Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia
Identifieur interne : 000E88 ( Pmc/Corpus ); précédent : 000E87; suivant : 000E89Evaluation of serological assays available in a biosafety level 2 laboratory and their application for survey of Middle East respiratory syndrome coronavirus among livestock in Ethiopia
Auteurs : Takurou Teramichi ; Shuetsu Fukushi ; Yuma Hachiya ; Simenew Keskes Melaku ; Keisuke Oguma ; Hiroshi SentsuiSource :
- The Journal of Veterinary Medical Science [ 0916-7250 ] ; 2019.
Abstract
A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was conducted among dromedary camels and herbivorous animals sharing the same pasturage in Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level (BSL)-2 laboratory. The results were similar to those obtained from the VN test using live MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment as the antigen as well as the competitive ELISA performed using a monoclonal antibody against MERS-CoV. According to the comprehensive results of the four types of serodiagnosis methods, positive antibodies were detected only in dromedary camels and the remaining herbivorous animals were not infected with the virus. Moreover, using the present procedure, serological tests for MERS-CoV can be conducted even in BSL 2 laboratory.
Url:
DOI: 10.1292/jvms.19-0436
PubMed: 31685722
PubMed Central: 6943330
Links to Exploration step
PMC:6943330Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Evaluation of serological assays available in a biosafety level 2 laboratory
and their application for survey of Middle East respiratory syndrome coronavirus among
livestock in Ethiopia</title><author><name sortKey="Teramichi, Takurou" sort="Teramichi, Takurou" uniqKey="Teramichi T" first="Takurou" last="Teramichi">Takurou Teramichi</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Fukushi, Shuetsu" sort="Fukushi, Shuetsu" uniqKey="Fukushi S" first="Shuetsu" last="Fukushi">Shuetsu Fukushi</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Hachiya, Yuma" sort="Hachiya, Yuma" uniqKey="Hachiya Y" first="Yuma" last="Hachiya">Yuma Hachiya</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Melaku, Simenew Keskes" sort="Melaku, Simenew Keskes" uniqKey="Melaku S" first="Simenew Keskes" last="Melaku">Simenew Keskes Melaku</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Oguma, Keisuke" sort="Oguma, Keisuke" uniqKey="Oguma K" first="Keisuke" last="Oguma">Keisuke Oguma</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Sentsui, Hiroshi" sort="Sentsui, Hiroshi" uniqKey="Sentsui H" first="Hiroshi" last="Sentsui">Hiroshi Sentsui</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31685722</idno><idno type="pmc">6943330</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943330</idno><idno type="RBID">PMC:6943330</idno><idno type="doi">10.1292/jvms.19-0436</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000E88</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E88</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Evaluation of serological assays available in a biosafety level 2 laboratory
and their application for survey of Middle East respiratory syndrome coronavirus among
livestock in Ethiopia</title><author><name sortKey="Teramichi, Takurou" sort="Teramichi, Takurou" uniqKey="Teramichi T" first="Takurou" last="Teramichi">Takurou Teramichi</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Fukushi, Shuetsu" sort="Fukushi, Shuetsu" uniqKey="Fukushi S" first="Shuetsu" last="Fukushi">Shuetsu Fukushi</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Hachiya, Yuma" sort="Hachiya, Yuma" uniqKey="Hachiya Y" first="Yuma" last="Hachiya">Yuma Hachiya</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Melaku, Simenew Keskes" sort="Melaku, Simenew Keskes" uniqKey="Melaku S" first="Simenew Keskes" last="Melaku">Simenew Keskes Melaku</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Oguma, Keisuke" sort="Oguma, Keisuke" uniqKey="Oguma K" first="Keisuke" last="Oguma">Keisuke Oguma</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Sentsui, Hiroshi" sort="Sentsui, Hiroshi" uniqKey="Sentsui H" first="Hiroshi" last="Sentsui">Hiroshi Sentsui</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Veterinary Medical Science</title><idno type="ISSN">0916-7250</idno><idno type="eISSN">1347-7439</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was
conducted among dromedary camels and herbivorous animals sharing the same pasturage in
Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of
MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level
(BSL)-2 laboratory. The results were similar to those obtained from the VN test using live
MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment
as the antigen as well as the competitive ELISA performed using a monoclonal antibody
against MERS-CoV. According to the comprehensive results of the four types of
serodiagnosis methods, positive antibodies were detected only in dromedary camels and the
remaining herbivorous animals were not infected with the virus. Moreover, using the
present procedure, serological tests for MERS-CoV can be conducted even in BSL 2
laboratory.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Alagaili, A N" uniqKey="Alagaili A">A. N. Alagaili</name></author><author><name sortKey="Briese, T" uniqKey="Briese T">T. Briese</name></author><author><name sortKey="Mishra, N" uniqKey="Mishra N">N. Mishra</name></author><author><name sortKey="Kapoor, V" uniqKey="Kapoor V">V. Kapoor</name></author><author><name sortKey="Sameroff, S C" uniqKey="Sameroff S">S. C. Sameroff</name></author><author><name sortKey="Burbelo, P D" uniqKey="Burbelo P">P. D. Burbelo</name></author><author><name sortKey="De Wit, E" uniqKey="De Wit E">E. de Wit</name></author><author><name sortKey="Munster, V J" uniqKey="Munster V">V. J. Munster</name></author><author><name sortKey="Hensley, L E" uniqKey="Hensley L">L. E. Hensley</name></author><author><name sortKey="Zalmout, I S" uniqKey="Zalmout I">I. S. Zalmout</name></author><author><name sortKey="Kapoor, A" uniqKey="Kapoor A">A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Vet Med Sci</journal-id><journal-id journal-id-type="iso-abbrev">J. Vet. Med. Sci</journal-id><journal-id journal-id-type="publisher-id">JVMS</journal-id><journal-title-group><journal-title>The Journal of Veterinary Medical Science</journal-title></journal-title-group><issn pub-type="ppub">0916-7250</issn><issn pub-type="epub">1347-7439</issn><publisher><publisher-name>The Japanese Society of Veterinary Science</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31685722</article-id><article-id pub-id-type="pmc">6943330</article-id><article-id pub-id-type="publisher-id">19-0436</article-id><article-id pub-id-type="doi">10.1292/jvms.19-0436</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virology</subject><subj-group><subject>Full Paper</subject></subj-group></subj-group></article-categories><title-group><article-title>Evaluation of serological assays available in a biosafety level 2 laboratory
and their application for survey of Middle East respiratory syndrome coronavirus among
livestock in Ethiopia</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>TERAMICHI</surname><given-names>Takurou</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>FUKUSHI</surname><given-names>Shuetsu</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>HACHIYA</surname><given-names>Yuma</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>MELAKU</surname><given-names>Simenew Keskes</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>OGUMA</surname><given-names>Keisuke</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>SENTSUI</surname><given-names>Hiroshi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref rid="cor1" ref-type="corresp"><sup>*</sup></xref></contrib><aff id="aff1"><label>1)</label>Laboratory of Veterinary Epizootiology, Department of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan</aff><aff id="aff2"><label>2)</label>Department of Virology I, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan</aff><aff id="aff3"><label>3)</label>Addis Ababa Science and Technology University, Akaky Kaliti Sub-city, Kilinto area, Addis Ababa, Ethiopia</aff><aff id="aff4"><label>4)</label>Present Address: Isahaya Meat Inspection Center, 79-20, Sakaimachi 79-20, Isahaya, Nagasaki 854-0022, Japan</aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>Correspondence to: Sentsui, H.: <email xlink:href="sentsui.hiroshi@nihon-u.ac.jp">sentsui.hiroshi@nihon-u.ac.jp</email></corresp></author-notes><pub-date pub-type="epub"><day>05</day><month>11</month><year>2019</year></pub-date><pub-date pub-type="ppub"><month>12</month><year>2019</year></pub-date><volume>81</volume><issue>12</issue><fpage>1887</fpage><lpage>1891</lpage><history><date date-type="received"><day>07</day><month>8</month><year>2019</year></date><date date-type="accepted"><day>24</day><month>10</month><year>2019</year></date></history><permissions><copyright-statement>©2019 The Japanese Society of Veterinary Science</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>This is an open-access article distributed under the terms of the Creative
Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0:
<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by-nc-nd/4.0/">https://creativecommons.org/licenses/by-nc-nd/4.0/</ext-link>)</license-p></license></permissions><abstract><p>A serological survey of Middle East respiratory syndrome coronavirus (MERS-CoV) was
conducted among dromedary camels and herbivorous animals sharing the same pasturage in
Ethiopia. The pseudotyped vesicular stomatitis virus coated with the spike protein of
MERS-CoV was used in virus neutralization (VN) tests performed in a biosafety level
(BSL)-2 laboratory. The results were similar to those obtained from the VN test using live
MERS-CoV and were more sensitive than the ELISA performed using synthetic MERS S1 fragment
as the antigen as well as the competitive ELISA performed using a monoclonal antibody
against MERS-CoV. According to the comprehensive results of the four types of
serodiagnosis methods, positive antibodies were detected only in dromedary camels and the
remaining herbivorous animals were not infected with the virus. Moreover, using the
present procedure, serological tests for MERS-CoV can be conducted even in BSL 2
laboratory.</p></abstract><kwd-group><kwd>camel</kwd><kwd>coronavirus</kwd><kwd>Ethiopia</kwd><kwd>Middle East respiratory syndrome</kwd><kwd>serological survey</kwd></kwd-group></article-meta></front><body><p>Middle East respiratory syndrome (MERS), first confirmed in Saudi Arabia in 2012, is an
emerging viral infection that causes severe respiratory symptoms in humans [<xref rid="r1" ref-type="bibr">1</xref>, <xref rid="r5" ref-type="bibr">5</xref>, <xref rid="r19" ref-type="bibr">19</xref>]. The pathogen, MERS-coronavirus (MERS-CoV), is
identified as a novel coronavirus species [<xref rid="r5" ref-type="bibr">5</xref>], and the
dromedary camel is considered a natural host [<xref rid="r10" ref-type="bibr">10</xref>, <xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r14" ref-type="bibr">14</xref>, <xref rid="r15" ref-type="bibr">15</xref>, <xref rid="r18" ref-type="bibr">18</xref>]. Patients
with MERS have mainly been identified in and near the Arabian Peninsula [<xref rid="r2" ref-type="bibr">2</xref>]. However, several cases have been detected in other countries among the
travelers who have visited the Arabian Peninsula, and MERS-CoV has been reported to spread
from patients to others via close contact [<xref rid="r3" ref-type="bibr">3</xref>, <xref rid="r4" ref-type="bibr">4</xref>, <xref rid="r7" ref-type="bibr">7</xref>, <xref rid="r12" ref-type="bibr">12</xref>]. Therefore, MERS-CoV has been classified as a biosafety
level (BSL)-3 pathogen and its handling requires a high-containment laboratory that ensures
biosecurity. Therefore, developing a serological diagnostic method that can be used even in a
BSL-2 laboratory and exhibits the same specificity and sensitivity as the conventional
serological test is crucial. A neutralization test using a pseudotyped virus integrated with
green fluorescent protein (GFP) gene and coated with the spike protein of MERS-CoV
(VSV-MERS/GFP) has recently been developed [<xref rid="r8" ref-type="bibr">8</xref>]. In the
present study, the specificity and sensitivity of the neutralization test using VSV-MERS/GFP
were confirmed using serum obtained from livestock that share a grazing area with the
dromedary camel in Ethiopia. The results were compared with three other antibody
tests—neutralization test using live MERS-CoV [<xref rid="r16" ref-type="bibr">16</xref>],
S1-ELISA [<xref rid="r17" ref-type="bibr">17</xref>], and competitive ELISA using a monoclonal
antibody against MERS-CoV (cELISA) [<xref rid="r9" ref-type="bibr">9</xref>]. Based on these
serological tests, the MERS-CoV susceptibility of other herbivores sharing pasture lands with
the dromedary camel to infection and to become a source of infection to humans was
examined.</p><sec sec-type="materials|methods" id="s1"><title>MATERIALS AND METHODS</title><sec><title>Pseudovirus</title><p>VSV-MERS/GFP, a recombinant vesicular stomatitis virus pseudotype, integrated with the
GFP gene and coated with the spike protein of MERS-CoV was used in the neutralization test
[<xref rid="r8" ref-type="bibr">8</xref>]. The infectivity of VSV-MERS/GFP was detected
based on GFP expression observed using fluorescence microscopy. GFP-expressing cells in
photographic images were counted using VH analyzer. The VSV-MERS/GFP titers were then
expressed as focus forming units (FFU) per m<italic>l</italic>.</p></sec><sec><title>Cell culture</title><p>293T and Vero cells were cultivated in Dulbecco’s modified Eagle’s medium supplemented
with 5% fetal bovine serum (FBS), 100 U/m<italic>l</italic> of penicillin, and 100
<italic>µ</italic>g/m<italic>l</italic> of streptomycin at 37°C [<xref rid="r8" ref-type="bibr">8</xref>]. 293T cells were used to prepare VSV-MERS/GFP and MERS-CoV
recombinant receptor binding domain (RBD) for the cELISA antigen. Vero cells were used for
neutralization tests by live MERS-CoV and VSV-MERS/GFP, respectively.</p></sec><sec><title>Serum samples</title><p>Serum samples were collected from dromedary camels (n=38; mean age, 4.3 years; range,
1–13 years), goats (n=25; mean age, 3.9 years; range, 1–8 years), sheep (n=25; mean age,
2.7 years; range, 1–4 years), and cattle (n=15; mean age, 6.7 years; range, 1–11 years)
from two herds in Bati district, Amhara region, and one herd in Fafen district, Somali
region, Ethiopia. All animals appeared healthy, shared the same pasturage during the day,
and stayed in barns or small grounds specific for each animal species surrounded by fences
at night.</p><p>Transportation of the serum samples to Japan was conducted with the permission of the
Japanese government (Animal quarantine inspection number NFIB070602-011) and followed the
rules and regulations of the OIE/FAO for biological sample transportation.</p><p>Serum from a rabbit immunized with recombinant MERS-CoV S protein was used as a positive
control for neutralization [<xref rid="r8" ref-type="bibr">8</xref>]. Sera from animals (5
cattle, 5 sheep and 5 goats) reared on the attached farm of Nihon University were used as
negative controls.</p></sec><sec><title>Neutralization test</title><p>The neutralization test using VSV-MERS/GFP was performed as previously described [<xref rid="r8" ref-type="bibr">8</xref>]. A medium composed of Eagle’s MEM supplemented with
5% FBS was used for virus and serum dilution. Serially diluted 0.05 m<italic>l</italic> of
test sera were mixed with equal volumes of 3,000 FFU of VSV-MERS/GFP and incubated at 37°C
for 1 hr. The mixture was inoculated to Vero cells seeded on a 96-well culture plate and
incubated at 37°C for 24 hr in a CO<sub>2</sub> incubator. GFP-positive cells were then
detected using a fluorescence microscope. The number of positive GFP cells was counted as
described above. The neutralization titer was determined as the highest serum dilution
showing ≤50% of the number of GFP-positive cells compared with the no serum control.</p><p>Neutralization test using live MERS-CoV was performed as previously described except
using Vero cells instead of Vero-TMPRSS2 cells [<xref rid="r16" ref-type="bibr">16</xref>]. Briefly, 0.05 m<italic>l</italic> of serially diluted test sera was mixed
with an equal volume of 100 TCID<sub>50</sub> of MERS-CoV (EMC isolate) in a 96-well
culture plate and incubated at 37°C for 1 hr; thereafter,Vero cells were added in each
well and cultivated at 37°C. Cytopathic effects (CPE) on the Vero cells were observed at 3
days after infection. The neutralization titer was determined as the highest serum
dilution showing at least 50% CPE on the inoculated cells.</p></sec><sec><title>S1-ELISA</title><p>Synthetic S1 fragment of MERS-CoV was obtained from Sino Biolobical Inc. (Beijing, China)
and used as the antigen [<xref rid="r17" ref-type="bibr">17</xref>]. ELISA microplates
were coated with 50 <italic>µl</italic> of 50 <italic>n</italic>g/m<italic>l</italic>antigen per well at 4°C overnight, following which the wells were incubated with PBS
containing 2% Block Ace and 0.05% Tween 20 for 2 hr at 37°C. Following the removal of
blocking reagent, diluted serum samples were added and incubated for 1 hr at 37°C. After
washing the wells thrice with 0.05% Tween 20 in PBS (PBS-T), a peroxidase-labeled protein
AG (Thermo Fisher Scientific, Waltham, MA, U.S.A.) was added and incubated for 1 hr at
37°C. Following further washing thrice with PBS-T, 100 <italic>µl</italic> of
2,2′-azinobis-3-ethylbenzthiazolinesulfonic acid (ABTS) substrate solution (Roche Applied
Science, Penzberg, Germany) was added and incubated for 20 min at room temperature. The
optical density (O.D.) of each well was measured at 450 nm using a microplate reader, and
mean absorbance was determined for each serum sample. One of camel serum that showed a
high antibody titer in the neutralization test by live MERS-CoV was treated as a positive
control.</p></sec><sec><title>Competitive ELISA</title><p>The MERS-CoV RBD was used as the antigen of the cELISA. For the preparation of
recombinant RBD, the mammalian expression plasmid pCAGGS-RBD, which encodes
histidine-tagged MERS-CoV RBD (amino acid 358–588), was transfected to 293T cells. At 2
days after transfection, the recombinant RBD was purified from the supernatant of
transfected cells using His-Bind Purification Kit (Merck, Damastadt, Germany). The cELISA
was performed as described by Fukushi <italic>et al</italic>. [<xref rid="r9" ref-type="bibr">9</xref>]. Briefly, MERS-CoV recombinant RBD with pre-determined optimal
quantity was coated on a 96-well ELISA plate at 4°C for overnight, following which the
wells were incubated with PBS containing 2% bovine serum albumin and 0.05% tween 20
(blocking reagent) for 2 hr at 37°C. Following the removal of the blocking reagent, 100
<italic>µl</italic> of a biotin-labeled monoclonal antibody mixed with serially diluted
serum samples was added and incubated for 1 hr at 37°C. One of camel serum that showed a
high antibody titer in the neutralization test by live MERS-CoV was treated as a positive
control. After washing the wells, a streptavidin-HRP (Thermo Fisher Scientific) was added
and incubated for 1 hr at 37°C. Following further washing, 100 <italic>µl</italic> of ABTS
substrate solution was added and incubated for 20 min at room temperature. The O.D. at 405
nm was measured against a reference of 490 nm using a microplate reader (Model 680
Microplate Reader; Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.).</p><p>Percent inhibition at each serum dilution was calculated as follows:</p><p>Percent inhibition=100−[O.D. (405–490 nm) of test sample]/[O.D. (405–490 nm) of no serum
control] × 100</p></sec></sec><sec sec-type="results" id="s2"><title>RESULTS</title><sec><title>Neutralization test using VSV-MERS/GFP</title><p>According to the results of the previous study, antibody titers of ≥16 are treated as
positive in neutralization test using VSV-MERS/GFP. In dromedary camels and cattle, 31 out
of 38 and 1 out of 15, respectively, were MERS antibody positive. Goats and sheep were all
MERS antibody negative (<xref rid="tbl_001" ref-type="table">Table l</xref><table-wrap id="tbl_001" orientation="portrait" position="float"><label>Table 1.</label><caption><title>Distribution of antibody titers in the neutralization test using VSV-MERS/GFP
<sup>a)</sup> among animals sharing grazing area with camels</title></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="3" align="center" valign="middle" colspan="1">Antibody titer</th><th colspan="4" valign="middle" align="center" rowspan="1">Animals (test sample numbers)</th></tr><tr><th colspan="4" rowspan="1"><hr></hr></th></tr><tr><th valign="middle" align="center" rowspan="1" colspan="1">Camel (38)</th><th valign="middle" align="center" rowspan="1" colspan="1">Cattle (15)</th><th valign="middle" align="center" rowspan="1" colspan="1">Goat (25)</th><th valign="middle" align="center" rowspan="1" colspan="1">Sheep (25)</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1"> <16</td><td align="center" valign="top" rowspan="1" colspan="1">7</td><td align="center" valign="top" rowspan="1" colspan="1">14</td><td align="center" valign="top" rowspan="1" colspan="1">20</td><td align="center" valign="top" rowspan="1" colspan="1">20</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">16</td><td align="center" valign="top" rowspan="1" colspan="1">15</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">64</td><td align="center" valign="top" rowspan="1" colspan="1">7</td><td align="center" valign="top" rowspan="1" colspan="1">1</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">≥256</td><td align="center" valign="top" rowspan="1" colspan="1">9</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td></tr></tbody></table><table-wrap-foot><p>a) Pseudotyped vesicular stomatitis virus coated with the spike protein of Middle
East respiratory syndrome coronavirus.</p></table-wrap-foot></table-wrap>). Moreover, in 15 camels, the antibody titer was 16 or 32; in 7, it was 64
or 128; and in 9 and control positive rabbit serum, it was ≥256. The antibody titer and
the positive rate of antibody against MERS-CoV increased with the age of the camels. The
antibody titer in one cow was 64. All sera collected from animals on the attached farm of
Nihon University were negative.</p></sec><sec><title>Comparison of other serological tests with the neutralization test using
VSV-MERS/GFP</title><p>Overall, 31 camels and control positive rabbit serum were found to be antibody positive
in the neutralization test using MERS-CoV, and 16 camels were positive in S1-ELISA. In the
cELISA, 26 camels and 1 cow were positive (<xref rid="tbl_002" ref-type="table">Table
2</xref><table-wrap id="tbl_002" orientation="portrait" position="float"><label>Table 2.</label><caption><title>Comparison of other serological tests with the neutralization test using
VSV-MERS/GFP<sup>a)</sup></title></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" valign="middle" rowspan="1" colspan="1">Animals<break></break>(test sample numbers)</th><th align="center" valign="middle" rowspan="1" colspan="1">NT<sup>b)</sup><break></break>(VSV-MERS/GFP)</th><th align="center" valign="middle" rowspan="1" colspan="1">NT<break></break>(MERS-CoV)</th><th align="center" valign="middle" rowspan="1" colspan="1">S1-ELISA</th><th align="center" valign="middle" rowspan="1" colspan="1">cELISA<sup>c)</sup></th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">Camel (38)</td><td align="center" valign="top" rowspan="1" colspan="1">31<sup>d)</sup></td><td align="center" valign="top" rowspan="1" colspan="1">31</td><td align="center" valign="top" rowspan="1" colspan="1">16</td><td align="center" valign="top" rowspan="1" colspan="1">26</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Cattle (15)</td><td align="center" valign="top" rowspan="1" colspan="1">1</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Goat (25)</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">ND<sup>e)</sup></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Sheep (25)</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">0</td><td align="center" valign="top" rowspan="1" colspan="1">ND</td></tr></tbody></table><table-wrap-foot><p>a) Pseudotyped vesicular stomatitis virus coated with the spike protein of Middle
East respiratory syndrome coronavirus. b) Neutralization test. c) Competitive ELISA.
d) Positive sample numbers. e) Not done.</p></table-wrap-foot></table-wrap>). The sera collected as negative controls from animals on the attached farm
of Nihon University were all negative in S1-ELISA and cELISA.</p><p>Each camel that was antibody positive in S1-ELISA or in neutralization tests using
MERS-CoV was found to be positive in neutralization tests using VSV-MERS/GFP. Cows that
were antibody positive in the neutralization test using VSV-MERS/GFP or cELISA were
different animals and both were antibody negative in the neutralization test using
MERS-CoV.</p><p>A comparison of the antibody titers detected in the neutralization tests using
VSV-MERS/GFP with those detected using live MERS-CoV showed a high correlation between
both antibody titers, with a correlation coefficient of 0.9753.</p></sec></sec><sec sec-type="discussion" id="s3"><title>DISCUSSION</title><p>All camel sera that were positive in any one of the tests—the S1-ELISA, cELISA, or
neutralization tests using live MERS-CoV—were positive in the neutralization test using
VSV-MERS/GFP. One cow serum was positive for cELISA and another cow was positive in the
neutralization test using VSV-MERS/GFP, but negative in all other tests. Because the
neutralization test using live MERS-CoV is considered the most accurate serological test and
because the two positive reactions were observed in only one serological test among the
four, these two bovine sera should be considered as nonspecific reactors. The exact reason
of nonspecific reaction of these sera remains unclear. The nonspecific reaction may result
from differences in immunological conditions of animals or in the degree of hemolysis during
the preparation of samples. For example, a nonspecific reaction sometimes appears in cattle
after injection with a certain inactivated vaccine in the ELISA kit for Johne’s disease
diagnosis in Japan. Some rhabdovirus cross react with VSV [<xref rid="r6" ref-type="bibr">6</xref>] and such a virus might be subclinically infected in Ethiopian cattle.</p><p>There are reports that MERS-CoV specific antibodies were not detected in sera from cattle,
goat and sheep in Jordan and Saudi Arabia, the MERS-CoV prevalence region [<xref rid="r11" ref-type="bibr">11</xref>, <xref rid="r13" ref-type="bibr">13</xref>]. However,
they were kept indoor or did not share the pastureland. Present results indicate that
MERS-CoV infects only dromedary camels and is unlikely to infect other domestic animals even
sharing the pastureland. Since the antibody positive rate and antibody titer of MERS
increased with age, it is considered that MERS-CoV establishes an infection cycle and
inapparently present only among dromedary camels [<xref rid="r11" ref-type="bibr">11</xref>]. In present studies, the limited numbers of samples were tested because the
import of animal sera from the foot-and-mouth disease endemic region is regulated in Japan.
Further studies using additional samples from other countries would be required to clarify
the role of other animal on the ecology and epidemiology of MERS-CoV.</p><p>A comparison of the two neutralization tests using VSV-MERS/GFP and MERS-CoV showed a high
correlation between both antibody titers. The former showed an antibody titer of ≥256,
whereas the latter showed an antibody titer of ≥512. S1-ELISA was not sensitive compared to
other tests because only 16 serum samples were positive and they required an antibody titer
of ≥64 in VSV-MERS/GFP. However, the sensitivity of S1-ELISA would be improved by examining
the cut-off value and dilution of the test serum. In cELISA, five serum samples were
negative out of the 31 positive samples in the neutralization tests, and an antibody titer
of 16 was observed in both neutralization tests. Therefore, neutralization test using
VSV-MERS/GFP exhibits sensitivity similar to the neutralization test using MERS-CoV and is
more sensitive compared with the S1-ELISA as well as cELISA.</p><p>The present study shows that the neutralization test using VSV-MERS/GFP, S1-ELISA, and
cELISA are as specific to MERS-CoV infection as the serological tests, although their
sensitivities slightly differ. The detection of antibody and epidemiological survey of
MERS-CoV could be possible by appropriately selecting these tests according to the desired
purpose, without using a BSL-3 laboratory.</p></sec></body><back><ack><p>This work was partly supported by JSPS KAKENHI (grant number 17H04642),
Japan Agency for Medical Research and Development (grant number JP18fk0108058) and the
Academic Frontier Project for Private Universities (S1491007) from the Ministry of
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