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Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms

Identifieur interne : 000255 ( Pmc/Corpus ); précédent : 000254; suivant : 000256

Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms

Auteurs : Qian Xie ; Yujuan Cao ; Juan Su ; Jie Wu ; Xianbo Wu ; Chengsong Wan ; Mingliang He ; Changwen Ke ; Bao Zhang ; Wei Zhao

Source :

RBID : PMC:5506503

Abstract

Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.

Electronic supplementary material

The online version of this article (doi:10.1007/s00705-017-3361-x) contains supplementary material, which is available to authorized users.


Url:
DOI: 10.1007/s00705-017-3361-x
PubMed: 28421366
PubMed Central: 5506503

Links to Exploration step

PMC:5506503

Le document en format XML

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<title level="j">Archives of Virology</title>
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<p id="Par1">Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.</p>
<sec>
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<p>The online version of this article (doi:10.1007/s00705-017-3361-x) contains supplementary material, which is available to authorized users.</p>
</sec>
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<journal-id journal-id-type="nlm-ta">Arch Virol</journal-id>
<journal-id journal-id-type="iso-abbrev">Arch. Virol</journal-id>
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<issn pub-type="ppub">0304-8608</issn>
<issn pub-type="epub">1432-8798</issn>
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<publisher-name>Springer Vienna</publisher-name>
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<article-id pub-id-type="pmid">28421366</article-id>
<article-id pub-id-type="pmc">5506503</article-id>
<article-id pub-id-type="publisher-id">3361</article-id>
<article-id pub-id-type="doi">10.1007/s00705-017-3361-x</article-id>
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<subject>Annotated Sequence Record</subject>
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<xref ref-type="aff" rid="Aff4">4</xref>
</contrib>
<aff id="Aff1">
<label>1</label>
<institution-wrap>
<institution-id institution-id-type="ISNI">0000 0000 8877 7471</institution-id>
<institution-id institution-id-type="GRID">grid.284723.8</institution-id>
<institution>Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health,</institution>
<institution>Southern Medical University,</institution>
</institution-wrap>
No. 1023 Shatai Road, Guangzhou, 510515 People’s Republic of China</aff>
<aff id="Aff2">
<label>2</label>
<institution-wrap>
<institution-id institution-id-type="ISNI">0000 0000 8803 2373</institution-id>
<institution-id institution-id-type="GRID">grid.198530.6</institution-id>
<institution>Medical Key Laboratory for Repository and Application of Pathogenic Microbiology, Research Center for Pathogens Detection Technology of Emerging Infectious Diseases,</institution>
<institution>Guangdong Provincial Center for Disease Control and Prevention,</institution>
</institution-wrap>
Guangzhou, China</aff>
<aff id="Aff3">
<label>3</label>
<institution-wrap>
<institution-id institution-id-type="ISNI">0000 0004 1792 6846</institution-id>
<institution-id institution-id-type="GRID">grid.35030.35</institution-id>
<institution>Department of Biomedical Sciences,</institution>
<institution>City University of Hong Kong,</institution>
</institution-wrap>
Hong Kong, China</aff>
<aff id="Aff4">
<label>4</label>
<institution-wrap>
<institution-id institution-id-type="ISNI">0000 0000 8877 7471</institution-id>
<institution-id institution-id-type="GRID">grid.284723.8</institution-id>
<institution>Guangzhou Key Laboratory of Drug Research for Emerging Virus Prevention and Treatment, School of Public Health,</institution>
<institution>Southern Medical University,</institution>
</institution-wrap>
Guangzhou, 510515 China</aff>
</contrib-group>
<pub-date pub-type="epub">
<day>18</day>
<month>4</month>
<year>2017</year>
</pub-date>
<pub-date pub-type="pmc-release">
<day>18</day>
<month>4</month>
<year>2017</year>
</pub-date>
<pub-date pub-type="ppub">
<year>2017</year>
</pub-date>
<volume>162</volume>
<issue>8</issue>
<fpage>2445</fpage>
<lpage>2449</lpage>
<history>
<date date-type="received">
<day>5</day>
<month>2</month>
<year>2017</year>
</date>
<date date-type="accepted">
<day>27</day>
<month>3</month>
<year>2017</year>
</date>
</history>
<permissions>
<copyright-statement>© The Author(s) 2017</copyright-statement>
<license license-type="OpenAccess">
<license-p>
<bold>Open Access</bold>
This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (
<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>
), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.</license-p>
</license>
</permissions>
<abstract id="Abs1">
<p id="Par1">Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.</p>
<sec>
<title>Electronic supplementary material</title>
<p>The online version of this article (doi:10.1007/s00705-017-3361-x) contains supplementary material, which is available to authorized users.</p>
</sec>
</abstract>
<kwd-group xml:lang="en">
<title>Keywords</title>
<kwd>Infectious Bronchitis Virus</kwd>
<kwd>ORF5 Gene</kwd>
<kwd>Inverted Repeat Sequence</kwd>
<kwd>Deletion Variant</kwd>
<kwd>Virus Packaging</kwd>
</kwd-group>
<funding-group>
<award-group>
<funding-source>
<institution>Guangdong Provincial Science and Technology </institution>
</funding-source>
<award-id>2010A040302003</award-id>
<principal-award-recipient>
<name>
<surname>Zhang</surname>
<given-names>Bao</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
<funding-group>
<award-group>
<funding-source>
<institution> Guangdong Provincial Science and Technology </institution>
</funding-source>
<award-id>2011B031800163</award-id>
<principal-award-recipient>
<name>
<surname>Zhang</surname>
<given-names>Bao</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
<funding-group>
<award-group>
<funding-source>
<institution-wrap>
<institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001809</institution-id>
<institution>National Natural Science Foundation of China</institution>
</institution-wrap>
</funding-source>
<award-id>NO.31670168</award-id>
<principal-award-recipient>
<name>
<surname>Zhang</surname>
<given-names>Bao</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
<funding-group>
<award-group>
<funding-source>
<institution>The 12th five-year-major-projects of China’s Ministry of Public Health</institution>
</funding-source>
<award-id>2012zx10004-213</award-id>
<principal-award-recipient>
<name>
<surname>Zhao</surname>
<given-names>Wei</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
<custom-meta-group>
<custom-meta>
<meta-name>issue-copyright-statement</meta-name>
<meta-value>© Springer-Verlag GmbH Austria 2017</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
</front>
<body>
<sec id="Sec1">
<title>Introduction</title>
<p id="Par2">Middle East respiratory syndrome coronavirus (MERS CoV) has been reported in more than 23 countries [
<xref ref-type="bibr" rid="CR1">1</xref>
] since the first case was identified in 2012 [
<xref ref-type="bibr" rid="CR2">2</xref>
]. Infection with this virus leads to a mortality rate of about 40%, but its origin is still not known [
<xref ref-type="bibr" rid="CR3">3</xref>
<xref ref-type="bibr" rid="CR7">7</xref>
]. MERS CoV belongs to lineage C of the betacoronaviruses and has a single-stranded, positive-sense, 30.1-kb RNA genome. The viral genomic RNA encodes four structural proteins, i.e., spike glycoprotein (S), envelope (E), matrix (M) and nucleocapsid (N), as well as several nonstructural proteins, including ORF3-5 and ORF8b [
<xref ref-type="bibr" rid="CR8">8</xref>
].</p>
<p id="Par3">Recently, 186 individuals were confirmed to be infected with MERS CoV in Korea. During the epidemic, one person who was in close contact with a MERS CoV patient started to show MERS symptoms shortly after he traveled to Guangdong Province of China and was confirmatively diagnosed with MERS CoV by lab tests. The patient was cured after 31 days of treatment with antiviral, anti-infection, and immune-enhancing agents. In order to better understand the transmission and evolution of this virus [
<xref ref-type="bibr" rid="CR9">9</xref>
], viral RNA was isolated from a nasopharyngeal swab sample of the Korean patient and sequenced. In addition to the wild-type (WT) virus, two deletion variants of MERS CoV were detected in this patient.</p>
</sec>
<sec id="Sec2">
<title>Materials and methods</title>
<p id="Par4">The cDNA was amplified using 24 pairs of primers (Supplemental Table 1). Each fragment amplified by RT-PCR was about 1500 bp in length. After electrophoresis, PCR products were recovered using a PCR purification kit and sequenced on an AB3730 sequencer (Life Technologies, Guangzhou, China). The sequences obtained from PCR products were assembled into a full-length genome sequence using DNAstar (version 7.0, DNASTAR Inc., Madison, WI, USA). [
<xref ref-type="bibr" rid="CR10">10</xref>
]. RNA was extracted from nasopharyngeal swab specimens collected on days 4, 5, 10, and 13 after onset of fever. Reverse transcription of RNA into cDNA was performed as described previously. The cDNA was used as the template for PCR amplification with LA-Taq mix (TaKaRa) and primer pair no. 22. PCR products were analyzed by 1% agarose gel electrophoresis. Protein sequences were aligned using MEGA (version 6.0) [
<xref ref-type="bibr" rid="CR11">11</xref>
]. TransMembrane software was used to predict the transmembrane domain of the ORF5 protein (
<ext-link ext-link-type="uri" xlink:href="http://www.cbs.dtu.dk/services/TMHMM/">http://www.cbs.dtu.dk/services/TMHMM/</ext-link>
) [
<xref ref-type="bibr" rid="CR12">12</xref>
]. RNA secondary structure was predicted using RNAfold software, available at
<ext-link ext-link-type="uri" xlink:href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</ext-link>
[
<xref ref-type="bibr" rid="CR13">13</xref>
].</p>
</sec>
<sec id="Sec3">
<title>Results and discussion</title>
<p id="Par5">All products yielded usable sequences except those produced using primer pair no. 22. Two specific products obtained by nested PCR (Fig. 
<xref rid="Fig1" ref-type="fig">1</xref>
A) were purified, cloned and sequenced. The lower-molecular-weight band was composed of two variants that differed by 5 bp. Variant 2 was longer than variant 1, with the sequence TATGG adjacent to the sequence CTCATGG). The upper band (WT) was 414 bp longer than variant 2 after the sequence CTCATGGTATGG. All fragments of the sequences were assembled into three contigs of WT, variant 1 and variant 2. The genomic sequences have been uploaded to GenBank as KT036372 [
<xref ref-type="bibr" rid="CR14">14</xref>
], KT036373 and KT036374, and the main differences in their nucleotide sequences are shown in Fig. 
<xref rid="Fig1" ref-type="fig">1</xref>
B.
<fig id="Fig1">
<label>Fig. 1</label>
<caption>
<p>Schematic diagram of WT, variant 1 and variant 2 of MERS CoV.
<bold>A.</bold>
PCR product electrophoresis of the variant fragment. M, DNA marker; lane 1, no. 22 PCR product of the sample.
<bold>B.</bold>
Comparison of three sequences in two bands (A).
<bold>C.</bold>
Protein changes of ORF5 and the E protein in the three genomes (WT, variant 1 and variant 2)</p>
</caption>
<graphic xlink:href="705_2017_3361_Fig1_HTML" id="MO1"></graphic>
</fig>
</p>
<p id="Par6">The predicted changes in the primary structures of the ORF5 and E proteins are shown in (Fig. 
<xref rid="Fig1" ref-type="fig">1</xref>
C). Variant 2 encodes a fusion protein of the ORF5 and E proteins (ORF5-E) with an 81-amino-acid (aa) deletion at the C-terminus of ORF5 and a 31-aa deletion at the N-terminus of the E protein. Variant 1 encodes two truncated proteins: a 143-aa fragment of the N-terminus of ORF5 with an additional 5 aa (FPYGY), and a 52-aa fragment of the C-terminus of the E protein. Until now, no such variant has been found in the NCBI database.</p>
<p id="Par7">Although the function of the S protein has been examined previously [
<xref ref-type="bibr" rid="CR15">15</xref>
<xref ref-type="bibr" rid="CR19">19</xref>
], our knowledge of ORF5 and E protein functions in MERS CoV is limited [
<xref ref-type="bibr" rid="CR20">20</xref>
]. Moreover, the effects of ORF5 and E protein mutations on viral packaging, infection and disease development have not been evaluated. Based on studies of other coronaviruses, it is believed that the E protein is important for virus packaging and replication [
<xref ref-type="bibr" rid="CR20">20</xref>
<xref ref-type="bibr" rid="CR22">22</xref>
]. The conserved hydrophobic transmembrane N-terminal domain of the E protein is necessary for CoV to be implanted in the membrane. Even single point mutations in the transmembrane protein of the infectious bronchitis virus (IBV) E protein [
<xref ref-type="bibr" rid="CR23">23</xref>
], or amino acid changes in the N-terminus of the SARS-CoV E protein can result in attenuation of virulence [
<xref ref-type="bibr" rid="CR24">24</xref>
]. To predict the function of the E protein of MERS CoV, we aligned the E and ORF5-E protein sequences of MERS CoV with those of two other coronaviruses, SARS-CoV and China Rattus coronavirus HKU24, using MEGA software (version 6.0) [
<xref ref-type="bibr" rid="CR11">11</xref>
]. The results showed that the E protein of MERS CoV shares high similarity with the other two coronavirus (45% for SARS CoV; 60% for HKU24 CoV) in the N-terminal, C-terminal and transmembrane domains (Fig. 
<xref rid="Fig2" ref-type="fig">2</xref>
A). The truncated E protein with a deletion of aa 1-30 lacks the N-terminus and a major part of the hydrophobic transmembrane domain in MERS CoV variant 1, which might directly impair virus packaging and replication [
<xref ref-type="bibr" rid="CR24">24</xref>
]. The putative fusion ORFF5-E protein (Fig. 
<xref rid="Fig2" ref-type="fig">2</xref>
B) encoded by variant 2 is predicted to have three transmembrane regions (TransMembrane Hidden Markov Models [
<xref ref-type="bibr" rid="CR12">12</xref>
]), and it remains unclear whether it is able to function like the wild-type E protein.
<fig id="Fig2">
<label>Fig. 2</label>
<caption>
<p>Structure analysis of the E proteins of the wild type and two variants of MERS CoV.
<bold>A.</bold>
E protein sequence alignment of related coronavirus. The sequences of SARS CoV (NP_828854), HKU24 (NC026011), MERS CoV (NC_019843), and ORF5-E of variant 1
<bold>B.</bold>
ORF5 protein transmembrane domain predicted with TMHMM at
<ext-link ext-link-type="uri" xlink:href="http://www.cbs.dtu.dk/services/TMHMM/">http://www.cbs.dtu.dk/services/TMHMM/</ext-link>
.
<bold>C.</bold>
RNA secondary structure prediction of the deletion region. RNA secondary structure was predicted using RNAfold software (
<ext-link ext-link-type="uri" xlink:href="http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi">http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi</ext-link>
). The input sequence was based on KT036372, nt 26963-27610. The solid and broken lines indicate the inverted repeat sequence of nt 27131-GTCATACACACCAA-27144 and nt 27527-TTGGTGTGTATGGC-27540, respectively</p>
</caption>
<graphic xlink:href="705_2017_3361_Fig2_HTML" id="MO2"></graphic>
</fig>
</p>
<p id="Par8">Almazán et al. reported that MERS CoV with a deletion in the E gene produced replication-competent but propagation-defective virus particles and proposed that this defective virus should be a potential vaccine candidate for preventing MERS CoV infection [
<xref ref-type="bibr" rid="CR25">25</xref>
]. The two variants identified in this study carried mutations in the N-terminal domain, which is dispensable for the function of the E protein. However, variations in this region lead to changes in the location of this protein, and therefore, the virulence of these two variants might be impaired to some extent. This needs to be investigated using a recombinant virus.</p>
<p id="Par9">The ORF5 gene of both variants of MERS CoV in this study was truncated and fused with the E protein. The effect of these variations on the virus could not be predicted because the function of the ORF5 gene is not well understood. However, Scobey et al. found that the effect of ORF5 deletions on the viral replication is minimal, but deletion of the whole ORF5 gene significantly enhances S protein expression [
<xref ref-type="bibr" rid="CR26">26</xref>
]. More investigations are required to determine the effects of the ORF5 mutant in these two variants.</p>
<p id="Par10">Intragenomic sequence deletions have been found in some coronavirus [
<xref ref-type="bibr" rid="CR27">27</xref>
,
<xref ref-type="bibr" rid="CR28">28</xref>
]. It has been proposed that this occurs by a copy-choice or template-strand-switching mechanism [
<xref ref-type="bibr" rid="CR29">29</xref>
]. One important condition is for there to be a specific leader sequence flanked by the deletion region and a stem-loop structure [
<xref ref-type="bibr" rid="CR30">30</xref>
]. Leader sequences corresponding to the UCUAAAC sequence of murine hepatitis virus (MHV) or the CUUAACA sequence of infectious bronchitis virus (IBV) were not found in MERS CoV in this study. Maori et al. have found that inverted repeats facilitate looping out of the middle genomic sequences during RNA replication, resulted in a defective RNA genome [
<xref ref-type="bibr" rid="CR31">31</xref>
]. An RNA secondary structure predicted using the RNAfold webserver [
<xref ref-type="bibr" rid="CR13">13</xref>
] suggested that the inverted repeat sequence contains long complementary sequences at each end and forms a strong stem-loop structure in the deletion region (Fig. 
<xref rid="Fig2" ref-type="fig">2</xref>
C). The deleted sequence was closely adjoined, characterized by a 14-bp nearly complete inverted repeat sequence consisting of 27131-GTCATACACACCAA-27144 and 27527-TTGGTGTGTATGGC-27540, which would result in RNA replicase jumping from one segment to another distant segment. Whether this feature is linked to RNA intramolecular recombination remains to be investigated.</p>
<p id="Par11">Wild-type MERS CoV and two variants were isolated for the first time from a patient who had traveled from Korea to China. Genomic sequencing revealed 414-bp and 419-bp deletions between ORF5 and the E protein that would result in partial fusion or truncation of these proteins. Whether this finding is a special case or not needs to be investigated by sequencing more samples. Based on previous studies of E protein localization [
<xref ref-type="bibr" rid="CR23">23</xref>
<xref ref-type="bibr" rid="CR25">25</xref>
,
<xref ref-type="bibr" rid="CR32">32</xref>
,
<xref ref-type="bibr" rid="CR33">33</xref>
], we conclude that the two variants might affect virus packaging, which could result in the attenuation of virulence and therefore be relevant for studies related to vaccine development, pathogenesis and viral evolution.</p>
</sec>
<sec sec-type="supplementary-material">
<title>Electronic supplementary material</title>
<sec id="Sec4">
<p>Below is the link to the electronic supplementary material.
<supplementary-material content-type="local-data" id="MOESM1">
<media xlink:href="705_2017_3361_MOESM1_ESM.doc">
<caption>
<p>Supplementary material 1 (DOC 100 kb)</p>
</caption>
</media>
</supplementary-material>
</p>
</sec>
</sec>
</body>
<back>
<fn-group>
<fn>
<p>Qian Xie, Yujuan Cao, Changwen Ke, Bao Zhang, Wei Zhao authors contributed equally to this article.</p>
</fn>
</fn-group>
<ack>
<title>Acknowledgements</title>
<p>We thank Dr. Zhu Hong (Zhejiang University) for scholarly advice, and Dr. Xu Jin (Southern Medical University) for language editing. This study was partially supported by grants from Guangdong Provincial Science and Technology (Nos. 2010A040302003 and 2011B031800163), National Natural Science Foundation of China (No. 31670168) and the 12th five-year-major-projects of China’s Ministry of Public Health, Grant No. 2012zx10004-213.</p>
</ack>
<notes notes-type="ethics">
<title>Compliance with ethical standards</title>
<notes notes-type="COI-statement">
<title>Conflict of interest</title>
<p id="Par12">The author of this study has no conflict of interest.</p>
</notes>
<notes>
<title>Ethical approval</title>
<p id="Par13">The MERS patient was informed of an urgent clinical test. This study was approved by the Medical Ethics Committee of Southern Medical University on May 28 and is in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.</p>
</notes>
</notes>
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