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CORD-19:12dd53397f3ddd026cfbfe6dfd1aaefc565b6aeb JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-169 Sentence denotes Simultaneous Detection of Antibodies to Mouse Hepatitis Virus Recombinant Structural Proteins by a Microsphere-Based Multiplex Fluorescence Immunoassay ᰔ Downloaded from
T2 171-179 Sentence denotes Abstract
T3 180-371 Sentence denotes We describe a new microsphere-based multiplex fluorescent immunoassay (MFI) using recombinant mouse hepatitis virus (MHV) proteins to detect antibodies to coronaviruses in mouse and rat sera.
T4 372-652 Sentence denotes All the recombinant proteins, including nucleocapsid (N) and 3 subunits of spike protein, S1, S2, and Smid, showed positive reactivity in MFI with mouse antisera to 4 MHV strains (MHV-S, -A59, -JHM, and -Nu67) and rat antiserum to a strain of sialodacryoadenitis virus (SDAV-681).
T5 653-896 Sentence denotes The MFI was evaluated for its diagnostic power, with panels of mouse sera classified as positive or negative for anti-MHV antibodies by enzyme-linked immunosorbent assay (ELISA) using MHV virion antigen and indirect fluorescent antibody assay.
T6 897-1099 Sentence denotes The reactivities of 236 naturally infected mouse sera were examined; 227 samples were positive by MFI using S2 antigen (96% sensitivity), and 208 samples were positive using N antigen (88% sensitivity).
T7 1100-1254 Sentence denotes Based on the assessment by MFI using the S2 and N antigens, only 3 serum samples showed double-negative results, indicating a false-negative rate of 1.3%.
T8 1255-1428 Sentence denotes In 126 uninfected mouse sera, including 34 ELISA false-positive sera, only 7 samples showed false-positive results by MFI using either the S2 or N antigen (94% specificity).
T9 1429-1559 Sentence denotes Similarly, the S2 and N antigen-based MFI was 98% sensitive and 100% specific in detecting anticoronavirus antibodies in rat sera.
T10 1560-1825 Sentence denotes Thus, this MFI-based serologic assay using the S2 and N antigens promises to be a reliable diagnostic method, representing a highly sensitive and specific alternative to traditional ELISA for detection of coronavirus infections in laboratory mouse and rat colonies.
T11 1827-1921 Sentence denotes Mouse hepatitis virus (MHV) is one of the most prevalent infectious agents of laboratory mice.
T12 1922-2108 Sentence denotes At present, the most widely used methods for diagnosis of MHV infections are serological tests by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody assay (IFA).
T13 2109-2221 Sentence denotes Although IFA is highly specific, it is subjective and is not suitable for screening of large numbers of samples.
T14 2222-2304 Sentence denotes ELISA has better throughput but requires larger amounts of serum samples than IFA.
T15 2305-2681 Sentence denotes Moreover, the MHV virion antigen currently used in ELISA is propagated in mouse cell lines, but purified viral antigens have some disadvantages, such as poor yields of purified viruses, less reproducibility of antigens, and contamination with cellular proteins, resulting in difficulties in quality control of antigens and frequent false-positive results in serological tests.
T16 2682-2862 Sentence denotes The recombinant antigen-based antibody detection tests are well known to provide high reproducibility, are easy to standardize, and do not require cultivation of infectious agents.
T17 2863-3097 Sentence denotes Although the use of recombinant antigens in serological tests has been widely reported for a variety of infectious diseases (1, 4, 7, 13, 14, 17, 19, 21, 23) , no serological test using recombinant MHV antigens is currently available.
T18 3098-3220 Sentence denotes MHV is composed of three major structural proteins: nucleocapsid protein (N), spike protein (S), and membrane protein (M).
T19 3221-3295 Sentence denotes The N, S, and M proteins all contribute to inducing host immune responses.
T20 3296-3429 Sentence denotes The N protein is the most abundant protein in the MHV virion, with an amino acid sequence that is highly conserved among MHV strains.
T21 3430-3555 Sentence denotes The S protein is the major target of the neutralizing antibodies and induces the earliest antibody responses (2, 5, 11, 18) .
T22 3556-3702 Sentence denotes Therefore, we chose the N and S proteins as candidates to generate recombinant antigens for the detection of antibody responses to MHV infections.
T23 3703-3909 Sentence denotes The microsphere assay technology developed by Luminex Corp. is suited to a high-throughput immunoassay for simultaneous detection of antibodies to multiple pathogens and antigens (4, 8, 9, 10, 12, 24, 25) .
T24 3910-4074 Sentence denotes In a microsphere-based multiplex fluorescent immunoassay (MFI), microspheres function as the solid phase to which protein antigens from various pathogens are bound.
T25 4075-4227 Sentence denotes One hundred sets of color-coded beads are used, and each bead set is distinguishable by its fluorescent emission when excited by a classification laser.
T26 4228-4501 Sentence denotes Since each of the bead sets can be conjugated with a unique antigen, each bead represents a separate antibody reaction after incubation with the serum samples to be tested, followed by a fluorescently labeled detection reagent and fluorescent emission by a detection laser.
T27 4502-4624 Sentence denotes Another distinct advantage of the MFI method is that a trace amount of serum sample is sufficient for multiplex detection.
T28 4625-4753 Sentence denotes Here we describe the usefulness of the MFI method using recombinant N and S antigens in serological diagnosis of MHV infections.
T29 4754-4961 Sentence denotes The sensitivity and specificity of recombinant antigen-based MFI were evaluated in comparison with those of the cell culture virion-based ELISA and IFA using experimentally and naturally infected mouse sera.
T30 4962-5129 Sentence denotes Furthermore, our MFI system was applied to serological testing of coronavirus infections in rats, including sialodacryoadenitis virus (SDAV) and rat coronavirus (RCV).
T31 5130-5229 Sentence denotes The MHV-S strain was obtained from ATCC and propagated in a mouse astrocytoma cell line (DBT) (6) .
T32 5230-5374 Sentence denotes DBT cells were grown in Eagle's minimum essential medium (MEM) containing 10% fetal bovine serum at 37°C with 5% CO 2 in a humidified incubator.
T33 5375-5401 Sentence denotes Animals and serum samples.
T34 5402-5627 Sentence denotes Antisera to the MHV-S, -A59, -JHM, and -Nu67 (20) strains were prepared from 7-week-old female ICR mice inoculated oronasally with 1 ϫ 10 6 to 1 ϫ 10 7 50% tissue culture infective doses (TCID 50 )/100 l of MHV culture stock.
T35 5628-5775 Sentence denotes Antiserum to SDAV-681 was prepared from 7-week-old female Wistar rats oronasally inoculated with 1 ϫ 10 6 TCID 50 /100 l of SDAV-681 culture stock.
T36 5776-5911 Sentence denotes At 4 weeks postinfection, serum was collected from the experimentally infected mice, and rats were euthanized by isoflurane inhalation.
T37 5912-6055 Sentence denotes For serial serum collection, serum samples were obtained from the tail veins of mice on days 0, 7, 14, 21, and 28 after inoculation with MHV-S.
T38 6056-6221 Sentence denotes The animal studies were approved by the institutional animal care and use committees of the University of Tsukuba and the Central Institute for Experimental Animals.
T39 6222-6391 Sentence denotes Uninfected control sera were collected from ICR, C3H, C57BL/6, and BALB/cA mice, and Wistar rats were purchased from CLEA Japan Inc. (Tokyo, Japan) at 6 to 30 weeks old.
T40 6392-6569 Sentence denotes Their source colonies were specified to be free of MHV or SDAV/RCV, and the sera collected were confirmed to be negative for MHV or SDAV/RCV by ELISA and IFA as described below.
T41 6570-6993 Sentence denotes To evaluate the specificity and sensitivity of the microsphere-based multiplex fluorescent immunoassay (MFI) method for detecting antibodies in mice and rats naturally infected with MHV or SDAV/RCV, mouse and rat sera from a variety of sources submitted to the Central Institute for Experimental Animals for health screening were tested by MFI as well as ELISA and IFA for detection of anti-MHV or anti-SDAV/RCV antibodies.
T42 6994-7041 Sentence denotes Viral RNA preparation and RT-PCR amplification.
T43 7042-7102 Sentence denotes DBT cells were infected with MHV-S and incubated for 2 days.
T44 7103-7211 Sentence denotes The infected cells were collected, and RNA was extracted with RNAiso Plus reagent (Takara Bio, Otsu, Japan).
T45 7212-7333 Sentence denotes For cDNA synthesis, aliquots of 100 ng of RNA were incubated at 65°C for 5 min with 0.5 M reverse oligonucleotide primer.
T46 7334-7629 Sentence denotes After chilling on ice, 200 U of SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) was added, followed by incubation at 50°C for 1 h in the presence of 0.5 mM (each) deoxynucleoside triphosphates (dNTPs), 5 mM dithiothreitol, and 1ϫ firststrand buffer in a reaction volume of 20 l.
T47 7630-7864 Sentence denotes Aliquots of 2 l of cDNA reaction mixture were added to PCR mixtures containing 0.6 M (each) forward and reverse primers, 0.2 mM (each) dNTP, 1ϫ Pyrobest buffer II, and 2.5 U of Pyrobest DNA polymerase (Takara Bio) in a volume of 50 l.
T48 7865-8040 Sentence denotes PCR was carried out for 35 cycles of 15 s at 94°C for denaturation, 30 s at 60°C for annealing, and 2 min at 72°C for extension using a PCR thermal cycler (TP600; Takara Bio).
T49 8041-8681 Sentence denotes The sequences of oligonucleotide primers used for reverse transcription-PCR (RT-PCR) were as follows: S1-FW, 5Ј-CCGGATCCATGTTGTCCGTGTTTATT T-3Ј (BamHI site in italics); S1-RV, 5Ј-TAGTCGACTCACGTGCGCGCGCG GTGAGCA-3Ј (SalI site in italics); S2-FW, 5Ј-CCGGATCCTCTGTCAGTAC GGGTTATA-3Ј (BamHI site in italics); S2-RV, 5Ј-GGTCGACCTCAATCCTC ATGGGAGGAAAT-3Ј (SalI site in italics); Smid-FW, 5Ј-ATGGATCCGGAT TTAATGATGTGGC-3Ј (BamHI site in italics); Smid-RV, 5Ј-CCGTCGACTC ATAGAATTTCTTGTA-3Ј (SalI site in italics); N-FW, 5Ј-CTAGAATTCATG TCTTTTGTTCCTGGG-3Ј (EcoRI site in italics); and N-RV, 5Ј-GCGCTCGA GTTACACATTAGAGTCATC-3Ј (XhoI site in italics).
T50 8682-8744 Sentence denotes PCR products were separated by 1% agarose gel electrophoresis.
T51 8745-8869 Sentence denotes Bands were excised from the gels, and the products were purified using a QIAquick gel extraction kit (Qiagen, Valencia, CA).
T52 8870-8893 Sentence denotes Cloning and sequencing.
T53 8894-9158 Sentence denotes Purified PCR products were cloned into the plasmid vector pGEM-T Easy (Promega, Madison, WI), and the sequences were determined using a BigDye PCR sequencing kit (Applied Biosystems, Carlsbad, CA) and the ABI Prism 3100-Avant genetic analyzer (Applied Biosystems).
T54 9159-9317 Sentence denotes The full region of the N gene was subcloned into the EcoRI and XhoI sites of the expression vector pGEX-5X-1 (GE Healthcare, Little Chalfont, United Kingdom).
T55 9318-9492 Sentence denotes The partial regions of the S gene, S1, S2, and Smid (Fig. 1A) , were subcloned into the BamHI and SalI sites of the expression vector pET28a(ϩ) (Novagen, Darmstadt, Germany).
T56 9493-9685 Sentence denotes After a ligation reaction carried out using the Ligation High reagent (Toyobo, Osaka, Japan), Escherichia coli BL21 or BL21(DE3) competent cells were transformed with the recombinant plasmids.
T57 9686-9738 Sentence denotes Expression and purification of recombinant proteins.
T58 9739-9944 Sentence denotes To produce the glutathione S-transferase (GST)-fused recombinant N protein, transformed E. coli BL21 was cultured in 1 liter of Luria-Bertani (LB) medium containing 100 g/ml ampicillin at room temperature.
T59 9945-10188 Sentence denotes When the culture media had reached an optical density at 600 nm (OD 600 ) of 0.6 to 0.8, isopropyl ␤-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.8 mM, followed by incubation for an additional 2 h at room temperature.
T60 10189-10268 Sentence denotes Cultures were placed on ice for 10 min and centrifuged at 2,000 ϫ g for 10 min.
T61 10269-10388 Sentence denotes Next, the supernatant was removed, and the pellet was suspended in 50 ml of Dulbecco's phosphate-buffered saline (PBS).
T62 10389-10558 Sentence denotes Then, Triton X-100 at a final concentration of 1% and dithiothreitol at a final concentration of 1 mM were added, and the mixture was stored at Ϫ80°C until purification.
T63 10559-10642 Sentence denotes The bacterial suspension was thawed and sonicated on ice 15 times with 10-s pulses.
T64 10643-10802 Sentence denotes The soluble fraction was collected after centrifugation at 8,000 ϫ g for 20 min, and then 1 ml of 50% glutathione Sepharose 4B equilibrated with PBS was added.
T65 10803-10950 Sentence denotes After incubation with gentle inversion at room temperature for 1 h, the matrix was transferred to a disposable column and washed with 20 ml of PBS.
T66 10951-11089 Sentence denotes Protein was eluted 4 times by incubation with 1 ml of 50 mM Tris-HCl (pH 8.2) containing 20 mM glutathione at room temperature for 20 min.
T67 11090-11201 Sentence denotes Usually, only fractions 1 and 2 were pooled for further use because they showed OD 280 values of 0.8 or higher.
T68 11202-11409 Sentence denotes In addition, E. coli BL21 carrying the pGEX-5X-1 vector, which contains the full-length N gene of Sendai virus (SeV), was utilized to prepare the GST-fused SeV N protein as an irrelevant GST control antigen.
T69 11410-11601 Sentence denotes To produce the His-tagged recombinant S1, S2, and Smid proteins, each transformed E. coli BL21(DE3) clone was cultured in 500 ml of LB medium containing 60 g/ml kanamycin at room temperature.
T70 11602-11779 Sentence denotes When the culture medium had reached an OD 600 of 0.4 to 0.5, IPTG was added to a final concentration of 0.4 mM, followed by incubation for an additional 4 h at room temperature.
T71 11780-11859 Sentence denotes Cultures were placed on ice for 10 min and centrifuged at 2,000 ϫ g for 10 min.
T72 11860-12066 Sentence denotes Next, the supernatant was removed, and the pellet was suspended in 25 ml of PBS containing 1% Triton X-100, 1 mg/ml lysozyme, and 1 mM dithiothreitol, and the mixture was stored at Ϫ80°C until purification.
T73 12067-12150 Sentence denotes The bacterial suspension was thawed and sonicated on ice with 30-s pulses 10 times.
T74 12151-12317 Sentence denotes The insoluble fraction was collected by centrifugation at 8,000 ϫ g for 20 min, and the pellet was suspended in 2.5 ml of PBS containing 1 M urea and 1% Nonidet P-40.
T75 12318-12535 Sentence denotes After sonication with 30-s pulses 10 times on ice, the pellet was collected by centrifugation at 13,000 ϫ g for 20 min, resuspended in 2.5 ml of PBS containing 1 M urea, and sonicated with 30-s pulses 10 times on ice.
T76 12536-12710 Sentence denotes The pellet was then collected again by centrifugation at 13,000 ϫ g for 20 min, dissolved in 500 l of PBS containing 8 M urea, and sonicated with 30-s pulses 10 times on ice.
T77 12711-12831 Sentence denotes After centrifugation at 13,000 ϫ g for 10 min, the supernatant was collected as partially purified recombinant proteins.
T78 12832-12960 Sentence denotes The concentration of the recombinant proteins was determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).
T79 12961-13102 Sentence denotes To assess the quality of the recombinant protein, protein samples were subjected to 8% SDS-PAGE and stained with BioSafe Coomassie (Bio-Rad).
T80 13103-13415 Sentence denotes Immunological reactivity of the recombinant protein was confirmed by immunoblotting analysis with mouse antiserum to the MHV-S strain (1:1,000 dilution), horseradish peroxidase (HRPO)-conjugated anti-mouse IgG (1:10,000 dilution; GE Healthcare), and an ECL Advance Western blotting detection kit (GE Healthcare).
T81 13416-13471 Sentence denotes Coupling of viral proteins to fluorescent microspheres.
T82 13472-13745 Sentence denotes Carboxylated fluorescent microspheres (Luminex, Austin, TX) were suspended in 80 l of 0.1 M NaH 2 PO 4 (pH 6.2) and activated by adding 10 l (each) of 50 mg/ml N-hydroxysuccinimide (Pierce, Rockford, IL) and 50 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Pierce).
T83 13746-13899 Sentence denotes The microsphere mixture was incubated for 20 min at room temperature in the dark, centrifuged at 8,000 ϫ g for 2 min, and washed twice with 250 l of PBS.
T84 13900-14078 Sentence denotes Then, recombinant N antigen and inactivated MHV virion antigen were coupled to microspheres by incubation for 2 h at room temperature in 500 l of PBS using a rotator in the dark.
T85 14079-14252 Sentence denotes Recombinant S1, S2, and Smid antigens were coupled to microspheres by incubation for 2 h at room temperature in 500 l of PBS containing 8 M urea using a rotator in the dark.
T86 14253-14496 Sentence denotes Antigen-coupled microspheres were washed three times with 500 l of 0.05% Tween 20 in PBS and resuspended in 250 l of 1% bovine serum albumin (BSA), 0.05% sodium azide, and 1ϫ Complete Mini protease inhibitor (Roche, Basel, Switzerland) in PBS.
T87 14497-14561 Sentence denotes After blocking, the microspheres were stored at 4°C in the dark.
T88 14562-14566 Sentence denotes MFI.
T89 14567-14645 Sentence denotes MFI was performed to detect antibodies to coronaviruses in mouse and rat sera.
T90 14646-14806 Sentence denotes Approximately 2,000 beads (each) of antigen-coupled microsphere sets were added to each well of a multiscreen HTS 96-well filter plate (Millipore, Bedford, MA).
T91 14807-14871 Sentence denotes Beads were washed with 200 l per well of 0.05% Tween 20-PBS VOL.
T92 14872-14918 Sentence denotes 18, 2011 RECOMBINANT MHV ANTIGEN-BASED MFI 759
T93 14919-15030 Sentence denotes on March 19, 2020 by guest http://cvi.asm.org/ by removing the fluid using a plate vacuum manifold (Millipore).
T94 15031-15108 Sentence denotes Serum samples were diluted with 1% BSA-PBS, and 100 l was added to each well.
T95 15109-15192 Sentence denotes The beads were incubated on a plate shaker in the dark for 1 h at room temperature.
T96 15193-15367 Sentence denotes After incubation, the beads were washed twice by adding 200 l of 0.05% Tween 20-PBS, shaking for 1 min on a plate shaker, and removing the fluid with a plate vacuum manifold.
T97 15368-15658 Sentence denotes Next, the beads were resuspended in 100 l of biotinylated anti-mouse IgG(HϩL) (Vector Laboratories, Burlingame, CA) diluted 1:2,000 or biotinylated anti-rat IgG(HϩL) (Vector Laboratories) diluted 1:1,000 in 1% BSA-PBS and incubated on a plate shaker in the dark for 1 h at room temperature.
T98 15659-15880 Sentence denotes The beads were washed twice with 0.05% Tween 20-PBS as described above and then resuspended in 100 l of 1 g/ml R-phycoerythrin (R-PE)conjugated streptavidin (streptavidin-R-PE; Molecular Probes, Eugene, OR) in 1% BSA-PBS.
T99 15881-16104 Sentence denotes After incubation on a plate shaker in the dark for 30 min at room temperature, the beads were washed three times with 0.05% Tween 20-PBS, resuspended in 100 l of 0.05% Tween 20-PBS, and analyzed in a Luminex-200 instrument.
T100 16105-16123 Sentence denotes MFI data analysis.
T101 16124-16222 Sentence denotes A minimum of 100 gated events were acquired for each bead set by using the Luminex-200 instrument.
T102 16223-16350 Sentence denotes Results of antibody detection are reported as the median fluorescent intensity of R-PE measured from 100 or more counted beads.
T103 16351-16490 Sentence denotes For each antigen, the optimal protein concentration for coupling and the cutoff value for designating positive or negative were determined.
T104 16491-16706 Sentence denotes To set the cutoff value, an average median fluorescent intensity for more than 30 normal mouse or rat sera was calculated for each antigen, and three times the standard deviation (SD) value was added to the average.
T105 16707-16931 Sentence denotes To determine the optimal protein concentration, the signal/noise (S/N) ratio was calculated as the median fluorescent intensity of anti-MHV-S-or anti-SDAV-681-positivecontrol serum diluted 1:100, divided by the cutoff value.
T106 16932-17054 Sentence denotes The protein concentra-tion at which the highest S/N ratio was obtained was used as the optimal concentration for coupling.
T107 17055-17061 Sentence denotes ELISA.
T108 17062-17239 Sentence denotes A commercial ELISA kit (Monilisa MHV; Wakamoto Pharmaceutical, Tokyo, Japan) was used to detect anti-MHV mouse antibody and anti-SDAV/RCV rat antibody in 1:40-diluted test sera.
T109 17240-17370 Sentence denotes Serum samples with OD 492 values of Ն0.4 were classified as positive by measurement with a microplate reader (model 680; Bio-Rad).
T110 17371-17381 Sentence denotes IFA assay.
T111 17382-17575 Sentence denotes MHV-Nu67-infected DBT cells were mixed with equal numbers of uninfected DBT cells, spotted onto 12-well Teflon printed slides, fixed in acetone at 4°C for 15 min, and stored at Ϫ20°C until use.
T112 17576-17692 Sentence denotes Aliquots of 10 l of test sera at a dilution of 1:10 in PBS were added to each well and incubated at 37°C for 15 min.
T113 17693-17948 Sentence denotes The slides were then washed in PBS and incubated with 10 l of fluorescein-conjugated anti-mouse IgG (MP Biomedicals, Irvine, CA) or fluorescein-conjugated anti-rat IgG (MP Biomedicals) diluted 1:100 in PBS containing 1% Evan's blue dye at 37°C for 15 min.
T114 17949-18052 Sentence denotes After the slides were washed, specific fluorescence was analyzed using a UV epifluorescence microscope.
T115 18053-18090 Sentence denotes Nucleotide sequence accession number.
T116 18091-18227 Sentence denotes The complete nucleotide sequence of the S gene cloned from the MHV-S strain has been submitted to DDBJ/EMBL/ GenBank under accession no.
T117 18228-18237 Sentence denotes AB593383.
T118 18238-18276 Sentence denotes Cloning of MHV-S strain S and N genes.
T119 18277-18405 Sentence denotes The N gene sequence obtained was identical to that of the MHV-S strain registered in GenBank with the accession number M35255. .
T120 18406-18654 Sentence denotes Partial-length S proteins were expressed as His-tagged proteins, S1-His (87 kDa), S2-His (68 kDa), and Smid-His (70 kDa). (C) Immunoblot detection of the recombinant proteins, N-GST, S1-His, S2-His, and Smid-His, with anti-MHV-S-strain mouse serum.
T121 18655-18860 Sentence denotes The N-GST protein was affinity purified using immobilized glutathione, and the S1-His, S2-His, and Smid-His proteins were partially purified from insoluble extracts by serial treatment with urea solutions.
T122 18861-18917 Sentence denotes The arrows indicate the major recombinant protein bands.
T123 18918-18945 Sentence denotes MWM, molecular size marker.
T124 18946-19168 Sentence denotes The S gene sequence obtained here did not show perfect identity to that of the MHV-S strain registered in GenBank with the accession number GU593319, with 4 nucleotide substitutions all resulting in amino acid alterations.
T125 19169-19338 Sentence denotes The S gene of the MHV-S strain showed a high degree of similarity with the S genes of the MHV-Y, -TY, -DVIM, -2 and -1 strains, with amino acid identities of 95% to 91%.
T126 19339-19512 Sentence denotes The degree of S gene similarity was also high between MHV-S and the rat coronavirus strains SDAV-681 and RCV-Parker, with amino acid identities of 92% and 91%, respectively.
T127 19513-19676 Sentence denotes On the other hand, the degree of S gene similarity between MHV-S and the MHV-3, -RI, -JHM, or -A59 strain was relatively low (amino acid identities of 80% to 82%).
T128 19677-19905 Sentence denotes The S2 region, i.e., the carboxyterminal half of the S protein, is highly conserved among MHV strains and rat coronaviruses, with identities of 88% to 99%, which were comparable to the degrees of N protein identity (91% to 96%).
T129 19906-20093 Sentence denotes However, the degrees of identity of the S1 region, i.e., the amino-terminal half of the S protein, among MHV strains and rat coronaviruses was 73% to 94%, which was lower than that of S2.
T130 20094-20281 Sentence denotes After sequencing, the full-length N gene was subcloned into the pGEX-5X-1 vector, and partial regions of the S gene, S1, S2, and Smid (Fig. 1A) , were subcloned into the pET28a(ϩ) vector.
T131 20282-20467 Sentence denotes In E. coli clones transformed with these recombinant plasmids, 4 recombinant proteins, the GST-fused N protein and the His-tagged S1, S2, and Smid proteins, were expressed successfully.
T132 20468-20593 Sentence denotes At first, we attempted to prepare the recombinant S protein as a soluble GST fusion protein in the same way as the N protein.
T133 20594-20837 Sentence denotes Since the S protein is too large to be produced efficiently in E. coli, we cloned each DNA fragment encoding the N-terminal S1 subunit, which forms the globular head of the peplomer, and the C-terminal S2 subunit, which forms the stalk region.
T134 20838-20950 Sentence denotes In addition, the middle part of the S protein near the proteolytic site, which we designated Smid, was prepared.
T135 20951-21154 Sentence denotes Since all the truncated recombinant S proteins in the GST fusion form were expressed in only small amounts, we finally produced the recombinant S1, S2, and Smid proteins as insoluble His-tagged proteins.
T136 21155-21298 Sentence denotes Unexpectedly, immobilized metal affinity chromatography did not enable purification of a large amount of these His-tagged proteins efficiently.
T137 21299-21403 Sentence denotes Therefore, we partially purified the insoluble His-tagged proteins by washing them in 1 M urea solution.
T138 21404-21515 Sentence denotes Finally, the recombinant S proteins were solubilized in 8 M urea-PBS and used as antigens for further analysis.
T139 21516-21577 Sentence denotes SDS-PAGE and immunoblotting analysis of recombinant proteins.
T140 21578-21645 Sentence denotes Recombinant proteins produced by E. coli were analyzed by SDS-PAGE.
T141 21646-21885 Sentence denotes As shown in Fig. 1B , the GST-fused N protein and the His-tagged S1, S2, and Smid proteins were detected as major recombinant protein bands, with estimated molecular masses of approximately 76 kDa, 87 kDa, 68 kDa, and 70 kDa, respectively.
T142 21886-22017 Sentence denotes On immunoblotting analysis, all the major recombinant MHV proteins showed strong reactivity with anti-MHV-S mouse serum (Fig. 1C) .
T143 22018-22190 Sentence denotes Several bands that do not correspond to the expected molecular masses of the recombinant MHV proteins were detected, and some of those were immunoreactive (Fig. 1B and C) .
T144 22191-22377 Sentence denotes It is likely that the smaller proteins present in the affinity-purified GST-N protein preparation are degraded recombinant N proteins, since those are immunoreactive with anti-MHV serum.
T145 22378-22583 Sentence denotes Multiple protein bands present in the His-tagged S protein preparations may be degraded proteins originated from the recombinant S proteins or E. coli proteins contaminating the insoluble inclusion bodies.
T146 22584-22633 Sentence denotes Development of recombinant MHV antigen-based MFI.
T147 22634-22781 Sentence denotes The reactivities of microspheres coupled with 5 to 40 g/ml of each recombinant antigen were analyzed using serially diluted anti-MHV-S mouse serum.
T148 22782-22881 Sentence denotes As shown in Fig. 2 , antigen dose-dependent reactions were observed for all of the antigens tested.
T149 22882-23139 Sentence denotes Although the median fluorescent intensity value for MHV virion antigen decreased sharply at low antigen concentrations, all of the recombinant antigens exhibited consistently high median fluorescent intensities even at the lowest antigen concentration used.
T150 23140-23391 Sentence denotes Specificity of the GST-fused MHV N protein for detection of anti-MHV antibody was confirmed by the lack of cross-reactivity of anti-MHV S serum with the GST-fused SeV N protein, used as an irrelevant GST control antigen ( Fig. 2 ; see also Table 2 ).
T151 23392-23535 Sentence denotes Furthermore, linear median fluorescent intensity results across a broad range of antibody concentrations were obtained for all of the antigens.
T152 23536-23681 Sentence denotes This linear reaction property of the MFI and low background binding of microspheres allowed the MFI to be performed at a serum dilution of 1:100.
T153 23682-23869 Sentence denotes The optimal protein concentration of each antigen for mouse serum testing was determined based on reactivity in MFI with 1:100-diluted anti-MHV mouse serum and negativecontrol mouse sera.
T154 23870-24038 Sentence denotes As shown in Table 1 , all the recombinant antigens provided the highest S/N ratio when used at 10 g/ml; therefore, the optimal protein concentration was set at 10 g/ml.
T155 24039-24421 Sentence denotes The cutoff value to distinguish positive samples was set at a median fluorescent intensity of 750 for the N antigen, 850 for the S1 antigen, 800 for the S2 antigen, and 1,000 for the Smid antigen according to the background median fluorescent intensity values, calculated as the mean plus 3 standard deviations after rounding up to the nearest 50 median fluorescent intensity units.
T156 24422-24573 Sentence denotes For MHV virion antigen, the optimal protein concentration and cutoff value were set at 40 g/ml and a median fluorescent intensity of 900, respectively.
T157 24574-24686 Sentence denotes In the same manner, the antigen concentration and cutoff value were optimized for rat serum testing ( Table 1 ).
T158 24687-24892 Sentence denotes The optimal protein concentration of the S1, S2, and Smid antigens was 10 g/ml, which was the same in mice and rats, while the N antigen was optimal at 5 g/ml and the virion antigen was optimal at 20 g/ml.
T159 24893-25115 Sentence denotes The cutoff value was set at a median fluorescent intensity of 1,000 for the N antigen, 750 for the S1 antigen, 450 for the S2 antigen, 500 for the Smid antigen, and 600 for virion antigen to measure antibodies in rat sera.
T160 25116-25367 Sentence denotes To evaluate cross-reactivities of the recombinant antigens prepared from the MHV-S strain, each recombinant antigen and MHV-S virion antigen was analyzed with mouse antisera raised to several MHV strains and rat antiserum to SDAV-681 by MFI (Fig. 3) .
T161 25368-25442 Sentence denotes Antisera to MHV-S, MHV-A59, and MHV-JHM showed a similar reaction pattern.
T162 25443-25612 Sentence denotes These sera reacted strongly with the recombinant N and virion antigens, moderately with the recombinant S2 and Smid antigens, and weakly with the recombinant S1 antigen.
T163 25613-25783 Sentence denotes Antiserum against MHV-Nu67 showed high reactivity with the S2 antigen, moderate reactivity with the Smid antigen, and weak reactivity with the virion, N, and S1 antigens.
T164 25784-25888 Sentence denotes High cross-reactivity of anti-SDAV rat serum was observed with the MHV virion, N, S2, and Smid antigens.
T165 25889-26098 Sentence denotes These results indicated that the recombinant antigens cross-react with antibodies generated to a variety of MHV strains and SDAV, although the strength of reactivity to each antigen varies among viral strains.
T166 26099-26270 Sentence denotes A time course of seroconversion to MHV detected by MFI, ELISA, and IFA was compared using serially collected serum samples from 10 mice experimentally infected with MHV-S.
T167 26271-26455 Sentence denotes As shown in Table 2 , only 2 or 3 of 10 mice seroconverted to MHV on day 7 after inoculation, and all mice seroconverted on day 14, according to the N MFI, S2 MFI, virion MFI, and IFA.
T168 26456-26540 Sentence denotes In contrast, all mice tested positive for anti-MHV antibodies on day 7 in the ELISA.
T169 26541-26670 Sentence denotes We also found nonspecific reactivity with day zero serum samples in the ELISA analysis but not in either the MFI or IFA analysis.
T170 26671-26857 Sentence denotes These results suggest that ELISA is superior in detecting antibodies to MHV at the early stage of infection and that MFI has high sensitivity and specificity, equivalent to those of IFA.
T171 26858-26893 Sentence denotes Sensitivity and specificity of MFI.
T172 26894-27207 Sentence denotes To evaluate the sensitivity of the recombinant MHV antigen-based MFI, 236 mouse sera a By measuring the median fluorescent intensity values of 32 negative mouse sera at a dilution of 1:100, background intensity was defined as the mean ϩ 3 SD after rounding up to the nearest 50 median fluorescent intensity units.
T173 27208-27367 Sentence denotes Signal/noise (S/N) ratio was calculated as the median fluorescent intensity of 1:100-diluted anti-MHV-S-strain mouse serum divided by the background intensity.
T174 27368-27490 Sentence denotes The optimal protein concentration of each antigen was determined at the concentration that provided the highest S/N ratio.
T175 27491-27689 Sentence denotes The background median fluorescent intensity at the optimal concentration was used as a cutoff value to differentiate between the positive and negative samples (indicated by underlined bold letters).
T176 27690-28042 Sentence denotes b The optimal protein concentration of each antigen and cutoff median fluorescent intensity value for rat sera were determined in the same manner using 30 negative rat sera. naturally infected with MHV and 50 rat sera naturally infected with SDAV or RCV, confirmed by double-positive results in a commercial MHV-ELISA and IFA, were tested with the MFI.
T177 28043-28218 Sentence denotes In MHV-infected mouse sera, as shown in Table 3 , the highest sensitivity was obtained with S2 (96%), followed by the Smid (89%), N (88%), virion (76%), and S1 antigens (65%).
T178 28219-28483 Sentence denotes In SDAV-infected rat sera, as shown in Table 4 , seropositive rates for S2, Smid, and virion were markedly high, with the same sensitivity (98%), followed by the N antigen with a similar high sensitivity (94%), but S1 antigen provided the lowest (60%) sensitivity.
T179 28484-28730 Sentence denotes With regard to the specificity of the recombinant MHV antigen-based MFI, non-MHV-infected mouse sera and non-SDAV/RCV-infected rat sera, confirmed by negative results in IFA, were tested and compared with a commercial MHV ELISA (Tables 3 and 4 ).
T180 28731-28855 Sentence denotes In MFI for detecting mouse and rat antibodies, all the antigens revealed high specificity (Ն90% for mice and 100% for rats).
T181 28856-28945 Sentence denotes In contrast, the specificity of a commercial MHV-ELISA was 73% for mice and 90% for rats.
T182 28946-29081 Sentence denotes Figure 4 shows a comparison of S2 antigen-based MFI and N-antigen-based MFI results for 236 sera from mice naturally infected with MHV.
T183 29082-29220 Sentence denotes Of 236 mouse sera tested, 9 serum samples were negative in S2-antigen-based MFI and 28 serum samples were negative in N-antigen-based MFI.
T184 29221-29304 Sentence denotes Only three serum samples (1.3%) showed double-negative results for S2 and N by MFI.
T185 29305-29428 Sentence denotes Of 126 sera from MHV-uninfected mice, 10 serum samples (7.9%) showed false-positive results for either the S2 or N antigen.
T186 29429-29530 Sentence denotes Taken together, these results indicated that the MFI using the recombinant S2 and N antigens has FIG.
T187 29531-29533 Sentence denotes 3.
T188 29534-29636 Sentence denotes Reactivities of recombinant MHV antigens with mouse antisera to MHV strains and rat antiserum to SDAV.
T189 29637-30133 Sentence denotes Recombinant MHV antigens (N, S1, S2, and Smid) and MHV virion antigen were coupled onto microspheres at the optimal protein concentration for each antigen, and their reactivities with serially diluted mouse antisera to MHV strains and rat antiserum to SDAV were analyzed by microsphere fluorescence immunoassay. }, N antigen; Ⅺ, S1 antigen; F, S2 antigen; ‚, Smid antigen; ‫,ء‬ virion antigen. Ն98% sensitivity and Ն92% specificity for detecting MHV and SDAV/RCV antibodies in mouse and rat sera.
T190 30134-30322 Sentence denotes In this study, the recombinant S and N proteins of MHV produced in E. coli were tested as a substitute for purified virion antigens for the detection of antibodies to rodent coronaviruses.
T191 30323-30611 Sentence denotes Major disadvantages of virion antigen are the variation in purities of virion proteins, which affects the specificity and sensitivity, laborious work to prepare large amounts of antigen, the possibility of remaining infectivity, and the loss of reactivity during the inactivation process.
T192 30612-30689 Sentence denotes First, we prepared the recombinant N protein as a soluble GST fusion protein.
T193 30690-30847 Sentence denotes However, we found that the sensitivity of the N protein-based ELISA was lower than that of the traditional ELISA using virion antigen in a preliminary study.
T194 30848-30939 Sentence denotes Therefore, we attempted to prepare recombinant S proteins in GST-fused and Histagged forms.
T195 30940-31165 Sentence denotes Because the S protein is too large to be produced efficiently in E. coli, we prepared truncated molecules of the S protein, including the N-terminal S1 subunit, the C-terminal S2 subunit, and the middle part, designated Smid.
T196 31166-31476 Sentence denotes In previous studies of MHV (3, 16) , bovine coronavirus (22) , and severe acute respiratory syndrome coronavirus (15, 26) , this middle region was identified as an immunodominant linear neutralization domain by analysis using a prokaryotic protein expression system and monoclonal antibodies or patients' sera.
T197 31477-31635 Sentence denotes As a result, large amounts of the His-tagged S1, S2, and Smid proteins were produced as insoluble proteins and were solubilized in a high-molar urea solution.
T198 31636-31815 Sentence denotes All of the recombinant S proteins that were solubilized under denaturing conditions using 8 M urea showed immunoreactivities with anti-MHV antibodies on immunoblotting and in MFI.
T199 31816-31967 Sentence denotes We found that the MFI reactivity of the microspheres coupled with S proteins in 8 M urea-PBS was stronger than that of the microspheres coupled in PBS.
T200 31968-32130 Sentence denotes These observations indicated that the recombinant S proteins solubilized with 8 M urea were capable of being cou-pled efficiently to microspheres in 8 M urea-PBS.
T201 32131-32303 Sentence denotes These findings may reflect the appearance of insoluble aggregates of the recombinant S proteins in PBS, which leads to a decrease in coupling efficiency and MFI reactivity.
T202 32304-32383 Sentence denotes All the recombinant proteins were expressed from the genes of the MHV-S strain.
T203 32384-32525 Sentence denotes Cross-reactivity of these recombinant proteins in detection of antibodies to other MHV strains and SDAV rat coronavirus was confirmed by MFI.
T204 32526-32703 Sentence denotes According to the calibration curves constructed with the experimentally infected sera to MHV strains and SDAV, cross-reactivity levels of the recombinant proteins were compared.
T205 32704-32820 Sentence denotes The median fluorescent intensity values corresponding to the 4 recombinant proteins were similar among the antisera.
T206 32821-32891 Sentence denotes However, antiserum to MHV-JHM reacted very weakly with the S1 antigen.
T207 32892-33219 Sentence denotes This weak reactivity was unlikely to be a consequence of the relatively low amino acid sequence identity (76%) of S1 between MHV-JHM and MHV-S, because the level of antibody to S1 in anti-MHV-A59 serum was equivalent to or higher than that in anti-MHV-S serum despite the even lower S1 identity (73%) between MHV-A59 and MHV-S.
T208 33220-33352 Sentence denotes Furthermore, antiserum to MHV-Nu67 was remarkable for its lower level of antibody to the N antigen than to the S2 and Smid antigens.
T209 33353-33497 Sentence denotes Such low reactivity to the N antigen may be reflective of the slow growth of MHV-Nu67, a low-pathogenicity strain isolated from nude mice (20) .
T210 33498-33716 Sentence denotes This may be correlated with the observation that the S protein is the earliest recognized antigen by the host immune system, whereas the N protein elicits antibody responses during the late stage of infection (2, 15) .
T211 33717-33945 Sentence denotes Nevertheless, the possibility that the differences in reaction pattern among the antisera to the virus strains described above were simply due to individual variation among the experimentally infected animals cannot be excluded.
T212 33946-34185 Sentence denotes The MFI calibration curve of each antigen constructed with anti-MHV-S serum suggested that MFI provides a broad dynamic range of antibody concentration, which allows accurate analysis of antibody levels at a single point of serum dilution.
T213 34186-34428 Sentence denotes We assessed 32 mouse sera and 30 rat sera from MHV or SDAV/RCV-free commercial breeder animals at a 1:100 dilution and established an optimal antigen concentration and a cutoff median fluorescent intensity value corresponding to each antigen.
T214 34429-34653 Sentence denotes The protein concentration providing the highest S/N ratio was determined as the optimal antigen concentration, and the mean median fluorescent intensity plus 3 SD value was set as a cutoff median fluorescent intensity value.
T215 34654-34942 Sentence denotes According to these criteria, serial serum samples from experimentally infected mice and a large number of serum samples from naturally infected laboratory mice were analyzed to select the favorable antigens in MFI for detecting antibodies to MHV and to evaluate diagnostic predictability.
T216 34943-35094 Sentence denotes An experimental infection study showed that seroconversion in the MFI took place from 7 to 14 days post-MHV infection in the majority of infected mice.
T217 35095-35165 Sentence denotes In contrast, all of the infected mice seroconverted in ELISA by day 7.
T218 35166-35327 Sentence denotes For early detection of MHV antibodies, ELISA is more sensitive than MFI, whereas the sensitivities of N-based MFI and S2-based MFI are equivalent to that of IFA.
T219 35328-35550 Sentence denotes Because IFA is the primary method used as confirmatory testing for ELISA and MFI-based screening as-says, special attention should be paid to false-negative results in MHV antibody detection within 2 weeks after infection.
T220 35551-35753 Sentence denotes With regard to the reactivity of sera from naturally infected mice, our results indicated that the S2 antigen has the highest sensitivity, but 4% (9 of 236) of samples still show false-negative results.
T221 35754-35881 Sentence denotes The recombinant N antigen resembles the MHV virion antigen in immunoreactivity with naturally and experimentally infected sera.
T222 35882-36007 Sentence denotes While the sensitivity of the N MFI was 88%, 6 of 9 false-negative samples in the S2 MFI showed positive results in the N MFI.
T223 36008-36161 Sentence denotes These results indicated that multiplex serological screening, consisting of S2 MFI and N MFI, has 98% sensitivity for detection of MHV infection in mice.
T224 36162-36308 Sentence denotes Naturally infected animal sera used in this study were retrospective serum samples screened for positivity for antibodies to MHV by ELISA and IFA.
T225 36309-36416 Sentence denotes Therefore, it is likely that the sensitivity of MFI is equivalent to that of the traditional ELISA and IFA.
T226 36417-36560 Sentence denotes Thirty-four sera that were negative for anti-N or anti-S2 antibodies may represent samples collected from mice at the early stage of infection.
T227 36561-36926 Sentence denotes It is not likely that the failure to detect antibodies based on the MFI using the N or S2 on March 19, 2020 by guest http://cvi.asm.org/ antigen resulted from a higher serum dilution in MFI (1:100 dilution) than that in ELISA (1:40 dilution), because no significant correlation was seen between ELISA OD values and median fluorescent intensity values in these sera.
T228 36927-37007 Sentence denotes Many of the singly positive sera showed definite reactivity with either N or S2.
T229 37008-37251 Sentence denotes Thus, production of antibody to each antigen varies among individuals or strains of animals as well as virus strains, and consequently, multiplex analysis using polyvalent antigens is recommended in recombinant antigen-based serologic testing.
T230 37252-37468 Sentence denotes Based on the assessment of 126 mouse sera and 59 rat sera from animals uninfected with MHV or SDAV/RCV, which were confirmed by IFA, we found that the MFI had a significantly lower false-positive rate than the ELISA.
T231 37469-37617 Sentence denotes The lower false-positive rate in MFI may be due to the lower nonspecific binding of serum antibodies to the microspheres than to ELISA plates (12) .
T232 37618-37850 Sentence denotes In the case of the recombinant antigenbased MFI, the purified recombinant proteins are covalently coupled to microspheres, and antigen purity eliminates nonspecific reactions that often cause high background problems in ELISA (25) .
T233 37851-37979 Sentence denotes Furthermore, the linear reaction and high sensitivity allow MFI to be used for analysis of serum samples at a dilution of 1:100.
T234 37980-38111 Sentence denotes Since the MHV ELISA was performed using 1:40-diluted sera, the higher serum dilution in MFI definitely affected the low background.
T235 38112-38324 Sentence denotes In summary, the MFI method developed with recombinant MHV S2 and N antigens was as sensitive as and more specific than the traditional MHV ELISA for screening of antibodies to coronaviruses in mouse and rat sera.
T236 38325-38462 Sentence denotes MFI-format assays have been used for the simultaneous detection of antibodies to a variety of pathogens in laboratory rodents (4, 8, 9) .
T237 38463-38675 Sentence denotes Although MHV virion antigen was successfully used to develop the MFI system for serodetection of 10 mouse pathogens (9) , this is the first report of using a recombinant antigen-based MFI to detect MHV infection.
T238 38676-38757 Sentence denotes There are many advantages to MFI over ELISA when screening large rodent colonies.
T239 38758-38897 Sentence denotes MFI allows screening of animals for serum antibodies to multiple pathogens or antigens simultaneously in one reaction well for each animal.
T240 38898-38993 Sentence denotes This markedly reduces the time and labor required to perform a separate ELISA for each antigen.
T241 38994-39123 Sentence denotes The MFI format also requires much less serum than ELISA, since multiple agents can be screened using only 1 l of undiluted serum.
T242 39124-39257 Sentence denotes In this regard, MFI is suitable for serological screening of small laboratory animals, such as mice, often through survival bleeding.
T243 39258-39471 Sentence denotes The amounts of antigens required for coupling microspheres for 200 analytes are equal to those used for coating a single well of an ELISA plate, thereby greatly saving cost, time, and labor for preparing antigens.
T244 39472-39627 Sentence denotes In our experience, antigencoupled microspheres can be stored in the refrigerator for at least 6 months without a significant reduction in immunoreactivity.
T245 39628-39793 Sentence denotes The multiplexing feature of the MFI method enables high throughput and automated screening for serological detection of many infectious agents in laboratory animals.