severe acute respiratory syndrome ( sars ) 
 is a new emerging infectious disease ,   which was first reported in china in 2002 
 and was rapidly spreading all over the world in 2003 [ 1 , 2 ] .
patients develop persistent fever , dry cough , progressive 
 radiographic changes of chest , and lymphopenia once infected . despite 
 treatment , about 1015% of the patients would die due to the acute respiratory 
 distress [ 36 ] .
a novel coronavirus ( sars - cov ) was isolated from 
 sars patients [ 79 ] . sars - cov is a positive - stranded rna virus with an envelop . 

comparative genomic studies using the in silico annotated proteins have 
 suggested that sars virus belongs to a new group of coronavirus . according to the genomic sequence of 
 sars - cov , it is predicted that there are several structural proteins can be 
 produced by sars - cov including spike ( s ) , envelop ( e ) , membrane ( m ) , and 
 nucleocapsid .
spike protein is very important in the binding and fusion of 
 coronavirus to the host cells [ 11 , 12 ] .
the s protein of sars - cov has 1255 amino 
 acids in length and 23 potential n - linked glycosylation sites .
the amino 
 terminus of the sars - cov s protein contains a short type 1 signal sequence 
 composed of hydrophobic amino acids that are presumably removed during 
 cotranslational transport through the endoplasmic reticulum .
the carboxyl 
 terminus consists of a transmembrane domain and a cytoplasmic tail rich in 
 cysteine residues . the majority of protein ( residues 121195 ) is outside the virus 
 particle , which can be divided into amino - terminal s1 and carboxyl - terminal s2 
 domain .
the s1 domain ( residues 12672 ) binds to the host cell receptor , angiotensin - converting enzyme 2 ( ace2 ) , while the 
 s2 domain is responsible for membrane fusion [ 1315 ] .
monoclonal 
 antibodies against s1 domain can block the receptor binding and contain potent 
 neutralization activity against sars - cov .
however , peptides derived 
 from s2 domain can also inhibit sars - cov infection . molecular mimicry , which is defined as similar structures shared by 
 molecules from dissimilar genes or by their protein products , is a 
 general strategy for pathogens to infect host cells and has been proposed as a pathogenic mechanism for autoimmune disease . 

therefore , identification of the molecular mimic regions of pathogen may be 
 helpful to understand the disease induced by that pathogen . at present , it is 
 unclear whether molecular mimicry occurs between sars - cov s proteins and human 
 peptides .
we have approached this question using computer to analyze the 
 sequence of spike protein of sars - cov and select regions that share the sequence 
 homology with human proteins .
the criteria for the selection of potential 
 regions include antigenic analysis and surface accessibility . in this study , we 
 find that several regions of the s protein share sequence homology with human 
 proteins .
synthetic peptides , which represent some of these regions , were 
 synthesized to understand their roles in sars - cov infection .
publically available human and coronavirus genome sequences at the national center for biotechnology information ( md , usa ) 
 were used for in silicoprediction .
algorithms predicting immunogenicity , second 
 structure prediction , protein topology analysis , and hydrophobicity were 
 conducted to design the tested peptides .
immunogenic viral peptides were 
 calculated based on the algorithm developed by kolaskar and tongaonkar .
the 
 algorithm is based on a table constructed from the occurrence of amino acid 
 residues in experimentally known antigenic epitopes .
secondary structural analyses of spike protein were 
 performed based on phd   and predator   algorithms .
similarity searches between s protein and 
 human genome database were performed by using blastp   with blosum 62 .
extra amino acid residues were added at either 
 n- or c - terminus to keep the hydrophobic amino acid content below 50%
. 
 peptides with high hydrophobicity are difficult to be tested in biochemical experiments 
 since most of in vitro assays 
 are conducted in aqueous buffers .
also , 
 on average , one charged residue is added for every five amino acids .
multiple antigen peptides were synthesized by 
 cytomol corp ( mountain view , calif , usa ) .
in addition , bradykinin and 
 angiotensin i ( ang i ) were purchased from sigma ( st . louis , mo , usa ) .
sars patient sera were collected by the center for disease control , department of 
 health ( taipie , taiwan ) from march to june , 2003 .
diagnosis of sars was based on the clinicalcriteria 
 established by the world health organization ( who ) .
patients with 
 sars - cov were confirmed by laboratory methods , including viral antigen detection , 
 rt - pcr , and serologic methods .
ten sars patient sera collected at the convalescent stage ( 20 days after disease onset )   were included in this study .
antibodies against peptides in human sera were detected by solid - phase capture technique 
 using individual peptide - coated plates .
elisa plate was coated with or without 50  l 
 peptides ( 100  g / ml ) per well and blocked by 1% bovine serum albumin ( bsa ) in 0.05% tween-20 in phosphate - buffered saline ( pbs ) 
 for 1 hour at room temperature .
test 
 serum samples were 1:100 diluted and added to the plate for 2 hours at room 
 temperature .
after incubation , the elisa plate was washed with 0.05% tween-20 in pbs for three times .
the 
 bound antibodies were detected by horseradish peroxidase- ( hrp- ) conjugated antihuman 
 immunoglobulin antibodies ( sigma aldrich , st . louis , mo , usa ) and peroxidase substrate , 
 tmb ( promega , madison , wiss , usa ) .
the absorbance was measured using the vmax microplate 
 reader ( molecular devices corporation , sunnyvale , calif , usa ) at 450  nm . 

antibodies against peptides in mouse sera were assayed by elisa as in human 
 sera except hrp - conjugated antimouse immunoglobulin antibodies ( sigma aldrich ) 
 was used to detect bound antibodies .
human lung adenocarcinoma cell line a549 and vero cells were grown in dmem supplemented 
 with 10% heat - inactivated fcs , 2  mm l - glutamate , and 50  ng / ml gentamycin .
cells were incubated in co2 incubator at 37c with 5% co2 in a humidified atmosphere . for immunofluorescent microscopy 
 observation ,
six- to eight - week - old female balb / c mice were used in this study .
these mice were 
 originally purchased from jackson laboratory ( bar harbor , me , usa ) and bred in 
 the laboratory animal center , national cheng kung university ( tainan , taiwan ) . 

synthetic peptides ( 1  mg / ml ) were emulsified with complete freund 's adjuvant 
 and injected intraperitoneally into balb / c mice .
mice were boosted with the 
 same peptide in pbs ( 50  g / mouse ) intraperitoneally two weeks 
 after priming .
sera were collected from the axially plexus of the mice at 
 different time intervals and tested for the presence of antibody against 
 peptides by elisa as mentioned above .
significant increase of antibody titer ( greater 
 than 4 folds ) against immunized - peptide was found in mouse hyperimmune sera as 
 compared to normal sera after boosting .
mouse hyperimmune sera against peptide d08 were incubated with a549 cells 
 at 4c for 1 hour . after washing 
 three times with pbs
, cells were incubated with 1  ml of 1  g / ml fitc - conjugated antimouse igg ( jackson immunoresearch 
 laboratories inc . ,
west grove , pa , usa ) at 4c for 1 hour and washed again with pbs .
proteins in the cell lysate of a549 were separated by 12% sds - page 
 and transferred to nitrocellulose sheets as described previously .
proteins 
 recognized by normal or peptide d08 hyperimmune mice sera were detected using 
 hrp - conjugated antimouse immunoglobulin antibodies ( sigma aldrich ) and 
 substrate .
vero e6 ( 4104 ) and a549 cells ( 5103 ) were incubated with 
 different doses of synthetic peptides as indicated for 72 hours .
cell 
 proliferation was detected using commercial xtt assay ( roche diagnostics , 
 indianapolis , ind , usa ) .
the il-8 production was assessed by commercial elisa kits ( r&d systems , minneapolis , 
 minn , usa ) according to the manufacturer 's instructions .
briefly , a549 cells ( 1105 ) were cultured alone or with different doses of peptides for 48 
 hours .
culture supernatants were collected after incubation , added to precoated 
 elisa plates , and incubated for 2 hours at 37c .
the developed color was read by the vmax microplate 
 reader ( molecular devices , calif , usa ) .
the levels of 
 significance for the differences between groups were analyzed using student 's t - test . a value of p < .05 was considered to be significant .
the whole amino acid 
 sequence of spike protein was analyzed to find out the potential immunogenic 
 regions and the regions shared sequence homology with human proteins , which is defined 
 as the pathogenic regions .
region 1 ( residues 199254 ) , region 2 ( residues 658715 ) , region 3 
 ( residues 893951 ) , and region 4 ( residues 11271184 ) have shared sequence homology 
 with hydroxyacid oxidase , human golgi autoantigen , angrgm-52 , and pallidin , 
 respectively . among these regions , region 3 with homology to human angrgm-52 has 
 the highest score ( with 34% identities and 48% similarity of conservative 
 substitutions ) .
in addition , because des - arg bradykinin and ang i are the substrates for ace2 , we also compared the sequence of s protein against bradykinin ( rppgfspfr ) and ang i ( drvyihpfhl ) and 
 found that residues 490502 ( gyqpyrvvvlsfee ) of s protein showed sequence homology with bradykinin as 
 indicated by bold letters here and in figure 2(b ) .
the identity score is 27% ; and 
 the similarity score from conservative substitutions is 36% .
eleven peptides ( d01d11 , see table 1 ) , which represent those pathogenic regions were 
 synthesized as well as bradykinin and ang i were tested to see whether those peptides 
 can be recognized by sars patients ' sera . 

the peptides were designed based on the algorithms predicting immunogenicity , 
 second structure , protein topology , and hydrophobicity .
our goal is to select for peptides with high 
 immunogenicity , with location on the protein surface , and with low hydrophobicity .
the designed peptide sequences were 
 synthesized and tested with clinical samples of sars patient sera .
a significant increase of sars patients ' sera 
 binding to peptide d01 , d07 , and d08 was found as compared to the binding of 
 normal sera ( see figure 3 ) .
to test whether synthetic peptides d01 , d07 , and d08 can induce antibodies crossreacted with 
 human proteins , we immunized mice with these peptides to generate hyperimmune 
 sera against these peptides .
we found hyperimmune sera against d08 can bind to 
 the cytoplasmic region of human a549 cells as demonstrated by immunofluorescent 
 stain ( see figure 4 ) . using western blot analysis , hyperimmune sera against d08 
 could recognize more bands in a549 cell lysate as compared to normal mice sera 
 ( see figure 5 ) .
in addition , hyperimmune sera against d07 , but not d01 , showed 
 similar crossreactivity to a549 cells as hyperimmune sera against d08 did ( data 
 not shown ) . to test whether synthetic peptides d10 , indeed , can induce antibodies crossreactive with 
 bradykinin and ang i , we immunized mice with d10 peptides to generate 
 hyperimmune sera against this peptide . significant increase of antibodies 
 against d10 was found in d10 hyperimmune sera , which could crossreact with 
 bradykinin , but not with ang i - coated plates ( see figure 6 ) . to understand whether d10 has similar biological activity as ang i , we incubated 
 vero cells and lung epithelial a549 cells with d10 , ang 1 , or control peptide d11 . 

both vero and a549 cells were induced to proliferate in the presence of d10 and 
 ang i but not the control peptide ( see figure 7 ) .
in addition , d10 and ang i could 
 also induce chemokine il-8 production of a549 cells ( see figure 8) .
in this study , we have identified four 
 pathogenic regions of sars - cov s protein which share sequence homology with 
 different human proteins . among them , pathogenic region 3 ( residues 893941 ) , 
 which shares sequence homology with angrgm-52 ( genbank accession no .
aal62340 ) , a 
 novel gene upregulated in human mesangial cells stimulated by angiotensin ii , may 
 deserve further investigation .
peptides d07 and d08 of this region were 
 recognized by the sera of sars patient indicating that this region is 
 immunogenic and can be recognized by the immune system during sars - cov 
 infection .
murine hyperimmune sera against peptides d07 or d08 were able to 
 bind to recombinant s2 but not s1 domain of s protein ( data not shown ) . in 
 addition ,
hyperimmune sera against d07 or d08 also bounded to the cytoplasmic region of a549 cells 
 and recognized several proteins in the a549 cell lysate .
these results indicate 
 that regions represented by d07 and d08 are immunogenic and may induce 
 autoantibodies . however , further study is required to understand the biological 
 function of these regions and the role of their antibodies in the pathogenesis 
 of sars - cov infection .
in addition to d07 and d08 peptides , we also noticed that d10 
 peptide which represents residues 490502 of s1 domain contained some 
 interesting activities .
the d10 peptide , which shared sequence homology with 
 bradykinin , was able to generate antibodies crossreactive with bradykinin .
in addition , 
 d10 peptide could stimulate a549 to produce il-8 and proliferation as ang i did . 

these results suggest that the region of d10 in s protein may bind to ang i receptor , ace2 , and 
 may be involved in the binding of sars - cov to ace2 .
this is consistent with the 
 previous report , which indicates that residues 318510 of s1 domain can bind to ace2   and is similar to 
 the receptor binding domain of the hcov-229e , which is within a fragment 
 containing residues 407 to 547 .
therefore , region 490502 of s1 domain may 
 be involved in the receptor binding domain of sars - cov . in summary
, our results suggest that molecular mimicry occurs between 
 sars - cov and host proteins .
motifs shared sequence homology with host proteins of 
 sars - cov may be involved in the binding and fusion of sars - cov to host cells .
antibody against these motifs may contain 
 neutralization activity against sars - cov infection or participate in the 
 immunopathogenesis induced by sars - cov . 

as reported previously , sars - cov , like influenza , can inhibit the host 's 
 corticosteroid stress response via a molecular mimicry strategy .
our studies on the mimicry motifs of s protein , which is involved in the virus , entry may provide alternative approaches to disrupt the infection of sars - cov , similar to the previous studies on the virus entry 
 [ 28 , 29 ] .
 molecular mimicry , defined as similar structures shared by molecules from dissimilar genes or proteins , is a general strategy used by pathogens to infect host cells .   
 severe acute respiratory syndrome ( sars ) is a new human respiratory infectious disease caused by sars coronavirus ( sars - cov ) .   
 the spike ( s ) protein of sars - cov plays an important role in the virus entry into a cell .   in this study , eleven synthetic peptides from the s protein 
 were selected based on its sequence homology with human proteins .   
 two of the peptides d07 ( residues 927937 ) and d08 ( residues 942951 ) were recognized by the sera of sars patients . 
 murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells a549 . 
 another peptide d10 ( residues 490502 ) stimulated a549 to proliferate and secrete il-8 .   
 the present results suggest that the selected s protein regions , which share sequence homology with human proteins , may play important roles in sars - cov infection . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION