five helices,responsible for regulating the dimerization of Mpro through the salt bridge interaction between the two precursors.The compact dimer formed by SARSCoV-2 Mpro had a contact interface,which was mainly located between the structural domain II of molecule A and the NH2 terminal residues of molecule B [21].Jin et al.showed the X-ray crystal structure of SARS-CoV-2 Mpro combined with Carmofur,with the resolution of 1.6 Å.In vero E6 cells,Carmofur inhibited SARS-CoV-2 replication with an ECof 24.30

μ

M.The crystal structure of the complex of Mpro and Carmofur showed that the compound straightly modified the catalytic Cys145 of SARS-CoV-2 Mpro [22].This study not only clarified the structural basis of Carmofur’s inhibition of SARS-CoV-2 Mpro,but also provided a basis for designing Carmofur analogs with stronger inhibition to treat COVID-19.

The researchers analyzed the high-resolution crystal structure of SARS-CoV-2 endonuclease Nsp15/ NendoU,and found that the structure of SARS-CoV-2 Nsp15 monomer was similar to other Nsp15 of coronavirus.They compared the structure with previously reported Nsp15 models from SARSCoV and MERS-CoV.The results showed that the structure was 88% and 51% identical to the Nsp15 sequences of SARS-CoV and MERS-CoV,and it presented as a hexamer,the functionally active form of endonuclease.The active site residues were conserved in sequences and conformation.Structural comparisons indicated that SARS-CoV Nsp15 inhibitors also had the opportunity to inhibit SARS-CoV-2 homologs,but MERS-CoV NendoU inhibitors did not [23].

Non-structural protein 9 (Nsp9) is involved in the replication of viral genome RNA.Nsp9 is thought to bind to RNA by unique folding,but its molecular basis remains unclear.Researchers analyzed the crystal structure of Nsp9,revealing the high structural conservation in the Nsp9 family.The binding site of the exogenous peptide was close to the dimer interface and affected the relative juxtaposition of monomers in the homodimer[24].

The RNA-dependent RNA polymerase RdRp(also called nsp12) catalyzes the synthesis of viral RNA and is a key component of the coronavirus replication/ transcription mechanism.Taking RNA polymerase as the core,the virus uses other cofactors,such as nsp7 and nsp8,to replicate itself.Researchers analyzed the three-dimensional spatial structure of the composite of SARS-CoV-2 full length nsp12 and co-factors nsp7 and nsp8,with the resolution of 2.9 Å,and found that in addition to the conserved structure of the viral polymerase family polymerase core and the NiRAN (nidovirus RdRpassociated nucleotidyl transferase) structure peculiar to coronavirus RdRp,nsp12 also had a newly discovered

β

-hairpin domain at its N-terminus [25].Although the overall structure of the SARS-CoV-2 nsp12-nsp7-nsp8 complex is similar to that of SARSCoV,some key features are different.Elucidation of the three-dimensional structure of RdRp will help inhibit the replication of SARS-CoV-2.Recently,Yin et al.identified two cryo-EM structures of the SARS-CoV-2 RdRp complex,either in a complex with template primer RNA and Remdesivir or in the apo form [26].RdRp is a direct target of the antiviral drug Remdesivir.Their study revealed the structural basis of the antiviral activity of Remdesivir and provided accurate three-dimensional structural information for the design of antiviral drugs.Researchers have identified the crystal structure of the N-terminal RNA binding domain of SARSCoV-2 nucleocapsid protein with the resolution of 2.7 Å.Although the overall structure is similar to the N-terminal RNA binding domains of other coronavirus nucleocapsid proteins,the surface electrostatic potential features are different.Further comparison with the equivalent domain of mild virus type HCoV-OC43 revealed a unique RNA binding groove next to the

β

-sheet core.Combined with

in vitro

data,the results provided several atomic resolution characteristics of N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein and guided the design of a novel antiviral agent for SARS-CoV-2 [27].Papain-like protease (PLpro) is an important protease of SARS-CoV-2.In addition to co-acting with 3CLpro to produce polyproteins,PLpro protein can also be used to remove ubiquitin and interferonstimulating genes (ISG15) in host cells to escape host cell immune responses.At present,the crystal structure of PLpro has been resolved (PDB ID:6W9C),but the article has not been published yet.Recently,Shin et al.characterized the biochemical structure and function of SARS-CoV-2 PLpro,and explained the differences from SARS-COV PLpro in controlling host interferon and NF-

κ

B pathways [28].In addition,Gao et al.identified the nonligand structure of SARS-COV-2 PLpro mutant C111S,demonstrating the feasibility of applying crystallographic methods to fragment-based screening [29].

Starting with the virus’s unique structure,researchers have further explained why SARSCoV-2 is more contagious than SARS-CoV.Scientists have a growing understanding on how SARS-CoV-2 binds to hACE2,and they are studying more protein structures to identify all possible drug targets.This structural information helps researchers to identify and design drugs that block viral infections.

Table 1 Structural studies of several important proteins of SARS-COV-2

3 SARS-CoV-2 hosts

3.1 The natural hosts of SARS-CoV-2

In order to analyze the evolutionary origin and possible natural hosts of SARS-CoV-2,researchers conducted genetic and evolutionary analysis using SARS-CoV-2 and a large amount of coronavirus data.The results showed that the common ancestors of SARS-CoV-2 and SARS/ SARS-like coronavirus are viruses similar to HKU9-1.Since the evolutionary neighbors and outgroups of SARSCoV-2 were found in various bats,it was speculated that the natural host for SARS-CoV-2 might be bats.In addition,they also found that an unknown intermediate host for SARS-CoV-2 might exist during the transmission from bats to humans [30].

Shortly after that,some researchers conducted comprehensive analysis and comparison on the SARS-CoV-2 sequence of different animal species with the help of relative synonymous codon usage(RSCU) bias.SARS-CoV-2 showed the most similar genetic information to that of bat coronavirus and the most similar codon usage bias to that of snakes.In other words,snakes are the most likely wild animal repository for the outbreak of SARS-CoV-2 infection [31].However,considering that SARS-CoV or MERS-CoV is transmitted among mammals and snakes are not mammals,this conclusion has been questioned by some researchers [32].

It has been reported that SARS-CoV-2 is 88% identical with two bat-derived severe acute respiratory syndrome-like coronaviruses (BAT-SLCoVZC45 and BAT-SL-CoVZXC21) collected in zhoushan,eastern China in 2018,and about 79%and 50% identical with SARS-CoV and MERS-CoV,respectively [33].The team of Zhengli Shi compared the SARS-CoV-2 genome with some coronavirus gene sequences detected in their laboratory early and found that SARS-CoV-2 was genetically similar to a coronavirus derived from a bat sample(BatCoV RaTG13) [32].Researchers sequenced the bat sample to obtain the whole genome sequence of BatCoV RaTG13 and found that the sequence consistency of the two viruses was as high as 96% [34].This study provided evidence that SARSCoV-2 originated from bats.Another research team conducted a systematic evolutionary analysis on the complete genome of SARS-CoV-2 (29,903 nucleotides),and the results showed that the virus shared 89.1% nucleotide similarity with a group of SARS-like coronaviruses derived from bats,and the relationship is the closest.In addition,the amino acid similarity between SARS-CoV-2 and Bat-SL-CoVZC45 in nsp7 and E proteins was 100% [35].

In addition to the above results,another study showed that SARS-CoV-2 was closely related to coronavirus from five wild animals,namely Paguma larvata,Paradoxurus hermaphroditus,Civet,Aselliscus stoliczkanus and Rhinolophus sinicus,and belonged to the same branch of the phylogenetic tree.However,the homology of the genome and ORF1a indicated that the virus was different from the coronavirus derived from these five animals and had the highest homology with RaTG13 [36].In summary,the natural host for SARS-CoV-2 might be bats.

Recently,the reassembly and comprehensive analysis of previously published Malaysian Pangolin viroomics data revealed that these Malay pangolins carried SARS-CoV-2-like coronavirus (named Pangolin-CoV) [37].Its genomic similarity was determined to be 91.02% and 90.55% to SARSCoV-2 and BatCoV RaTG13.Although Pangolin-CoV is not as close to SARS-CoV-2 as it is to RaTG13,the S1 protein of Pangolin-CoV and SARSCoV-2 is highly consistent.The five key amino acid residues for the interaction of Pangolin-CoV and SARS-CoV-2 with the human ACE2 receptor were completely identical,and four mutations occurred in RaTG13,suggesting that Pangolin-CoV and SARSCoV-2 had similar host cell recognition ability [38].Besides,a furin protein recognition sequence at the S1/S2 restriction site was found in SARS-CoV-2,while Pangolin-CoV RaTG13 and SARS-CoV lacked this functional motif.These findings suggest that the pangolin species is a natural host for SARS-CoV-2-like coronavirus.

3.2 Intermediate hosts of SARS-CoV-2

It was reported previously that SARS-CoV was transmitted from civet cats to humans,while MERSCoV was transmitted from dromedary camels to humans [39,40].A virus host prediction (VHP)method was developed based on deep learning algorithms to predict potential intermediate host for SARS-CoV-2 [41].The study showed that infection pattern of bat coronavirus was more similar to SARS-CoV-2 than coronaviruses that infect other vertebrates,and that bats and minks might be two candidate reservoirs for SARS-CoV-2 because the infection patter of mink virus was similar to SARSCoV-2.

Xiao

et al.pointed out that SARS-CoV-2 might originate from the recombination of Pangolin-CoVlike virus and a Bat-CoV-RaTG13-like virus [42].They analyzed 25 kinds of Malayan pangolins and detected 17 species with Pangolin-CoV.Histological changes and clinical symptoms were observed in infected pangolins.More importantly,circulating antibodies against Pangolin-CoV could react with the S protein of SARS-CoV-2.A coronavirus highly associated with SARS-CoV-2 was isolated from pangolins,indicating that pangolin has the potential to serve as an intermediate host for SARS-CoV-2.

Guan’s team reported the identification of SARS-CoV-2-related coronavirus in the pangolin(Manis javanica) captured during anti-smuggling operation in South China [43].Genome sequencing showed that the newly discovered coronaviruse in pangolins was approximately 85.5% to 92.4%similar to SARS-CoV-2,and it belonged to two sublineages of SARS-CoV-2-related coronaviruses.The discovery of multiple lineages of Pangolin-CoV and their similarity to SARS-CoV-2 suggest that pangolin may be the intermediate host for SARSCoV-2.

Liu et al.found that pangolin coronaviruses(Pangolin-CoV) was genetically related to both SARS-CoV-2 and bat coronavirus,but phylogenetic analysis did not support the direct evolution of SARS-CoV-2 from Pangolin-CoV [44].Their research indicates that pangolin is the natural host for

β-

coronavirus and has the potential to infect humans.

Another study reported that genomic analysis of SARS-CoV-2 identified 96% similarity with RaTG13,but the receptor binding motif (RBM)of the two genomes exhibited low sequence similarity.They identified high sequence similarity in RBM between SARS-CoV-2 and the reconstructed coronavirus genome from the pangolin metagenomic data set,indicating the origin of SARS-CoV-2 [45].

Systematic comparison and analysis were used to predict the interaction between the receptor binding domain (RBD) of the S protein and ACE2 [46].The results indicated that turtles(Chrysemys picta bellii,Chelonia mydas,and Pelodiscus sinensis) might be the potential intermediate host for SARS-CoV-2.

Based on the structural information of the complex of hACE2 and SARS-CoV-2 RBD,researchers analyzed the affinity of 20 key residues of ACE2 to S protein in mammals,birds,and turtles [47].Their results indicate that neither the snake nor the turtle is the intermediate host for SARS-CoV-2,and the study recommends that Bovidae and Cricetidae be included in the selection of intermediate hosts for SARS-CoV-2.

In one study,researchers determined the insertion of a unique peptide (PRRA) in the human SARS-CoV-2 virus.They believed SARS-CoV-2 did not come directly from the pangolin because the coronavirus carried by the pangolin had no RRAR motif [48].

Chu et al.used molecular dynamics simulation and free energy calculation to speculate about the intermediate hosts of SARS-CoV-2 and found that tree shrew and ferret might be the two assumed intermediate hosts for SARS-CoV-2 zoonosis transmission [49].

Sun et al.analyzed the genetic conservation of ACE2 in mammals and collected more than 140 transcriptome data sets from human and common mammal species.Their results indicated that a wide range of mammalian species might be the hosts for SARS-CoV-2.They also observed that ACE2 was expressed in a species-specific manner in the mammals tested.High levels of expression in the skin and eyes of cats and dogs suggested that these animals might play a role in spreading SARS-CoV-2 to humans [50].

Wu et al.developed a new intermediate host mining method.82 representative ACE2 sequences were selected from 1000 sequences with the closest homology to human ACE2 protein and were modeled using homologous modeling.By calculating the binding free energy of ACE2 protein with SARS-CoV-2 RBD,the potential natural hosts,intermediate hosts and susceptible animals of SARS-CoV-2 were systematically analyzed.The results showed that Rhinolophus affinis might be the natural host for SARS-CoV-2,and the virus might be directly transmitted from bats to humans.Rhinolophus sinicus

might be another natural host for SARS-CoV-2,and the virus might be transmitted directly from bats to humans.Pangolins might not be intermediate hosts,nor are birds and reptiles.Mice,guinea pigs and rats were not sensitive to SARS-CoV-2.Primates,some wild felidae,civet,goats,spotted hyenas and golden hamsters might be the intermediate hosts [51].In order to study the potential intermediate host for SARS-CoV-2,Dabravolski et al.analyzed the sequences of S glycoproteins from different animals,especially the bats.They conducted a phylogenetic analysis,reconstructed the S glycoproteins,and performed alignment and comparison.Phylogenetic results showed that SARS-CoV-2 was more similar to

β

-coronavirus isolated from bats.They also found that the yak

β

-coronavirus strain YAK/ HY24/ CH/ 2017 best matched the S glycoprotein structural models [52].They pointed out that yak might be an intermediate host for SARS-CoV-2.

We present here a summary of the possible intermediate hosts studied to date (Table 2).These studies showed that bats might be the natural host for SARS-CoV-2,but the intermediate host is still unknown.Only when the natural and intermediate hosts of SARS-CoV-2 are found,the virus source can be cut off and transmission to humans can be prevented.

Table 2 Possible intermediate hosts

4 Virus source

Since the outbreak of COVID 19,a large number of studies on the source of SARS-CoV-2 have been carried out.Compete genome sequence has been reported,providing insight into the evolutionary origins of the the virus.There is a high degree of sequence similarity (＞99%) between the genome sequences of SARS-CoV-2 [53,54],but studies showed that the virus began to mutate in patients.Mutations of different SARS-CoV-2 isolates mainly occurred in ORF1ab,ORF3a,ORF8,S and N [55].In addition,studies have reported increased levels of viral diversity in some SARS-CoV-2 infected patients,suggesting that the viral genome has begun to evolve in the population,which may affect its virulence and infectivity [56].

Highly pathogenic MERS-CoV and SARSCoV was proved to originate from bats,but there is controversy on the origin of SARS-CoV-2 [57-59].With the deepening of research,people have got a further understanding of the origin of SARS-CoV-2.

The earliest genomic sequence data indicated that SARS-CoV-2 was a member of the genus

β

-coronavirus and belonged to a subgenus including SARS-CoV [33-35,60].The nucleotide similarity between SARS-CoV-2 and SARS-CoV is 79%,while that in S protein is only about 72%.In contrast,the main proteases are highly conserved between SARS-CoV-2 and SARS-CoV,with 96%homology.Although SARS-CoV-2 is closely related to SARS-CoV,the biological difference between them is obvious.

Although the source of SARS-CoV-2’s zoonotic disease has not been determined,its genome sequence is 88% homologous to the two batderived SARS-like coronaviruses (bat-SL-CoVZC45 and bat-SL-CoVZXC21) [33].Subsequent identification of two SARS-CoV-2 parental viruses greatly facilitated comparative genomic analysis.RaTG13,a bat coronavirus found in Rhinolophus affinis in Yunnan province,shares 96.2% sequence homology with SARS-CoV-2 [34].Despite this sequence similarity,SARS-CoV-2 and RaTG13 differ in many key genomic features,the most important of which is that SARS-CoV-2 contains a multiple base (furin) cleavage site insertion (PRRA residue) [61].In addition,the receptor binding domains of SARS-CoV-2 and RaTG13 are only 85% similar,and only one of the six key amino acid residues is identical [13].Bat coronavirus RmYN02 shares 93.3% nucleic acid homology with SARSCoV-2 in the range of complete virus genome.RmYN02 shares 97.2% homology with SARSCoV-2 in the longest coding gene region (1ab),while RmYN02 shares only 62.4% amino acid homology to SARS-CoV-2 in the RBD and may not bind to ACE2.However,like SARS-CoV-2,RmYN02 inserts multiple amino acids at the junction sites of the S1 and S2 subunits of S protein [62].This provides strong evidence that such insertions can occur in nature.Their research showed that SARSCoV-2 originated from a variety of naturally occurring recombination events in those viruses found in bats and other wildlife.The origin of SARSCoV also involved similar recombination events [63].In another study,researchers found that the newly discovered coronavirus in pangolins shared about 85.5% to 92.4% nucleotide sequence homology with SARS-CoV-2,and also had a receptor binding domain that was expected to interact with ACE2 [43].

Current research indicates that

Rhinolophus affinis

or

Malay pangolin

may be a potential natural source hosts for SARS-CoV-2.However,it has no been determined whether it is RaTG13,Pangolin-CoV or coronaviruses carried by other natural origin hosts,nor is it clear what intermediate hosts are involved in the transmission to humans.

Andersen et al.presented the salient features of the SARS-CoV-2 genome and discussed the possible scenarios where these features might appear.Most importantly,the analysis provides evidence for the argument that“SARS-CoV-2 is neither a laboratory product nor a virus intentionally manipulated”.Based on their genome sequencing analysis,Andersen et al.concluded that there were two possible situations for the origin of SARS-CoV-2.One is that the virus evolves to the current pathogenic state through natural selection in a non-human host and then is transferred to humans.Another is that the non-pathogenic version of the virus is transferred from the animal host to humans,and then evolves in humans into the current pathogenic state [64].Rehman et al.pointed out that SARS-CoV-2 used mutations and recombination as key strategies for different genomic regions such as spike glycoprotein,membrane,envelope,nucleocapsid,and membrane,that mutations in different genomic regions of SARSCoV-2 had specific effects on viral reproductive adaptability,and that SARS-CoV-2 had become a new virus in human society through natural selection strategies [65].

Genetic analysis of the 86 complete or nearly complete genomes of SARS-CoV-2 revealed many mutations and deletions in the coding and noncoding regions.These results provide evidence for the genetic diversity and rapid evolution of SARSCoV-2 [66].In order to better understand the evolution of SARS-CoV-2,researchers analyzed the codon usage pattern of SARS-CoV-2.The results showed that M protein and E protein tended to slowly evolve through the accumulation of nucleotide mutations in their genes,while genes encoding N protein,viral replicase and S protein tended to evolve faster [67].

The research results of Huang et al.showed that SARS-CoV-2 was a recombinant virus of bat and pangolin coronaviruses [68].Furthermore,genetic mutations and evolution of SARS-CoV-2 during its global transmission were found in their study.Their findings indicate that homologous recombination has occurred between the bat and pangolin coronaviruses,triggering the emergence and cross-species transmission of SARS-CoV-2,and SARS-CoV-2 is still developing due to its adaptability during the outbreak [68].The results of Tagliamonte et al.also indicate that SARS-CoV-2 may be caused by the recombination events of ancestral strains that circulate between the bat and the pangolin [69].

Through phylogenetic analysis of 160 complete SARS-CoV-2 genomes,Forster’s team identified three core variants of the virus:A,B,and C [70].Type A virus is the closest to the coronavirus found in bats and pangolins.It is the original virus type,with type B derived from type A,and type C derived from type B [69].Types A and C were found outside East Asia,mainly in Europe and the United States,while type B is the most common type in East Asia [70].

A recent study reported that a coronavirus isolated from Malayan pangolin had 90.7%,98.6%,100% and 97.8% amino acid homology with SARSCoV-2 in S,M,E and N genes,respectively.The receptor binding domain in the S protein of Pangolin-CoV is almost the same as that of SARS-CoV-2,with only one non-critical amino acid difference.Comparative genomic analysis indicated that SARSCoV-2 might originate from the recombination of Pangolin-CoV-like virus and Bat-CoV-RaTG13-like virus [71].

At present,the source of SARS-CoV-2 has not yet been completely determined,and further investigation is needed.

5 Pathogenesis

5.1 The receptors for SARS-CoV-2 entering the host cells

SARS-CoV-2 is the seventh member of the coronavirus family that infects humans.HCoV-229E,HCoV-NL63,HCoV-OC43 and HCoVHKU1 mainly cause mild respiratory infections,while SARS-CoV and MERS-CoV can cause severe pneumonia [72].Viral infection begins with the binding of viral particles to receptors on the host cell surface.Angiotensin converting enzyme 2(ACE2) and dipeptidyl peptidase 4 (DPP4) are the main receptors for S glycoproteins on the surface of SARS-CoV and MERS-CoV [73,74].S protein mediates the attachment of the virus to the host cell surface receptors and the fusion with the host cell membrane,thus promoting the virus entry into the host cell.S1 subunit binds to the receptor,and S2 subunit mediates the fusion of the virus with the host cell membrane and stimulates a series of immune responses,such as the production of neutralizing antibody and the activation of cytotoxic T lymphocytes.Wan et al.pointed out that the sequence of SARS-CoV-2 RBD,including the receptor binding motif (RBM) that directly contacts ACE2,was similar to that of SARS-CoV [75].In addition,several key residues in SARS-CoV-2 RBM had good interaction with human ACE2,suggesting that these two viruses might enter the cells through the same receptor.Zhou et al.demonstrated that ACE2 might be the cell receptor for SARS-CoV-2 to enter cells and that SARS-CoV-2 did not use other coronavirus receptors,such as aminopeptidase N(APN) and DPP4 [34].Hoffmann et al.demonstrated that SARS-CoV-2 entered the host cells through ACE2 and could be blocked by serine protease TMPRSS2 inhibitor [76].The binding affinity of SARS-CoV-2 S protein to human ACE2 is about 10-20 times that of SARS-CoV S protein [13].In most cases,S protein is cleaved into S1 and S2 subunits by the host protease.S1 subunit consists of N-terminal domain (NTD) and C-terminal domain(CTD),both of which act as the receptor binding entities.Wang et al.identified S1 CTD (SARSCoV-2-CTD) as the key region for the interaction between SARS-CoV-2 and hACE2,and resolved the crystal structure of SARS-CoV-2-CTD bound to hACE2 [18].The results showed that,compared with SARS-RBD,SARS-CoV-2-CTD had a stronger affinity for hACE2.

Bao et al.investigated the pathogenicity of SARS-CoV-2 using transgenic mice carrying hACE2 and infected with the virus.They found hACE2 mice infected with SARS-CoV-2 lost weight,observed the replication of SARS-CoV-2 in the lungs,and elucidated the typical histopathology of infected transgenic mice [77].This study clarified the pathogenicity of SARS-CoV-2 in hACE2 mice.The mouse model of SARS-CoV-2 infection is of great value for understanding the pathogenesis of COVID-19.

Studies have confirmed that ACE2 is the receptor of SARS-CoV-2,but it is puzzling that although ACE2 is widely expressed in a variety of organs,its expression in the lung,the main organ of infection is moderate but not high.Therefore,Cui et al.hypothesized that other genes might play a crucial role in the entry of SARS-CoV-2 into human cells [78].AGTR2 (angiotensin II receptor type 2) is a G protein-coupled receptor that interacts with ACE2 and is highly expressed in the lung with high tissue specificity.Simulation of proteinprotein interaction based on 3D structure showed that AGTR2 had higher binding affinity with SARSCOV-2 Spike protein than ACE2.The researchers believed that AGTR2 might be a novel putative gene for the entry of SARS-CoV-2 into human cells.This belief provides a different perspective for the study of SARS-COV-2 proteins and its receptors.

SARS-CoV-2 S protein recognizes the cellular receptor ACE2 and enters and infects host cells.Li et al.found that SAR2-CoV-2 infection further enhanced ACE2 and that EZH2-mediated H3K27me3 could inhibit the expression of ACE2 [79].EZH2 is the major enzyme that catalyzes H3K27me3.Their findings elucidate the pathogenesis of COVID-19,guide the targeted drug therapy of ACE2 expression,and provide a basis for the prevention and adjuvant treatment of COVID-19.

Lukassen et al.investigated the expression levels of ACE2 and TMPRSS2 as well as their distribution across cell types in lung tissues and from subsegmental bronchial branches by sequencing single nuclei and single cell RNA [80].The results showed that TMPRSS2 was strongly expressed in both tissues and ACE2 was mainly expressed as a transient secretory cell type in segmental bronchial branches.Besides,these transiently differentiating cells showed enrichment of pathways related to RHO GTPase function and viral processes,indicating increased susceptibility to SARS-CoV-2 infection [80].Their research provides important clues for the infection and pathogenesis of COVID-19.TMPRSS2 plays an important role in the transmission of the virus and the resulting immunopathology in the airways of mouse models infected with SARS-CoV and MERS-CoV [81].Matsuyama et al.found that a TMPRSS2-expressed vero E6 cell line was highly sensitive to SARSCoV-2 infection,and capable of effectively isolating and spreading SARS-CoV-2 [82].This study showed that,like SARS-CoV and MERS-CoV,SARS-CoV-2 infection could be enhanced by TMPRSS2.Zang et al.found that the expression of TMPRSS2 and TMPRSS4 enhanced the fusion activity of SARSCoV-2 S and promoted the virus entry into the host cells.In addition,they also pointed out that the intestine was a potential site for SARS-CoV-2 replication,which might lead to local and systemic diseases as well as overall disease progression [83].

A recent study has shown that SARSCoV-2 invades host cells through a novel CD147-spike protein (SP) pathway.S protein binds to the receptor CD147 on the host cell and mediates the invasion of the virus [84].CD147 is associated with viral infection and plasmodium invasion [85-87].Previous studies have confirmed that CD147 plays an important role in viral infection of host cells.The discovery of CD147-SP,a new pathway for SARSCoV-2 to invade host cells,provides an important target for the development of antiviral drugs.

5.2 Cytokine storm and immune injury

Cytokine storm is an excessive immune response to external stimuli (such as viruses and bacteria).When the virus attacks the body,it will stimulate the secretion of a large number of inflammatory factors,such as IL,IFN,and CXCL,resulting in cytokine cascade.With the exudation of inflammatory factors,cytokines will abnormally increased in tissues and organs,interfere with the immune system and cause excessive immune response in the body,leading to diffuse damage of lung cells,pulmonary fibrosis,multi-organ damage,and even death.Studies have shown that one of the main causes of acute respiratory distress syndrome(ARDS) and multiple organ failure is cytokine storm [88-91].ARDS is the leading cause of death in patients infected with SARS-CoV-2,SARS-CoV,or MERS-CoV [92,93].Analysis of the clinical features of 41 SARS-CoV-2 infected patients showed that 12 patients had ARDS [94].Another study elucidated the clinical characteristics of 99 patients with SARSCoV-2 infection,showing that 17 patients developed ARDS,11 of whom died of multiple organ failure [95].

Huang et al.found that a large number of IL1B,IP10,IFN,and MCP1 were expressed in patients infected with SARS-CoV-2,and these cytokines might activate T-helper-1 (Th1) cell response [94].Compared with patients who did not need ICU admission,patients who needed ICU admission had higher concentrations of IP10,MCP1,GCSF,TNFα,and MIP1A,which indicated that cytokine storm was related to the severity of the disease [94].Surprisingly,SARS-CoV-2 infection led to increased secretion of T-helper-2 (Th2) cytokines (such as IL-4 and IL-10) that suppressed inflammation,which was different from SARS-CoV infection [94,96].The expression levels of IL-2R and IL-6 in serum were positively correlated with the severity of COVID-19 [97].

Xiong et al.used RNA sequencing technology to sequence RNA isolated from bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cells (PBMC) specimens of COVID-19 patients.Their study demonstrated the persistence of inflammation and cytokine storms in patients,and highlighted the association between the pathogenesis of COVID-19 and excessive cytokine release (such as CCL2/ MCP-1,CXCL10/ IP-10,CCL3/ MIP-1A,CCL4/ MIP1B) [98].This study provides a valuable resource for understanding the pathogenesis of COVID-19.

There is a potential link between the changes of lymphocyte subsets and the pathogenesis of virus [99].Previous studies have shown that lymphocytes and subsets of CD4T cells,CD8T cells,B cells,and NK cells are essential for the maintenance of immune system function.A study of 140 hospitalized COVID-19 cases showed that lymphopenia (75.4%) was observed in most patients,and the number of blood eosinophils and the number of lymphocytes in severe patients after admission were positively correlated [100].

Some studies have indicated that lymphopenia may be associated with mortality,especially in patients with low levels of CD8,CD3and CD4T lymphocytes [101-104].It has been reported that SARS-CoV-2 can infect T cells through receptor-dependent S protein-mediated membrane fusion [105].The number of T cells in COVID-19 patients decreased significantly,and the surviving T cells failed [106].Notably,the expression level of hACE2 in T cells was very low [105].Therefore,the authors believed that SARS-CoV-2 might enter T cells through a novel receptor [105],which might trigger new ideas about pathogenic mechanisms and therapeutic interventions.

In addition,studies have shown a significant decrease in peripheral blood lymphocytes in patients with COVID-19 [95,107].Unfortunately,any changes in peripheral lymphocyte subsets in COVID-19 are still unknown.In a recent study,Wang et al.found that the total number of lymphocytes,CD4T cells,CD8T cells,B cells,and NK cells were reduced in patients with COVID-19 [108].The severer the case,the lower the number.These subsets were shown to be significantly correlated with inflammatory states in COVID-19,particularly CD8T cells and CD4/ CD8ratios.CD8T cells were independent predictors of COVID-19 severity and therapeutic effect [108].Their study shows that changes in peripheral lymphocyte subsets are closely related to the clinical characteristics of COVID-19,which provides insights into the pathogenesis of COVID-19 and the development of new biomarkers and therapeutic strategies for COVID-19.

Xu et al.found that the number of peripheral CD4 and CD8 T cells was significantly decreased,but the state was overactivated,the concentration of highly proinflammatory CCR6Th17 in CD4 T cells was increased,and CD8 T cells had high concentration of cytotoxic particles [109].In this study,the excessive activation of T cells,manifested by an increase in Th17 and high cytotoxicity of CD8 T cells,partially explained the patient’s severe immune impairment.This study provides new insights into the pathogenesis of SARS-CoV-2 associated pneumonia.Wu et al.proposed that TH17 response in cytokine storms of COVID-19 might be an emerging target for the JAK2 inhibitor Fedratinib [110].

5.3 Immune evasion

Innate immunity,the first line of defense against virus infection,plays a key role in the process of virus clearance.The innate immune system recognizes the virus invasion through pattern recognition receptors (PRRs),and then produces interferon (IFN) and a series of cytokines to resist or clear the virus.At the same time,in order to better survive in host cells,viruses have evolved various mechanisms to escape the host immune system.MERS-CoV and human coronavirus OC43 (HCoVOC43) can escape antiviral innate immunity by inhibiting the activation of NF-

κ

B [111].MERSCoV’s M,4a,4b proteins and PLpro inhibit the type I interferon and NF-

κ

B signaling pathways,thereby contributing to innate immune escape [112-115].Interferon type I imbalance caused fatal pneumonia in mice infected by SARS-CoV [115].Although it is not clear whether SARS-CoV-2 has a similar mechanism of action,the study on SARS-CoV and MERS-CoV provides us with new ideas.

Recently,Shang et al.found that SARS-COV-2 RBD had a higher binding affinity for hACE2 than SARS-CoV RBD,while the binding affinity of SARS-CoV-2 S protein for hACE2 was equal to or lower than that of SARS-CoV S protein.The authors pointed out that the dynamic state of RBD could explain this phenomenon.RBD of SARSCoV-2 is mostly lying down and not conducive to binding to the receptor,thus resulting in the lower binding affinity between SARS-CoV-2 and hACE2 than that of SARS-CoV.Notably,the cell entry of SARS-CoV-2 was pre-activated by furin,thereby reducing its dependence on target cell protease entry.The authors pointed out that the high affinity of the RBD for SARS-CoV-2 binding to hACE2,the latent RBD in S protein and the pre-activation of furin might help SARS-CoV-2 to avoid immune surveillance,while maintaining effective cell entry [71].The study reveals the mechanisms of SARS-CoV-2 infection in human cells and immune escape,providing guidance for the development of antibody drugs and vaccines.

6 Conclusion

The infection of SARS-CoV,MERSCoV and SARS-CoV-2 has posed a huge threat to human health.However,no specific drug or vaccine against SARS-CoV-2 has been identified or produced.Research on protein structure is essential for understanding the structural basis and functional characteristics of viruses entering target cells,identifying and optimizing antiviral drugs,and guiding vaccine design.Early investigation of the source,natural and intermediate hosts,and pathogenesis of SARS-CoV-2 is of great significance for the development of antiviral drugs,the control of virus transmission,the prevention of disease deterioration,and the reduction of complications and mortality.

At present,the research on SARS-COV-2 still faces the following difficulties.First of all,development of innovative drugs involves relatively long research and development cycle,high investment and high risk.Therefore,it may be the best choice to look for anti-SARS-COV-2 drugs from the drugs that are already on the market or in the clinical research stage.Secondly,the main difficulty in vaccine research lies in the fact that although its genome sequence has been determined,the pathogenic mechanism has not yet been fully elucidated.Therefore,it is particularly important to understand the structural and non-structural genes of the virus and their functions.The use of scientific resources is crucial for the control of the epidemic.It is important to coordinate global efforts to accelerate SARS-COV-2 research on major scientific issues related to the etiology,epidemiology,pathogenesis,disease prevention and treatment of SARS-COV-2 infection,and to carry out basic and prospective joint research.In addition,the establishment of animal disease model of SARS-COV-2 infection has been initiated to facilitate preclinical drug screening and new antiviral therapy,and to provide a convenient and effective animal model platform for further exploration of the pathogenesis of SARS-COV-2,the relationship between virus variation and host adaptation,and vaccine development.

Acknowledgement

This work was supported by funds from National Mega-project for Innovative Drugs (grant number 2019ZX09721001-004-007),National Natural Science Foundation of China (NSFC)(U1803122,81773637,81773594,and U1703111),the Fundamental Research Fund for the Central Universities (HUST COVID-19 Rapid Response Call,No.2020kfyXGYJ037),and the fellowship of China Postdoctoral Science Foundation(2020T130039ZX).

Conflicts of interest

The authors declare that they have no conflict of interest.