Patent Application: US-201314430010-A

Abstract:
the invention provides an isolated essentially mammalian positive - sense single stranded rna virus classifiable as belonging to the order : nidovirales ; family : coronaviridae ; subfamily : coronavirinae ; genus : betacoronavirus ; and non - lineage a , non - lineage b or non - lineage d , human betacoronavirus . the invention also provides a human virus having a receptor binding domain capable of binding to a dipeptidyl peptidase 4 . the invention also provides diagnostic means and methods , prophylactic means and methods and therapeutic means and methods to be employed in the diagnosis , prevention and / or treatment of disease , in particular of respiratory disease , in particular of mammals , more in particular in humans .

Description:
virus was isolated from a 60 - year old man with acute pneumonia and acute renal failure in saudi arabia . virus was isolated from sputum specimen in vero cells and llc - mk2 cells . five days after inoculation , cytopathic effects were observed , consisting of rounding of the cells , detachment of cells , and syncytium formation ( fig1 ). cells in the original sputum sample and infected cultured cells were also tested with specific antibodies against influenza a and b viruses , parainfluenza viruses types 1 - 3 , respiratory syncytial virus , and adenovirus , but such tests yielded negative results . sputum specimens and infected cell culture supernatants did not react in pcr - based assays specific for paramyxoviruses , enteroviruses , and adenoviruses . however , these samples did react with pcr - based assays to detect all coronaviruses . a 251 nucleotide fragment was amplified with one such test ( vijgen , l ., e . moes , e . keyaerts , s . li , and m . van ranst . 2008 . a pancoronavirus rt - pcr assay for detection of all known coronaviruses . methods mol biol 454 : 3 - 12 ). a second pcr - based assay to detect all coronaviruses ( drosten c , gunther s , preiser w , van der werf s , brodt h r , becker s , rabenau h , panning m , kolesnikova l , fouchier r a , berger a , burguière a m , cinatl j , eickmann m , escriou n , grywna k , kramme s , manuguerra j c , müller s , rickerts v , sturmer m , vieth s , klenk h d , osterhaus a d , schmitz h , doerr h w . identification of a novel coronavirus in patients with severe acute respiratory syndrome . n engl j med . 348 , 1967 - 76 ( 2003 )) also yielded positive results ( fig2 ). viral rna was isolated from infected cell culture supernatants using a high pure rna isolation kit ( roche ). extracted rna was copied to cdna by reverse transcriptase using random hexamers . pan - coronavirus polymerase chain reaction ( pcr ) was used to amplify a conserved region of open reading frame 1b ( drosten c , gunther s , preiser w , van der werf s , brodt h r , becker s , rabenau h , panning m , kolesnikova l , fouchier r a , berger a , burguiere a m , cinatl j , eickmann m , escriou n , grywna k , kramme s , manuguerra j c , müller s , rickerts v , sturmer m , vieth s , klenk h d , osterhaus a d , schmitz h , doerr h w . identification of a novel coronavirus in patients with severe acute respiratory syndrome . n engl j med . 348 , 1967 - 76 ( 2003 )). the pcr fragments of the pan - coronavirus pcrs were sequenced . to this end , pcr products were purified from the gel and sequenced using a bigdye terminator v3 . 1 cycle sequencing kit ( applied biosystems , nieuwerkerk a / d ijssel , the netherlands ) and a 3130xl genetic analyzer ( applied biosystems ), according to the instructions of the manufacturer . the sequence clearly corresponded with conserved region of open reading frame 1b of a coronavirus ( fig3 ). reference coronavirus genome sequences were downloaded from genbank and the part of the genomes that corresponded with the amplified fragment of hcov - sa1 were aligned . a maximum likelihood tree was constructed to infer the phylogenetic relationships ( fig4 ). this phylogenetic tree showed that the new hcov - sa1 belongs to lineage c of the genus betacoronavirus , along with the bat coronaviruses hku4 and hku5 . the betacoronavirus genus contains 3 additional lineages ( a , b , d ). hcov - hku1 and hcov - oc43 belong to lineage a while sars - cov belongs to lineage b . lineage d does not contain any human pathogens , and is represented in the tree by rousettus bat coronavirus hku9 . hcov - sa1 is thus clearly distinct from previously known human coronaviruses . hcov - nl63 and hcov - 229e are even more distinct from hcov - sa1 , since these two human pathogens belong to a different genus , the alphacoronavirus genus . to further characterize the virus genome , viral rna was extracted from infected cell culture supernatant using the high pure rna isolation kit ( roche ). rna was subjected to reverse transcriptase using circular permuted primers ( welsh , j . & amp ; mcclelland , m . fingerprinting genomes using pcr with arbitrary primers . nucleic acids res . 18 , 7213 - 7218 ( 1990 )) that were extended with random hexamer sequences . the amount of dna was amplified by polymerase chain reaction ( pcr ), using the circular permuted primers . the randomly amplified fragments were sequenced using the 454 / roche gs - flx sequencing platform . a fragment library was created according to the manufacturer &# 39 ; s protocol without dna fragmentation ( gs flx titanium rapid library preparation , roche ). the empcr ( amplification method lib - l ) and gs junior sequencing run was performed according to instructions of the manufacturer ( roche ). the sequence reads were trimmed at 30 nucleotides from the 3 ′ and 5 ′ ends to remove all primer sequences . sequence reads from the gs - flx sequencing data were assembled into contigs using clc genomics software 4 . 6 . 1 . using this “ deep - sequencing ” approach on the 454 - sequencing platform , approximately 80 % of the virus genome sequence was obtained . subsequently , specific primers were designed to amplify 30 overlapping fragments of approximately 1500 basepairs by pcr . each of these pcr products was sequenced using conventional sanger sequencing . to this end , pcr products were purified from the gel and sequenced using a bigdye terminator v3 . 1 cycle sequencing kit ( applied biosystems , nieuwerkerk a / d ijssel , the netherlands ) and a 3130xl genetic analyzer ( applied biosystems ), according to the instructions of the manufacturer . the nearly full - length sequence is presented in file hcov - sa1 . rtf . this sequence contains some uncertainties within the extreme 50 nucleotides of both ends . however , this information is not required to classify the coronavirus . the same figure also displays the full coding potential of hcov - sa1 . as a minimum , the hcov - sa1 virus genome encodes the open reading frames common to the virus of the betacoronavirus genus , including orf1ab that encodes many enzymatic products , the spike surface glycoprotein ( s ), the non - structural genes ns3a , ns3b , ns3c , ns3d , the small envelope ( e ) protein , the matrix ( m ) protein , and the nucleocapsid ( n ) protein . open reading frames are presented in files orf1ab . rtf , s . rtf , ns3a . rtf , ns3b . rtf , ns3c . rtf , ns3d . rtf , e . rtf , m . rtf , n . rtf . other open reading frames may be present . comparison of the orf1ab gene product of hcov - sa1 with those of the other members of the betacoronavirus genus , hku4 and hku5 was used to test if hcov - sa1 belongs to one of these known species or represents a new species within the genus . the international committee on the taxonomy of viruses ( ictv ) considers viruses that share more than 90 % aa sequence identity in the conserved replicase domains to belong to the same species . this 90 % identity threshold serves as the sole species demarcation criterion . since amino acid sequence identity of orf1ab between hcov - sa1 and hku4 and hku5 is below 74 % ( table 1 ), we conclude that hcov - sa1 represents a novel species of the betacoronavirus genus , although such classification requires ictv approval . a plasmid encoding hcov emc s1 fc was generated by ligating a fragment encoding the s1 region ( residues 1 747 ) into the pcaggs expression vector as an n terminal fusion with the fragment encoding the fc domain of human igg ( fig1 and 2 ). likewise , an s1 fc expression plasmid was made for the sars coronavirus s1 subunit ( strain urbani : residues 1 676 ) and the fipv s1 subunit ( strain 79 1146 ; residues 1 788 ). s1 fc proteins were expressed by transfection of the expression plasmids into 293t cells and affinity purified from the culture supernatant using protein a sepharose beads . a plasmid encoding the ectodomain of human dpp4 ( fig1 ) was generated by ligating a fragment encoding residues 39 766 of human dpp4 into a pcd5 expression vector encoding the signal sequence of cd5 and a onestrep affinity tag ( iba gmbh ). soluble dpp4 ectodomain was expressed by transfection of the expression plasmid into 293t cells and affinity purified from the culture supernatant using streptactin sepharose beads ( iba gmbh ). a plasmid encoding hcov emc s1 fc was generated by ligating a fragment encoding the s1 region ( residues 1 747 ) into the pcaggs expression vector as an n terminal fusion with the fragment encoding the fc domain of human igg separated by a thrombin cleavage site . likewise , an fc expression plasmid was made for the sars coronavirus s1 subunit ( isolate cuhk w1 : residues 1 676 ), the fipv s1 subunit ( isolate 79 1146 ; residues 1 788 ) and the ectodomain of human ace2 ( sace2 ; residues 1 614 ). fc chimeric proteins were expressed by transfection of the expression plasmids into 293t cells and affinity purified from the culture supernatant using protein a sepharose beads ( ge healthcare ). purified ace2 fc was cleaved with thrombin and soluble ace2 was purified by gel filtration . a plasmid encoding the ectodomain of human dpp iv ( sdpp iv ) was generated by ligating a fragment encoding residues 39 766 of human dpp iv into a pcd5 expression vector encoding the signal sequence of cd5 and the onestrep tag ( iba gmbh ). soluble dpp iv ectodomain was expressed by transfection of the expression plasmid into hek 293t cells and affinity purified from the culture supernatant using strep tactin sepharose beads ( iba gmbh ). the immunoprecipitation protocol was essentially carried out as described before with some modifications ( li et al ., 2003 , nature 426 : 450 , included herein by reference ). in short , huh 7 cells were washed twice with ice cold pbs , scraped off the plastic with a rubber policeman , pelleted and lysed in ice cold lysis buffer ( 0 . 3 % ddm in pbs ) containing protease inhibitors ( roche complete mini ) at a final density of − 2 . 5 × 107 cells / ml . cell lysates were precleared with protein a sepharose beads after which 10 micrograms of probe s1 fc was added to 1 ml of cell lysate and incubated for 1 hour at 4 ° c . under rotation . precipitates were washed thrice with lysis buffer and once with pbs and subjected to novex ® 4 12 % tris glycine gradient gel ( invitrogen ) under reducing and non reducing conditions . a distinct 110 kda band precipitated with emc s1 fc was visualized by gelcodeblue staining , excised from the gel , incubated with trypsin and analyzed by ms . results are shown in fig2 and results of target analyses are shown in fig2 . dpp4 cell surface expression was measured using s1 fc proteins . cells were washed twice with ice cold pbs , scraped off the plastic with a rubber policeman and suspended into single cells by pipetting cells up and down . s1 binding of cells was measured by incubating 2 . 5 × 105 cells with 15 μg / ml of s1 fc followed by incubation with the fluorescent dye alexa488 labeled goat anti human igg antibody and analyzed by flow cytometry . results are shown in fig2 . rna from 200 μl of supernatant was isolated with the magnapure lc total nucleic acid isolation kit ( roche ) external lysis protocol and eluted in 100 al . hcov emc rna was quantified on the abi prism 7700 , with use of the taqman reverse transcription reagents and taqman pcr core reagent kit ( applied biosystems ), using 20 μl isolated rna , lx taqman buffer , 5 . 5 mm mgcl2 , 1 . 2 mm dntps , 0 . 25 u amplitaq gold dna polymerase , 0 . 25 u multiscribe reverse transcriptase , 0 . 4 u rnase inhibitor , 200 nm primers , and 100 nm probe . amplification parameters were 30 minutes at 48 ° c ., 10 minutes at 95 ° c ., and 40 cycles of 15 seconds at 95 ° c ., and 1 minute at 60 ° c . rna dilutions isolated from a hcov emc stock were used as a standard . results are shown in fig1 , 24 , 25 and 26 . hcov emc and sars cov s1 fc proteins ( 2 . 5 μg ) were mock incubated or incubated with 12 . 5 μg soluble dpp iv ( sdpp iv ) or soluble ace2 ( sace2 ) in a total volume of 100 μl pbs . precipitates were washed thrice with lysis buffer and once with pbs , and subjected to a novex ® 4 12 % tris glycine gradient gel ( invitrogen ) under non reducing conditions . results are shown in fig2 . 1d sds page gel lanes were cut into ˜ 1 mm slices ( indicated as nr . 2 in fig3 ) using an automatic gel slicer and subjected to in gel reduction with dithiothreitol , alkylation with chloroacetamide and digestion with trypsin ( promega , sequencing grade ), essentially as described by van den berg et al . ( cell stem cell 6 : 369 , included herein by reference ). alternatively , immunoprecipitated proteins were reduced and alkylated on beads similarly as described above . nanoflow lc ms / ms was performed on either an 1100 series capillary lc system ( agilent technologies ) coupled to an ltq orbitrap xl mass spectrometer ( thermo ), or an easy nlc coupled to a q exactive mass spectrometer ( thermo ), operating in positive mode and equipped with a nanospray source . peptide mixtures were trapped on a reprosil c18 reversed phase column ( dr maisch gmbh ; column dimensions 1 . 5 cm × 100 μm , packed in house ) at a flow rate of 8 al / minute . peptide separation was performed on reprosil c18 reversed phase column ( dr maisch gmbh ; column dimensions 15 cm × 50 μm , packed in house ) using a linear gradient from 0 to 80 % b ( a = 0 . 1 % formic acid ; b = 80 % ( v / v ) acetonitrile , 0 . 1 % formic acid ) in 70 or 120 minutes and at a constant flow rate of 200 nl / minute . the column eluent was directly sprayed into the esi source of the mass spectrometer . mass spectra were acquired in continuum mode ; fragmentation of the peptides was performed in data dependent mode by cid or hcd . peak lists were automatically created from raw data files using the mascot distiller software ( version 2 . 3 ; matrixscience ) or proteome discoverer ( version 1 . 3 ; thermo ). the mascot algorithm ( version 2 . 2 ; matrixscience , uk ) was used for searching against a uniprot database ( release 2012 — 10 . fasta , taxonomy : homo sapiens , or macaca mulatta , or myotis lucifugus , or chlorocebus sabaeus , or felis catus , included herein by reference ). the peptide tolerance was set to 10 ppm and the fragment ion tolerance was set to 0 . 8 da for cid spectra ( ltq orbitrap ) or to 20 mmu for hcd ( q exactive ) spectra ). a maximum number of two missed cleavages by trypsin were allowed and carbamidomethylated cysteine and oxidized methionine were set as fixed and variable modifications , respectively . results are shown in fig2 . inhibition of hcov emc replication in huh7 cells by antibodies to dpp4 huh7 cells were incubated with 20 μg / ml goat polyclonal antiserum against dpp4 , a goat antiserum against ace2 , normal goat serum or left untreated . after 1 hour incubation , the cells were infected with hcov emc at a multiplicity of infection of 0 . 01 and left for 1 hour . cells were washed twice and again incubated with medium containing the respective antibodies . supernatant collected at 2 hours ( open bars ) and 20 hours ( closed bars ) was tested for presence of hcov using a taqman assay . results are shown as a ct in fig2 . serum from a macaque infected with hcov emc inhibits binding of recombinant s1 to huh7 cells . serum at a dilution of 1 : 20 , obtained from macaques at day 0 ( blue line ) and day 14 ( red line ) after infection with 5 × 107 tcid50 hcov emc , was preincubated for 1 hour at room temperature with 1 . 25 μg / ml recombinant s1 protein that was biotinylated and subsequently incubated on huh7 cells . after treatment with fitc labeled streptavidin , cells were analyzed for fluorescence . in gray background , binding using a control biotinylated protein is shown ( fig2 ). one aspect of the present invention relates to methods for forming crystals comprising fragments of dpp and viral protein as well as crystals comprising fragments of dpp and viral protein . crystallization of dpp is essentially known from , for example , u . s . pat . no . 7 , 344 , 852 or u . s . patent publication 2005 / 0260723 that are included herein by reference . in one embodiment of the present invention , a method for forming crystals comprising fragments of dppiv and viral protein is provided comprising forming a crystallization volume comprising fragments of dppiv and viral protein , one or more precipitants , optionally a buffer , optionally a monovalent and / or divalent salt and optionally an organic solvent ; and storing the crystallization volume in a container under conditions suitable for crystal formation . in yet another embodiment , a method for forming crystals comprising fragments of dppiv and viral protein is provided comprising forming a crystallization volume comprising fragments of dppiv and viral protein in solution comprising peg precipitant listed hereinbelow ; and storing the crystallization volume in a container under conditions suitable for crystal formation . peg precipitant 5 50 % w / v of precipitant , wherein the precipitant comprises one or more members of the group consisting of peg mme having a molecular weight range between 300 10000 , and peg having a molecular weight range between 100 10000 ph 5 9 . buffers that may be used include , but are not limited to , tris , bicine , cacodylate , acetate , citrate , mes and combinations thereof . additives optionally 0 . 05 to 0 . 8 m additives wherein the additives comprises sarcosine or 0 . 5 % to 25 % additives wherein the additives comprises xylitrol ; protein concentration 1 mg / ml 50 mg / ml ; temperature 1 ° c . to 25 ° c . in yet another embodiment , a method for forming crystals comprising fragments of dppiv and viral protein is provided comprising forming a crystallization volume comprising fragments of dppiv and viral protein ; introducing crystals comprising fragments of dppiv and viral protein as nucleation sites , and storing the crystallization volume under conditions suitable for crystal formation . crystallization experiments may optionally be performed in volumes commonly used in the art , for example , typically 15 , 10 , 5 , or 2 microliters or less . it is noted that the crystallization volume optionally has a volume of less than 1 microliter , optionally 500 , 250 , 150 , 100 , 50 or less nanoliters . it is also noted that crystallization may be performed by any crystallization method including , but not limited to , batch , dialysis and vapor diffusion ( e . g ., sitting drop and hanging drop ) methods . micro and / or macro seeding of crystals may also be performed to facilitate crystallization . in one variation , crystals comprising dppiv are formed by mixing a substantially pure dppiv fragment and a substantially pure s1 hcov emc fragment with an aqueous buffer containing a precipitant at a concentration just below a concentration necessary to precipitate the proteinaceous substance . one suitable precipitant for crystallizing fragments of dppiv and viral protein is polyethylene glycol ( peg ), which combines some of the characteristics of the salts and other organic precipitants ( see , for example , ward et al ., j . mol . biol . 98 : 161 , 1975 , and mcpherson , j . biol . chem . 251 : 6300 , 1976 . during a crystallization experiment , water is removed by diffusion or evaporation to increase the concentration of the precipitant , thus creating precipitating conditions for the protein . in one particular variation , crystals are grown by vapor diffusion in hanging drops or sitting drops . according to these methods , a protein / precipitant solution is formed and then allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration for producing crystals . the protein / precipitant solution continues to equilibrate until crystals grow . by performing submicroliter volume sized crystallization experiments , as detailed in u . s . pat . no . 6 , 296 , 673 , effective crystallization conditions for forming crystals of fragments of dppiv and viral protein complex are obtained . in order to accomplish this , systematic broad screen crystallization trials are performed on a dppiv / viral protein fragment complex using the sitting drop technique . one skilled in the art will recognize that the crystallization conditions provided herein can be varied and still yield protein crystals comprising fragments of dppiv and viral protein . as the conditions for the crystallization , physical and chemical factors such as a hydrogen ion concentration ( ph ), a kind of buffer used and a concentration thereof , a kind of a precipitant added and a concentration thereof , protein concentration , salt concentration , temperature and the like can be involved . a method for controlling and investigating the factors includes batch methods , dialysis methods , vapor diffusion methods ( hanging drop method , sitting drop method and the like ) and the like , described , for instance , in t . l . blundell et al ., protein crystallography , 59 82 ( 1976 ), published by academic press , or the like . the method for crystallization includes the batch methods , dialysis methods , vapor diffusion methods and the like . by the above method , physical and chemical factors such as a hydrogen ion concentration ( ph ), a kind and a concentration of the buffer used , and a kind and a concentration of the precipitant used , and physical and chemical factors such as protein concentration , salt concentration and temperature can be also determined . the hydrogen ion concentration ( ph ) can be adjusted with a buffer . it is desired that the buffer is a buffer having buffering action in a broad range of ph , and being capable of suppressing precipitation of a non proteinous crystal between the co existing ion in the solution used during crystallization and the precipitant or the like . the buffer includes tris hydrochloric acid buffer , phosphate buffer , cacodylate buffer , acetate buffer , citrate buffer , glycine buffer and the like . the precipitant may be a substance capable of elevating an effective concentration of the protein or changing a hydrogen ion concentration ( ph ) of the protein solution . generally , the precipitant includes salts such as ammonium sulfate , sodium sulfate , sodium phosphate , potassium phosphate , sodium citrate , ammonium citrate , sodium chloride , potassium chloride and ammonium chloride ; polyethylene glycols having various average molecular weights of about 200 , about 1000 , about 2000 , about 4000 , about 6000 , about 8000 , about 20000 or the like ; organic solvents such as 2 methyl 2 , 4 pentadiol , methanol , ethanol , isopropanol , butanol and acetone , and the like . the protein concentration may be a concentration suitable for crystallization , and it is desired that the protein concentration is , for example , 1 to 50 mg / ml , preferably 5 to 20 mg / ml , more preferably 7 to 15 mg / ml . it is desired that the temperature conditions are 3 ° c . to 25 ° c ., preferably 12 ° c . to 22 ° c . in the case where the crystallization is carried out by the batch method , the crystallization can be carried out by gradually adding a precipitant solution comprising a precipitant , buffer and the like , so as to form a layer on the top layer of the solution containing the dipeptidyl peptidase to give a mixture , or by gradually adding the solution comprising the dppiv / viral protein fragment complex , so that the solution is an upper layer of the precipitant solution to give a mixture . here , the mixture is allowed to stand in a tightly closed vessel or container . in the case where the crystallization is carried out by the dialysis method , the crystallization can be carried out by placing a solution comprising dppiv / viral protein fragment complex in a size exclusion semi permeable membrane , and placing a precipitant solution outside of the size exclusion semi permeable membrane as a reservoir solution , thereby diffusing the reservoir solution to the solution comprising the dppiv / viral protein fragment complex via the semi permeable membrane . in the case where the crystallization is carried out by the hanging drop method in the vapor diffusion method , the crystallization can be carried out by placing a mixed solution of a solution comprising the dppiv / viral protein fragment complex and a precipitant solution in a closed vessel allowing to be hanged at a position above the upper space of a reservoir in which the precipitant solution is contained as a reservoir solution , wherein the vapor pressure of the reservoir solution in the reservoir is set to be lower than that of the mixed solution . in the case where the crystallization is carried out by the sitting drop method in the vapor diffusion method , the crystallization can be carried out by placing a mixed solution comprising a solution comprising the dppiv / viral protein fragment complex and a precipitant solution in a closed vessel at a position higher than the liquid surface of a reservoir in which the precipitant solution is contained as a reservoir solution , wherein the vapor pressure of the reservoir solution in the reservoir is set to be lower than that of the mixed solution . the crystallization can be carried out by the sitting drop method from the viewpoint of obtaining excellent quality and large crystals . crystals comprising fragments of dppiv and viral protein have a wide range of uses . such crystals may , for example , be used to perform x ray or neutron diffraction analysis in order to determine the three dimensional structure of fragments of dppiv and viral protein and , in particular , to assist in the identification of its active site where fragments may bind . knowledge of the binding site region allows rational design and construction of ligands including inhibitors . crystallization and structural determination of fragments of dppiv mutants and / or viral protein mutants having altered bioactivity allows the evaluation of whether such changes are caused by general structure deformation or by side chain alterations at the substitution site . because dppiv protein levels may not always accurately reflect the levels of active dppiv enzyme , it may be useful to measure dppiv enzymatic activity in proteinaceous substances instead . use of a test system that is tested for dppiv assay in proteinaceous substances as diverse as plasma , serum , urine , saliva , tissue , live cells and cell extracts , and exudates is recommended . such a test system may be the dppiv / cd26 activity assay for biological samples provided by enzo ® life sciences ( on the world wide web at enzolifesciences . com ). a known dppiv inhibitor , such as p32 / 98 ( ki = 130 nm ) is preferably included for use as a control . to examine if cytokines decrease susceptibility to hcov emc infection through an effect on cell surface dpp4 expression , we analyzed dpp4 expression after treatment with different cytokines . all treatments were done in quadruplets ( 96 well experiments ) or triplicate ( 6 well and 24 well experiments ). cell cultures were grown for 24 to 48 hours and then changed to medium containing 1 % newborn calf serum , and treated with recombinant human ( r hu ) il 4 ( bd pharmingen ), r hu ifn y , r hu tnf a , r hu il 13 , r hu il 10 , r hu il 1 , r hu tgf beta ( peprotech inc .) and r hu ifn a ( roche ) at a concentration of 10 ng / ml , 48 hours before infection for a further evaluation of changes in dppiv surface protein expression and changes in susceptibility to hcov emc infection . in a first experiment , r hu tgf beta down regulates dpp4 expression and reduces the cells &# 39 ; susceptibility to virus infection and reduces virus replication . to examine if a compound decreases susceptibility to hcov emc infection through an effect on cell surface dpp4 expression , we analyze dpp4 expression after treatment with different compounds . huh 7 cells are grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ), supplemented with 10 % fetal bovine serum ( fbs ), sodium bicarbonate and 20 mm hepes buffer . all treatments are done in quadruplets ( 96 well experiments ) or triplicate ( 6 well and 24 well experiments ). cultures are grown for 24 to 48 hours and then changed to medium containing 1 % newborn calf serum , and treated with compound , i . e ., adenosine ( 300 μm ) or control vehicle for a further 48 hour evaluation of changes in dppiv surface protein expression and changes in susceptibility to hcov emc infection . in a first experiment , adenosine down regulates dpp4 expression and reduces the cells &# 39 ; susceptibility to virus infection and reduces virus replication . in a second experiment , inhibition of hcov emc replication in huh7 cells by soluble adenosine deaminase ( ada ) was demonstrated where inhibition with ace2 was negative . huh7 cells were incubated with different concentrations of recombinant soluble ada or recombinant soluble ace2 . after 1 hour incubation , the cells were infected with hcov emc at a multiplicity of infection of 0 . 01 . after 8 hours , cells were fixed and stained with a rabbit antiserum against hcov emc nsp4 and cells were counted . results are shown as number of infected cells per well . infection of huh7 cells is inhibited by recombinant soluble ada but not by recombinant soluble ace2 . the results are shown in fig2 . in a third experiment , inhibition of hcov emc replication in huh7 cells by soluble dpp4 was demonstrated . different concentrations of recombinant soluble dpp4 or recombinant soluble ace2 were incubated with hcov emc for 1 hour at 37 ° c . and used to infect huh7 cells . after 8 hours , cells were fixed and stained with a rabbit antiserum against hcov emc nsp4 and cells were counted . results are shown as number of infected cells per well . infection of huh7 cells is inhibited by recombinant soluble dpp4 but not by recombinant soluble ace2 . the results are shown in fig2 . the spike ( s ) protein of the recently emerged human coronavirus ( mers cov ) mediates infection by binding to the cellular receptor dipeptidyl peptidase 4 ( dpp4 ). here , we mapped the receptor binding domain in the s protein to a 231 amino acid fragment ( residues 358 588 ) by evaluating the interaction of spike truncation variants with receptor expressing cells and soluble dpp4 . antibodies to this domain much less so to the preceding n terminal region efficiently neutralize mers cov infection . it is herein now also shown by co immunoprecipitation and facs analyses that an internal region of the s1 of hcov emc consisting of 231 amino acids is sufficient to bind its receptor , dpp4 . it was also shown that the region elicits the most neutralizing antibodies against the virus . those results identified the receptor binding region of the s protein by convincing methods and the region contains major neuralization epitopes . additionally , the inventors herein further map the receptor binding domain ( rbd ) in the spike protein of the novel coronavirus emc ( hcov emc , now mers cov ). based on data obtained with bioinformatic tools they designed truncation variants of the s1 portion of hcov emc s ( emc s ) and showed that the s1 variant harboring residues 358 588 i ) co purifies with recombinant cd26 ( the hcov emc receptor ), binds to cellular cd26 in a facs based assay and elicits neutralizing antibodies in immunized rabbits with higher efficiency than the wt s1 subunit . just 10 years following the outbreak of the severe respiratory acute syndrome coronavirus ( sars cov ), the world is confronted with yet another deadly human coronavirus . the virus , first provisionally called human coronavirus emc ( hcov emc ) but now named mers cov , referring to its emergence in the middle east and to the respiratory syndrome it causes , belongs to the betacoronavirus genus lineage 2c . it has thus far been identified in over 50 patients from or linked to the arabian peninsula , approximately half of them being fatal . like with sars cov , patients affected by mers cov suffer from severe and often lethal lower respiratory tract infection . the epidemiology of mers cov is still enigmatic , but the geographical distribution of epidemiologically unlinked individuals points to intermittent , zoonotic transmission from a — so far unknown animal source , whereas a number of reported clusters indicate limited human to human spread . the main determinant of coronavirus tropism is the viral spike ( s ) protein as it mediates binding to a cell surface receptor . the mers cov s protein , a 1353 amino acid type i membrane glycoprotein , assembles into trimers that constitute the spikes or peplomers on the surface of the enveloped coronavirus particle . the protein combines the two essential entry functions , namely that of host receptor binding and membrane fusion , which are attributed to the n terminal ( s1 , residues 1 751 ) and c terminal ( s2 , residues 752 1353 ) half of the s protein , respectively . recently , we have identified dipeptidyl peptidase 4 ( dpp4 , also known as cd26 ), expressed in the human lung , as a functional receptor for mers cov . importantly , mers cov can also use the evolutionary conserved dpp4 of other species , most notably that of bats . coronaviruses bind to receptors via independently folded , generally about 150 300 residues long , receptor binding domains ( rbd ) present in their s1 subunit , of which the location within s1 can vary . thus , for the betacoronavirus mouse hepatitis virus ( mhv ), the binding to its ceacam receptor has been mapped to the n terminal ˜ 300 amino acids of the spike protein whereas for the sars cov of the same genus binding to the ace2 receptor maps to residues 323 502 of s1 . identification of the rbd can hence help the development of monoclonal antibodies and vaccines for the treatment and prevention of infection . the rbd is the most important target for neutralizing antibodies preventing virus receptor interaction . we previously used the s1 domain of mers cov fused to the fc region of human igg to demonstrate the interaction of s1 with dpp4 expressing cells and with soluble , i . e ., non membrane anchored dpp4 . to identify the receptor binding domain in the mers cov s1 subunit , we generated s1 fc protein chimeras with truncations at the c terminus and n terminus of the s1 domain . we considered a three domain structure of the mers cov s1 protein ( residues 1 357 , 358 588 and 589 747 ) based on the predicted location and structure of the rbd of two other betacoronaviruses , mhv and sars cov , of which the homologous regions for mers cov s map to the residues 18 351 and 379 580 , respectively . in addition , a soluble form of human dpp4 ( residues 39 766 ) was made , which was c terminally tagged with the fc region . these proteins were expressed in hek 293t cells after transfection of the expression plasmids and subsequently affinity purified from the cell culture supernatant using protein a sepharose beads as described . the fc region of purified sdpp4 fc was proteolytically removed using trypsin ( data not shown ). first , we analyzed the s1 fc proteins and c terminal si truncations thereof for their ability to interact with sdpp4 using a co purification assay . sdpp4 was efficiently co purified by the s1 fc variants encompassing residues 1 588 and 1 747 , whereas the 1 357 s1 fc variant was unable to bind sdpp4 . we next generated an s1 fc variant comprising residues 358 588 , a region homologous to the ace2 receptor binding domain in sars cov s1 . this s1 fc truncation variant efficiently bound soluble dpp4 , indicating that the dpp4 receptor binding domain is located within the 358 588 residues domain of the mers cov spike protein . we subsequently tested the ability of these s1 fc variants to bind to hek 293t cells transiently expressing dpp4 by using flow cytometry . the s1 fc variants encompassing residues 1 588 and 358 588 bound to dpp4 expressing hek 293t cells with efficiencies comparable to the full length s1 protein , whereas no binding was observed with the 1 357 s1 fc variant . these data show the 358 588 amino acids s1 region to be essential and sufficient for binding to dpp4 expressing cells , consistent with the results of the sdpp4 interaction study . finally polyclonal antibodies were raised in rabbits against the 1 747 , 1 357 and 358 588 s1 fc variants ( davids biotechnology gmbh , germany ). the sera , which displayed equal elisa titers towards its antigen ( 1 : 300 , 000 , data not shown ), were tested for their ability to neutralize virus infectivity . antibodies raised against the 358 588 s1 fc variant efficiently neutralized virus infectivity , superior to those raised against the 1 747 and 1 357 s1 fc variants . this indicates that neutralizing epitopes within s1 are primarily localized to the rbd region . the elicited antibodies are likely to block the interaction of the spike protein with dpp4 thereby neutralizing mers cov infectivity . the results demonstrate the preferred potential of s1 protein and of the 358 588 s1 polypeptide or functional fragments thereof reactive with the mers cov neutralizing antibody for use as subunit vaccines with a high biosafety profile compared to vaccines based on inactivated viruses or live attenuated virus . except for the betacoronavirus mhv , which binds to its ceacam receptor through a domain in the n terminal part of its s1 protein , the rbds of all other coronaviruses that engage protein receptors and that have been mapped occur in the c terminal portion of the s1 subunit . examples also include the alphacoronaviruses binding to ace2 ( hcov nl63 ) and apn ( e . g ., tgev , hcov 229e ). in this study , we have experimentally mapped the rbd of mers cov to a 231 amino acid fragment ( residues 358 588 ) within the spike protein . this domain nicely corresponds with the s1 region recently anticipated to interact with the dpp4 receptor on the basis of theoretical s1 structure predictions . the rbd in the mers cov s1 protein localizes in the same region where the sars cov s protein interacts with its ace2 receptor . the sars cov rbd structure displays a five stranded β sheet core structure ( β1 4 and β7 ) maintaining the overall domain conformation , and a long extended loop containing two anti parallel β sheets ( β5 and β6 ) responsible for receptor binding {{ }}. intriguingly , compared to sars cov , the rbd of mers cov contains a relatively conserved core domain but a highly variable loop region , tentatively explaining the differential receptor usage . crystallization and structure analysis of this mers cov rbd region in complex with dpp4 will give detailed insight into the virus receptor binding interface . here we show that mers coronavirus ( mers - cov ) replicates in cells of different species using dipeptidyl peptidase 4 ( dpp4 ) as a functional receptor . this suggests a broad host species tropism allowing zoonotic transmission from many animal species . here we show contrasting dpp4 receptor functionality in different animal species . resistance of ferrets to mers - cov infection was due to the inability to bind mers - cov as a result of amino acid variation in the ferret dpp4 β - propeller region . in contrast , dpp4 expressing respiratory epithelial cells in the lower — but not upper — respiratory tract of cynomolgus macaques were targeted by mers - cov , which resulted in relatively mild disease . variable dpp4 expression and adenosine deaminase ( ada )— shown to act as a natural antagonist for mers - cov infection — may potentially modulate mers - cov infection . our findings illuminate the role of dpp4 sequence and expression variability in host range restriction and outcome of respiratory mers - cov infection and lead us to conclude that mers - cov receptor sequence and expression variability determine host range restriction of lower respiratory mers - cov infection . coronaviruses ( covs ) usually cause common colds in humans but zoonotic transmission occasionally introduces more pathogenic viruses into the human population as was demonstrated by the severe acute respiratory syndrome ( sars ) outbreak . in 2012 a previously unknown human coronavirus ( cov ), now named middle east respiratory syndrome cov ( mers - cov ), was isolated from the sputum of a 60 - year - old man in saudi arabia who presented with acute pneumonia with a fatal outcome . to date , several infection clusters have been reported over a one - year period with around 50 % of the reported human cases being fatal . although limited human - to - human transmission has been observed , it is feared that by acquiring additional mutations mers - cov may spread more easily . mers - cov represents a novel betacoronavirus species with the closest known relatives being clade 2c bat covs , detected in diverse species of bats but not yet in any animal species from the arabian peninsula . although mers - cov replicates in cells of different species including bats , pigs and ( non -) human primates , its ability to infect different animal species may be restricted given the fact that hamsters were shown to resist mers - cov infection . therefore , a further understanding of factors that determine host restriction and viral transmission need to be revealed by studies in different animal species . herein we identified dipeptidyl peptidase 4 ( dpp4 ) as a functional mers - cov receptor in human and bat cells . to further analyse dpp4 usage by mers - cov in vivo , ferrets ( n = 4 ), known to be susceptible to several respiratory viruses including sars - cov and influenza viruses , were inoculated intratracheally with mers - cov . the animals did not seroconvert and only low levels of virus were detected by rt - qpcr in respiratory swabs at 1 - 2 days post infection ( dpi ). in vitro , ferret primary kidney cells could not be infected with mers - cov despite dpp4 surface expression , while transfection of these cells with human dpp4 ( hdpp4 ) rendered the cells susceptible , suggesting that ferret dpp4 ( fdpp4 ) does not efficiently bind mers - cov . consistently , mdck cells transfected with fdpp4 did not bind to synthetic mers - cov spike ( s1 ) protein and were not infected by the virus ( fig3 b , c ). dpp4 is an ectoenzyme that cleaves dipeptides from hormones , chemokines and cytokines by its conserved c - terminal ca - hydrolase domain of the protein , while its n - terminal eight - blade β - propeller domain contains more sequence variability . by constructing dpp4 chimeras we observed that the blades 4 and 5 containing hdpp4 domain ( residues 246 - 505 ) could confer to ferret dpp4 the ability to bind to s1 and to mediate mers - cov infection when expressed in non - susceptible cells ( fig3 b , c ). a quick change site - directed mutagenesis kit ( stratagene ) was used to construct different hdpp4 point mutants . the presence of the correct mutations and absence of undesired mutations was confirmed by sequencing analysis . plasmids were transfected into mdck cells in triplicate , after 24 h incubation individual wells were split to determine dpp4 cell surface expression , s1 - binding and susceptibility to mers - cov infection on the same transfected cell culture . consistently , substitution of selected solvent exposed residues present in blades 4 and 5 of hdpp4 by those occurring at these positions in fdpp4 , abrogated dpp4 &# 39 ; s capacity to bind to s1 and to mediate mers - cov cell susceptibility upon transfection , suggesting that these residues are involved in mers - cov binding and entry ( fig3 d , e ). reciprocal substitutions of these amino acids in fdpp4 however , did not confer s1 binding , demonstrating the complexity of the interaction in the face the highly polymorphic nature of these two blades . the identified residues also are critical in binding the human enzyme adenosine deaminase ( ada ), a natural dpp4 ligand that is involved in the development and maintenance of the immune system . using recombinant ada , significant inhibition of mers - cov infection and spike protein binding was demonstrated revealing a natural occurring antagonist able to block mers - cov infection . the data on the co - crystallization of the receptor binding domain of s1 and dpp4 are in line with the data presented . phylogenetic analysis of the virus binding region of dpp4 indicated that human , macaque , horse and rabbit dpp4 cluster together as do dpp4 &# 39 ; s from cattle , pig and bats , that are somewhat more distantly related . small animals including ferret , mice and most likely hamsters , shown to resist mers - cov infection , are more divergent in the dpp4 virus binding region , which at least in the case of ferrets has consequences for mers - cov binding . considering the highly conserved virus binding region in macaque dpp4 as compared to hdpp4 , we first confirmed the use of cynomolgus macaque dpp4 as a functional mers - cov receptor . dpp4 antibodies blocked mers - cov infection of macaque primary kidney cells in vitro . besides macaques , rabbits may be a potential animal model for mers - cov infection ; ex vivo inoculation of rabbit lung and kidney tissues revealed susceptibility to mers - cov . we subsequently inoculated ten young adult cynomolgus macaques intratracheally with mers - cov and euthanized them at 1 ( n = 4 , macaques 1 - 4 ), 4 ( n = 4 , macaques 5 - 8 ) and 28 dpi ( n = 2 , macaques 9 and 10 ). all animals remained free of severe clinical signs and maintained a rhythmic pattern of body temperatures fluctuating between 35 ° c . ( night ) and 39 ° c . ( day ) that seemed slightly elevated after inoculation . neutralizing antibodies with titers 40 - 80 were detected in the two mers - cov infected macaques that were euthanized at 28 dpi . upon necropsy , there were a few mild focal red - grey slightly depressed areas affecting less than 5 % of the lung tissue , although one lobe of macaque 7 had a dark red rim with evidence of suppurative bronchopneumonia , consistent with the detection of escherichia coli bacteria in this lobe . mers - cov mrna was detected at highly variable levels in pharyngeal and nasal swabs on 1 to 11 dpi and at low levels in rectal swabs on 2 and 3 dpi . in addition , mers - cov was detected by rt - qpcr in the lungs , nasal septum , serum and spleen and in one animal — macaque 1 — also in the kidney , liver , colon and urine at 1 dpi . infectious virus was detected only in one pharyngeal swab sample and to a limited extent in the lungs . using a probe that targets the mers - cov nucleocapsid gene , hybridization was observed in epithelial cells in bronchioles , and in moderate numbers of type 2 and few type 1 pneumocyte - resembling cells in the alveoli at 1 dpi while at 4 dpi very few cells were found positive . consistent with activation of cytokines like ccl3 , the lungs showed mild alveolitis , characterized by thickening of the alveolar septa with infiltration of few neutrophils and macrophages and moderate type 2 hypertrophy and hyperplasia at 4 dpi . in the alveolar lumina there were increased numbers of alveolar macrophages and occasionally small amounts of edematous fluid with fibrin and few neutrophils . consistent with the capacity of the virus to induce syncytia in vitro , syncytial cells were seen . by applying a technique that enables successive staining of the same tissue section , tropism of mers - cov for cells expressing dpp4 in vivo was demonstrated . thus , the experimental infection of young adult macaques with mers - cov revealed that macaque dpp4 positive cells in the lower respiratory tract can be infected with mers - cov but the associated pathological changes are relatively mild , indicating that young adult macaques are at best a suboptimal mers - cov animal model for the often fatal mers - cov infection in humans . abundant ace2 expression in the respiratory tract has been suggested to facilitate rapid spread of sars - cov , a critical factor in the rapid induction of innate immune responses that drive the acute respiratory distress syndrome . in non - infected macaques dpp4 expression was restricted to non - ciliated cells , type 2 cells and endothelial cells whereas no staining was observed in ciliated epithelial cells of the ( upper ) respiratory tract . the absence of dpp4 on the upper respiratory tract epithelial cells , consistent with the inability to detect viral antigen in these cells , therefore may limit efficient virus transmission through the upper respiratory route . kidneys , liver , intestine , and sub mucosal glands of the upper respiratory tract were found to contain varying levels of dpp4 , which mainly localized to the apical side of the cells . enhanced dpp4 expression was observed in the lungs of the bacterial co - infected macaque 7 , which excreted infectious virus in the pharyngeal swab and displayed a higher body temperature . we observed that lps stimulation of in vitro differentiated macrophages enhanced dpp4 expression . attempts to infect these cells were unsuccessful , likely due to ada production by these cells . interestingly , dpp4 was virtually absent in the lower respiratory tract epithelium of ferrets but could be visualized in the kidneys of these animals . contrastingly , relatively strong dpp4 expression was observed on different cell types in human lungs , including a mers - cov infected individual . in several pathological conditions such as viral infections and type 2 diabetes increased levels of ( soluble ) dpp4 have been demonstrated . thus , relatively low levels of dpp4 expression in the lungs of young adult macaques could partly explain the mild infection observed after mers - cov infection but further studies need to reveal the role of varying dpp4 and ada expression levels in regulating mers - cov replication in vivo . our findings demonstrate that the host range potential of the emerging novel human mers - cov is primarily determined by the mers - cov binding to and tissue distribution of dpp4 . the co - localisation of dpp4 with mers - cov in the lower respiratory tract of mers - cov infected non - human primates ( in bronchioles and alveoli ), and the inability to infect ferrets further supports the sole involvement of dpp4 as a functional receptor in mers - cov entry . variable levels of dpp4 expression in the lower respiratory tract may impose mers - cov host range restriction and explain why studies in rhesus macaques have not been successful to reproduce the severe disease seen in humans . future studies need to unravel the significance of variable dpp4 expression in mers - cov patients , for example as a result of co morbidities like microbial infections , type 2 diabetes or aging . the hdpp4 cdna was obtained as described . total rna was isolated from ferret primary kidney cells using rneasy mini kit ( qiagen ) and cdnas were synthesized by using the superscript reverse transcriptase ( life technologies ). the complete dpp4 genes were amplified using pfu ultra ii fusion hs dna polymerase ( stratagene ) and cloned into the pcdna 3 . 1 expression vector ( life technologies ). human to ferret dpp4 mutants of cdna constructs were made by utilizing unique restriction enzyme sites shared by human and ferret dpp4 . pst i can cut human and ferret dpp4 into three fragments ( human , amino acid 1 - 246 , 247 - 504 and 505 - 766 and ferret , amino acid 1 - 245 , 246 - 503 and 504 - 765 ). the middle fragment of human and ferret dpp4 was exchanged between human and ferret , the final plasmid constructs contained different combinations of fragments : human - ferret - human ( hfh ) or ferret - human - ferret ( fhf ). a quick change site - directed mutagenesis kit ( stratagene ) was used to construct different hdpp4 point mutants . the presence of the correct mutations and absence of undesired mutations was confirmed by sequencing analysis . plasmids were transfected into mdck cells in triplicate , after 24 h incubation individual wells were split to determine dpp4 cell surface expression , s1 - binding and susceptibility to mers - cov infection on the same transfected cell culture . s1 binding and infection were corrected for dpp4 cell surface expression as determined by the goat polyclonal antiserum against dpp4 ( r & amp ; d systems ), a secondary fitc conjugated rabbit anti goat serum followed by facs analysis . sequence alignment was performed by using clustalw in the mega5 . 0 software package ( www . megasoftware . net ), and the trees were constructed by using the neighbor - joining method with p - distance ( gap / missing data treatment ; complete deletion ) and 1 , 000 bootstrap replicates as in mega version 5 . 0 . a plasmid encoding mers - cov s1 - fc was generated by ligating a fragment encoding the s1 domain ( residues 1 - 747 ) 3 ′- terminally to a fragment encoding the fc domain of human igg into the pcaggs expression vector . likewise , an s1 - fc expression plasmid was made the fipv s1 domain ( isolate 79 - 1146 ; residues 1 - 788 ). fc chimeric proteins were expressed by transfection of the expression plasmids into hek - 293t cells and affinity purified from the culture supernatant using protein a sepharose beads ( ge healthcare ). s1 binding of cells was measured by incubating 105 cells with 15 mg / ml of s1 - fc followed by incubation with fitc or dylight - 488 - labelled goat - anti - human igg antibody and analysis by flow cytometry . virus stocks of mers - cov ( emc isolate ) were prepared . transfected cos - 7 cells , huh - 7 and primary ferret and macaque kidney cells were inoculated with mers - cov for 1 h with high moi . after washing the cells were incubated with medium containing 1 % fetal bovine serum . alternatively we used thin cut slices from the lungs and kidneys of rabbits that were incubated in culture medium with virus for 24 h . at 8 or 24 h after infection cells were fixed with formaldehyde and stained using rabbit - anti - sars - cov nsp4 antibodies that are cross - reactive for hcov - emc , according to standard protocols using a fitc conjugated swine - anti - rabbit antibody as a second step . primary ferret or macaque kidney cells were preincubated with antibodies to dpp4 ( polyclonal goat - anti dpp4 immunoglobulin , r & amp ; d systems ) at 20 μg / ml to block mers - cov infection . recombinant human ada ( r & amp ; d systems ) was preincubated with hdpp4 transfected cells or huh7 cells for 1 h after which the cells were infected with mers - cov for 8 h and processed . ten cynomolgus macaques ( macaca fascicularis ), 3 - 5 years old with active temperature transponders in the peritoneal cavity ( n = 3 ), were inoculated with 5 × 106 tcid50 of mers - cov via the intranasal and intratracheal route . in addition , four ferrets ( mustello fuoris ) were inoculated with 1 × 106 tcid50 of mers - cov via the intranasal and intratracheal route . animals were checked daily for clinical signs . just before infection and at different dpi , animals were anesthetized with ketamine and nasal , pharyngeal , and rectal swabs were taken , which were placed in 1 ml dulbecco &# 39 ; s modified eagle &# 39 ; s medium supplemented with 100 iu penicillin / ml and 100 □ g of streptomycin / ml ( virus transport medium ) and frozen at − 70 ° c . until rt - pcr analysis . the animals were euthanized at different days ( day 1 , 4 or 28 ) p . i . by exsanguination under ketamine anesthesia . approval for animal experiments was obtained from the institutional animal welfare committee ( nr emc 2808 ). necropsies were performed according to a standard protocol . for semi - quantitative assessment of gross pathology , the percentage of affected lung tissue from each lung lobe was determined at necropsy , recorded on a schematic diagram of the lung and the area of affected lung tissue was subsequently calculated ( gross pathology score ). one lung of each monkey was inflated with 10 % neutral - buffered formalin by intrabronchial intubation and suspended in 10 % neutral - buffered formalin overnight . samples were collected in a standard manner ( from the cranial , medial and caudal parts of the lung ), embedded in paraffin , cut at 3 om and used for immunohistochemistry ( see below ) or stained with hematoxylin and eosin ( h & amp ; e ). the lung , liver , spleen , kidney , intestine , trachea , and tracheobronchial lymphnode h & amp ; e sections were examined by light microscopy . the ish probes targeting the nucleocapsid gene of mers - cov were designed by advanced cell diagnostics ( hayward , calif .) and ish was performed according to the manufacturer &# 39 ; s instructions and ish staining was visualized using substrate fast red ( pink ). controls included probes against sars - cov nucleocapsid protein and tissues from non infected animals . family consent was granted for limited postmortem tissue retrieval from a mers - cov patient in the uk , consisting of a 20 - cm - long midline incision in lower chest and upper abdomen , from which tissue samples were collected from both lungs . archival paraffin - embedded human tissue sections were obtained from the department of pathology , erasmus m c . four historic macaque controls were used as mock ( pbs ) infected . for histological analysis , samples were placed in 10 % neutral - buffered formalin and further processed for routine immunohistochemistry . serial 3 μm lung sections were stained using according to standard protocols using antibodies to dpp4 ( polyclonal goat - anti dpp4 immunoglobulin , r & amp ; d systems . for phenotyping to test ddp4 expression of mers - cov infected cells , we used a destaining - restaining technique . briefly , the precipitate used for visualization of mers - cov antigen staining was dissolved in graded 100 %- 70 % alcohols . to detach the primary antibody and red immunohistochemistry signals , slides were soaked in eluding buffer ( 5 ml 0 . 1m hcl , 95 ml 0 . 1m nacl containing 1m glycine ) for 2 hours . the sections were treated with two 5 min intervals heating in citric acid buffer ph 6 . 0 to denature any undetached primary antibody . the slides were then incubated with antibodies against dpp4 in pbs / 0 . 1 % bsa for 1 hour at rt . after washing , sections were incubated with horseradishperoxidase labeled anti - goat igg 1 / 100 in pbs / 0 . 1 % bsa for 1 hour at rt . peroxidase activity was revealed by incubating slides in 3 , 3 ′- diaminobenzidine - tetrachlorhydrate ( dab ) ( sigma ) for 3 - 5 minutes , resulting in a brown precipitate , followed by counterstaining with hematoxylin . samples were analysed with the upe pcr and confirmed by a nucleocapsid specific pcr . rna from 200 □ 1 of culture supernatant was isolated with the magnapure lc total nucleic acid isolation kit ( roche ) and eluted in 100 □ 1 . mers - cov rna was quantified on the abi prism 7700 , with the taqman ® fast virus 1 - step master mix ( applied biosystems ) using 20 □ 1 isolated rna , 1 × taqman mix , 0 . 5 u uracil - n - glycosylase , 45 pmol forward primer ( 5 ′- gggtgtacctcttaatgccaattc - 3 ′), 45 pmol reverse primer ( 5 ′- tctgtcctgtctccgccaat - 3 ′) and 5 pmol probe ( 5 ′- fam - acccctgcgcaaaatgctggg - bhq1 - 3 ′). amplification parameters were 5 min at 50 ° c ., 20 sec at 95 ° c ., and 45 cycles of 3 s at 95 ° c ., and 30 sec at 60 ° c . rna dilutions isolated from an mers - cov stock were used as a standard . lung tissue samples ( 0 . 3 - 0 . 5 gram ) were taken for rt - pcr and microarray analysis in rna - later ( ambion , inc .). rna was isolated from homogenized post mortem tissue samples using trizol reagent ( invitrogen ) and the rneasy mini kit ( qiagen ). cdna synthesis was performed with ˜ 1 □ g total rna and superscript iii rt ( invitrogen ) with oligo ( dt ), according to the manufacturer &# 39 ; s instructions . semi - quantitative rt - pcr was performed as described previously to detect mers - cov and to validate cellular gene expression changes as detected with microarrays of ccl3 ( applied biosystems ). differences in gene expression are represented as the fold change in gene expression relative to a calibrator and normalized to a reference . gapdh ( glyceraldehydes - 3 - phosphate dehydrogenase ) was used as endogenous control to normalize quantification of the target gene . the samples from the mock - infected macaques were used as a calibrator . average results (□ s . e . m .) for groups were expressed as fold change compared to pbs - 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