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Article(id=1240633243620406235, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1240633237542851387, articleNumber=null, orderNo=null, doi=10.20043/j.cnki.MPM.202403475, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1711382400000, receivedDateStr=2024-03-26, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1773719615129, onlineDateStr=2026-03-17, pubDate=1716566400000, pubDateStr=2024-05-25, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1773719615129, onlineIssueDateStr=2026-03-17, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1773719615129, creator=13701087609, updateTime=1773719615129, updator=13701087609, issue=Issue{id=1240633237542851387, tenantId=1146029695717560320, journalId=1227665162245664772, year='2024', volume='51', issue='10', pageStart='1729', pageEnd='1920', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1773719613680, creator=13701087609, updateTime=1773720039302, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1240635022806405370, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1240633237542851387, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1240635022806405371, tenantId=1146029695717560320, journalId=1227665162245664772, issueId=1240633237542851387, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=1878, endPage=1883, ext={EN=ArticleExt(id=1240633244031448048, articleId=1240633243620406235, tenantId=1146029695717560320, journalId=1227665162245664772, language=EN, title=Genetic characteristics of the first imported monkeypox virus in the mainland, China, columnId=1228016573156360233, journalTitle=Modern Preventive Medicine, columnName=Disease Control and Prevention, runingTitle=null, highlight=null, articleAbstract=
Objective

Different library construction methods, combined with high-throughput sequencing methods, were used to understand the genetic characteristics of the virus of the first confirmed imported monkeypox case in mainland China, and to compare the advantages and disadvantages of different library types.

Methods

The herpetic fluids and nasopharyngeal swabs of the first imported monkeypox case in the Chinese mainland were used as samples. After nucleic acid extraction and quantification, the library was directly constructed or the amplicon library was constructed using the monkeypox virus whole genome capture kit, and the whole genome sequence of the virus was obtained by high-throughput sequencing. Combined with 34 monkeypox virus sequences downloaded from the NCBI and GISAID databases, a phylogenetic evolutionary tree was constructed using vaccinia, variola, and cowpox sequences as out groups.

Results

The whole genome sequence of the virus was obtained by both library construction methods, named hMpxV/China/CQ-CQCDC-001/2022, which belongs to the monkeypox virus branch IIb, located in the same branch as hMpxV/Germany/BE-ChVir28656/2022 belonging to the IIb B.1 branch. Metagenomic libraries have more uniform sequence coverage than amplicon libraries, but the sequencing depth is lower and the effective data volume accounts for less.

Conclusion

Clade IIb B.1 monkeypox viruses have already been introduced into the mainland China, and the sequencing of subsequently discovered monkeypox viruses should be based on the sample size and sequencing timeframe, sequencing cost-effectiveness ratio, and other factors to choose the appropriate library construction method.

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目的

通过不同文库构建方式，结合高通量测序方法，了解中国大陆首例输入性猴痘确诊病例病毒的基因特征，比较不同文库类型优缺点。

方法

以中国大陆首例输入性猴痘病例的疱疹液及鼻咽拭子为样本，提取核酸定量后直接进行文库构建或使用猴痘病毒全基因组捕获试剂盒进行扩增子文库构建，通过高通量测序，获得病毒全基因组序列。结合NCBI和GISAID数据库下载的34个猴痘病毒序列，以牛痘病毒、天花病毒和痘苗病毒序列为外群，构建系统发育进化树。

结果

两种文库构建方式均测序获得该病毒的全基因组序列，命名为hMpxV/China/CQ-CQCDC-001/2022，属于MPXV IIb分支，与属于IIb B.1分支的hMpxV/Germany/BE-ChVir28656/2022位于同一分支。宏基因组文库较扩增子文库所得序列覆盖度更均匀，但测序深度较低，有效数据量占比较少。

结论

IIb B.1分支猴痘病毒已经传入中国大陆境内，对后续发现的猴痘病毒测序时应根据样本量和测序时限、测序费效比等因素选择适合的文库构建方法。

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裴晓方，E-mail:
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唐云（1993—），女，硕士在读，主管技师，研究方向：微生物、公从健康与检验

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唐云（1993—），女，硕士在读，主管技师，研究方向：微生物、公从健康与检验

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BMJ, 2022, 377: o1239., articleTitle=Seven monkeypox cases are confirmed in England, refAbstract=null), Reference(id=1240633255624503923, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, doi=null, pmid=null, pmcid=null, year=2022, volume=3, issue=8, pageStart=e554, pageEnd=e555, url=null, language=null, rfNumber=[5], rfOrder=4, authorNames=Otu A, Ebenso B, Walley J, journalName=The Lancet. Microbe, refType=null, unstructuredReference=Otu A, Ebenso B, Walley J, et al. Global human monkeypox outbreak: atypical presentation demanding urgent public health action[J]. The Lancet. 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Viruses, 2023, 15(4): 995., articleTitle=Mpox virus: its molecular evolution and potential impact on viral epidemiology, refAbstract=null), Reference(id=1240633256597582506, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, doi=null, pmid=null, pmcid=null, year=2022, volume=28, issue=8, pageStart=1569, pageEnd=1572, url=null, language=null, rfNumber=[14], rfOrder=14, authorNames=Isidro J, Borges V, Pinto M, journalName=Nature Medicine, refType=null, unstructuredReference=Isidro J, Borges V, Pinto M, et al. Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus[J]. Nature Medicine, 2022, 28(8): 1569-1572., articleTitle=Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus, refAbstract=null), Reference(id=1240633256715023024, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[15], rfOrder=15, authorNames=World Health Organization, journalName=null, refType=null, unstructuredReference=World Health Organization. 2022-23 Mpox (Monkeypox) outbreak: Global trends[EB/OL].[2024-04-30]. https://mcc-covid.crc.pitt.edu/COVID19_official_websites/Global/monkeypox_who_report/2023-02-23_183451.html., articleTitle=2022-23 Mpox (Monkeypox) outbreak: Global trends, refAbstract=null), Reference(id=1240633256811492022, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, doi=null, pmid=null, pmcid=null, year=2019, volume=69, issue=null, pageStart=8, pageEnd=11, url=null, language=null, rfNumber=[16], rfOrder=16, authorNames=Sadeuh-Mba SA, Yonga MG, Els M, journalName=Infection, Genetics and Evolution, refType=null, unstructuredReference=Sadeuh-Mba SA, Yonga MG, Els M, et al. Monkeypox virus phylogenetic similarities between a human case detected in Cameroon in 2018 and the 2017-2018 outbreak in Nigeria[J]. 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journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Fig.1, caption=Summary of the reads generated by each of the two methods, figureFileSmall=a4GSj480a4KY6J83QVU1gQ==, figureFileBig=2TVBlXOxofrjrDT4qebdLA==, tableContent=null), ArticleFig(id=1240633251597971882, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=图1, caption=两种方法下机数据中reads的构成情况图, figureFileSmall=a4GSj480a4KY6J83QVU1gQ==, figureFileBig=2TVBlXOxofrjrDT4qebdLA==, tableContent=null), ArticleFig(id=1240633251820270012, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Fig.2, caption=Traces of two methods mapping to the Reference NC_063383.1, figureFileSmall=clHk5dqK7Y8gz0PIxcrNEw==, figureFileBig=q5s0QFrzWPzjM7dIdwo2KQ==, tableContent=null), ArticleFig(id=1240633251950293444, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=图2, caption=两种方法有参拼接时覆盖度情况, figureFileSmall=clHk5dqK7Y8gz0PIxcrNEw==, figureFileBig=q5s0QFrzWPzjM7dIdwo2KQ==, tableContent=null), ArticleFig(id=1240633252084511177, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Fig.3, caption=Phylogeny of monkeypox viruses based on complete genomes, figureFileSmall=ANzlXe+rYG4VycN6m2kWxw==, figureFileBig=GYYOGkmKlQ8xTMjfedlaIA==, tableContent=null), ArticleFig(id=1240633252227117524, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=图3, caption=基于全基因组的猴痘病毒进化比对（最大似然法，Bootstrap 值设定为1 000）, figureFileSmall=ANzlXe+rYG4VycN6m2kWxw==, figureFileBig=GYYOGkmKlQ8xTMjfedlaIA==, tableContent=null), ArticleFig(id=1240633252331975127, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Fig.4, caption=SNPs characterizing of the virus from the import case, figureFileSmall=/hNbH2NDxjZBmdNok6MVoQ==, figureFileBig=v5/DidvgWL9+X/UZpyLkbQ==, tableContent=null), ArticleFig(id=1240633252441027045, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=图4, caption=hMpxV/China/CQ-CQCDC-001/2022的变异位点示意图, figureFileSmall=/hNbH2NDxjZBmdNok6MVoQ==, figureFileBig=v5/DidvgWL9+X/UZpyLkbQ==, tableContent=null), ArticleFig(id=1240633252554273263, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Table 1, caption=

Genomes included in the analyze

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名称登录号国家/地区采集日期分支
猴痘病毒NC_063383.1IIb A
Zaire-96-I-16NC_003310.1扎伊尔I
痘苗病毒NC_006998.1外群
天花病毒NC_001611.1外群
牛痘病毒NC_003663.2外群
MpxV/cynomolgus_monkey/USA/un-WRAIR7-61-P2/1962EPI_ISL_13056556美国1962IIa
hMpxV/Liberia/CDC-184/1970EPI_ISL_13058405利比里亚1970IIa
hMpxV/Israel/IIBR-01/2018EPI_ISL_13056289以色列2018-10-04IIb A.1
hMpxV/Singapore/NCID-01/2019EPI_ISL_13058475新加坡2019-05-08IIb A.1
hMpxV/United_Kingdom/UKHSA-2091/2019EPI_ISL_13734230英国2019-12IIb A.1
hMpxV/India/KL-ICMR-16-5316-553/2022EPI_ISL_13953610印度2022-07-13IIb A.2.1
hMpxV/Thailand/CU-220016-FTV/2022EPI_ISL_14011193泰国2022-07-17IIb A.2
hMpxV/United_Kingdom/UKHSA-9000166/2022EPI_ISL_14752288英国2022-06-29IIb A.2.2
hMpxV/USA/PA-CDC-0016/2022EPI_ISL_15016116美国2022-07IIb A.2.2
hMpxV/Nigeria/CDC-M3021/2018EPI_ISL_13056285尼日利亚2018IIb A
hMpxV/Thailand/NIC-PKT-M1/2022EPI_ISL_14295679泰国2022-08-02IIb A
hMpxV/United_Kingdom/UKHSA-50/2022EPI_ISL_14439755英国2022-06-14IIb B.1.10
hMpxV/USA/WA-CDC-0009/2022EPI_ISL_15016106美国2022-07IIb B.1.11
hMpxV/Brazil/SP-IAL-07/2022EPI_ISL_13436658巴西2022-06-14IIb B.1.12
hMpxV/United_Kingdom/UKHSA-9000426/2022EPI_ISL_15158453英国2022-08IIB B.1.1
hMpxV/United_Kingdom/UKHSA-9000390/2022EPI_ISL_15158421英国2022-08IIb B.1.2
hMpxV/United_Kingdom/UKHSA-56/2022EPI_ISL_14439761英国2022-07-07IIb B.1.3
hMpxV/United_Kingdom/UKHSA-J6xzTO_9000287/2022EPI_ISL_14934687英国2022-08IIb B.1.4
hMpxV/Switzerland/ZH-UZH-IMV-3ba64538/2022EPI_ISL_13251723瑞士2022-06-02IIb B.1.5
hMpxV/Peru/LIM-INS-102/2022EPI_ISL_14584307秘鲁2022-08-03IIb B.1.6
hMpxV/Hong_Kong/HKU-220914-001/2022EPI_ISL_14945299香港2022-09-06IIb B.1.7
hMpxV/United_Kingdom/UKHSA-WOcPOH_9000297/2022EPI_ISL_14934692英国2022-08IIb B.1.7
hMpxV/Germany/un-RKI-103/2022EPI_ISL_13734238德国2022-07IIb B.1.8
hMpxV/Brazil/BA-IAL-92/2022EPI_ISL_14995619巴西2022-08-31IIb B.1.9
hMpxV/USA/IL-IDPH-021/2022EPI_ISL_15158335美国2022-07-05IIb B.1
hMpxV/USA/IL-IDPH-084/2022EPI_ISL_15158398美国2022-07-06IIb B.1
hMpxV/DRC/CDC-005/1978EPI_ISL_13053218刚果金1978I
hMpxV/DRC/OHSU-01-P5/1978EPI_ISL_13058456刚果金1978I
hMpxV/Taiwan/TCDC-110-364682/2022EPI_ISL_13908328中国台湾2022-07IIb B.1
hMpxV/Taiwan/CVDCDC-110-231642/2022EPI_ISL_13632071中国台湾2022-06-27IIb B.1.5
hMpxV/Germany/BE-ChVir28656/2022EPI_ISL_13889435德国2022-06-21IIb B.1
hMpxV/Germany/BE-ChVir28446/2022EPI_ISL_13890471德国2022-06-10IIb B.1
), ArticleFig(id=1240633252713656826, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=表1, caption=

本研究中使用的基因组序列信息

, figureFileSmall=null, figureFileBig=null, tableContent=
名称登录号国家/地区采集日期分支
猴痘病毒NC_063383.1IIb A
Zaire-96-I-16NC_003310.1扎伊尔I
痘苗病毒NC_006998.1外群
天花病毒NC_001611.1外群
牛痘病毒NC_003663.2外群
MpxV/cynomolgus_monkey/USA/un-WRAIR7-61-P2/1962EPI_ISL_13056556美国1962IIa
hMpxV/Liberia/CDC-184/1970EPI_ISL_13058405利比里亚1970IIa
hMpxV/Israel/IIBR-01/2018EPI_ISL_13056289以色列2018-10-04IIb A.1
hMpxV/Singapore/NCID-01/2019EPI_ISL_13058475新加坡2019-05-08IIb A.1
hMpxV/United_Kingdom/UKHSA-2091/2019EPI_ISL_13734230英国2019-12IIb A.1
hMpxV/India/KL-ICMR-16-5316-553/2022EPI_ISL_13953610印度2022-07-13IIb A.2.1
hMpxV/Thailand/CU-220016-FTV/2022EPI_ISL_14011193泰国2022-07-17IIb A.2
hMpxV/United_Kingdom/UKHSA-9000166/2022EPI_ISL_14752288英国2022-06-29IIb A.2.2
hMpxV/USA/PA-CDC-0016/2022EPI_ISL_15016116美国2022-07IIb A.2.2
hMpxV/Nigeria/CDC-M3021/2018EPI_ISL_13056285尼日利亚2018IIb A
hMpxV/Thailand/NIC-PKT-M1/2022EPI_ISL_14295679泰国2022-08-02IIb A
hMpxV/United_Kingdom/UKHSA-50/2022EPI_ISL_14439755英国2022-06-14IIb B.1.10
hMpxV/USA/WA-CDC-0009/2022EPI_ISL_15016106美国2022-07IIb B.1.11
hMpxV/Brazil/SP-IAL-07/2022EPI_ISL_13436658巴西2022-06-14IIb B.1.12
hMpxV/United_Kingdom/UKHSA-9000426/2022EPI_ISL_15158453英国2022-08IIB B.1.1
hMpxV/United_Kingdom/UKHSA-9000390/2022EPI_ISL_15158421英国2022-08IIb B.1.2
hMpxV/United_Kingdom/UKHSA-56/2022EPI_ISL_14439761英国2022-07-07IIb B.1.3
hMpxV/United_Kingdom/UKHSA-J6xzTO_9000287/2022EPI_ISL_14934687英国2022-08IIb B.1.4
hMpxV/Switzerland/ZH-UZH-IMV-3ba64538/2022EPI_ISL_13251723瑞士2022-06-02IIb B.1.5
hMpxV/Peru/LIM-INS-102/2022EPI_ISL_14584307秘鲁2022-08-03IIb B.1.6
hMpxV/Hong_Kong/HKU-220914-001/2022EPI_ISL_14945299香港2022-09-06IIb B.1.7
hMpxV/United_Kingdom/UKHSA-WOcPOH_9000297/2022EPI_ISL_14934692英国2022-08IIb B.1.7
hMpxV/Germany/un-RKI-103/2022EPI_ISL_13734238德国2022-07IIb B.1.8
hMpxV/Brazil/BA-IAL-92/2022EPI_ISL_14995619巴西2022-08-31IIb B.1.9
hMpxV/USA/IL-IDPH-021/2022EPI_ISL_15158335美国2022-07-05IIb B.1
hMpxV/USA/IL-IDPH-084/2022EPI_ISL_15158398美国2022-07-06IIb B.1
hMpxV/DRC/CDC-005/1978EPI_ISL_13053218刚果金1978I
hMpxV/DRC/OHSU-01-P5/1978EPI_ISL_13058456刚果金1978I
hMpxV/Taiwan/TCDC-110-364682/2022EPI_ISL_13908328中国台湾2022-07IIb B.1
hMpxV/Taiwan/CVDCDC-110-231642/2022EPI_ISL_13632071中国台湾2022-06-27IIb B.1.5
hMpxV/Germany/BE-ChVir28656/2022EPI_ISL_13889435德国2022-06-21IIb B.1
hMpxV/Germany/BE-ChVir28446/2022EPI_ISL_13890471德国2022-06-10IIb B.1
), ArticleFig(id=1240633252852068868, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Table 2, caption=

Ct value of the two kinds of samples

, figureFileSmall=null, figureFileBig=null, tableContent=
Ct值试剂1试剂2
疱疹液124.8226.33
疱疹液224.7426.16
鼻咽拭子121.7923.33
鼻咽拭子221.2423.03
), ArticleFig(id=1240633254374601232, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=表2, caption=

两种不同类型样本实时荧光PCR检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=
Ct值试剂1试剂2
疱疹液124.8226.33
疱疹液224.7426.16
鼻咽拭子121.7923.33
鼻咽拭子221.2423.03
), ArticleFig(id=1240633254479458841, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=EN, label=Table 3, caption=

Molecular analysis of the hMpxV/China/CQ-CQCDC-001/2022

, figureFileSmall=null, figureFileBig=null, tableContent=
变异核苷酸位点编码基因氨基酸变异蛋白质结构域的结构、定位和功能
G1262AOPG001S105LPIE 结构域，趋化因子结合蛋白（Cop-C23L）
G2591AOPG002S54FT TNF-α 受体同源物和 PIE 结构域，趋化因子结合蛋白，CrmB
G3111AOPG003-
G3522AOPG003-
C3818TOPG003D264NANK 和 PRANC 结构域，与 NF-κB p105 亚基和 SCP1 结合
C7771TOPG019-
G14000TOPG025A423DANK 和 PRANC 结构域，针对 IFIT 进行降解，抗 IFN
G15428ANA-
A18769GOPG031-
G21723AOPG037-
C23105TOPG038E19KPIE 结构域，阻止 SD28 介导的 T 细胞活化，分泌型
C23564TOPG039-
G25661ANA-
T28175COPG044-
G30367AOPG047-
G31053AOPG047R48CBTB和Kelch结构域，有助于毒力，更大的病变大小
G34459AOPG053P78S成熟病毒粒子（MV）膜蛋白，进入融合复合体（EFC）成分
G37202AOPG056-
G38360AOPG056E125K包裹的病毒粒子（WV）成分与E2蛋白复合，促进WV运输
C38662TOPG056-
C39119TOPG057-
C39139TOPG057E353K棕榈脂化包膜病毒（EV）糖蛋白，P-脂肪酶同源物，WV 形成
G52885AOPG071-
G54117AOPG071L108FDNA 聚合酶
G54635AOPG072D56N巯基氧化酶，S-S 键形成途径，病毒蛋白
C63921TOPG083E266K丝氨酸蛋白酶，病毒粒子形态发生
G64297AOPG083-
C72362TOPG092D196N病毒磷蛋白，7蛋白复合体，早期形态发生
C73066TOPG093S30L病毒晚转录因子1，（VLTF-1），PCNA同源物
G73239AOPG093D88N病毒晚转录因子1，（VLTF-1），PCNA同源物
G74205AOPG094M142IMV膜，EFC组分，肉豆蔻酸酯
G77383AOPG098E162KDNA 结合核心转录蛋白（VP8），I7 依赖性裂解
G81275AOPG105-
A81977GOPG105-
C82373TOPG105-
G82451AOPG105-
C83326TOPG105S734LDNA 依赖性 RNA 聚合酶亚基 rop147
C84587TOPG105-
G87230AOPG109H740YRNA 聚合酶相关蛋白（RAP94），早期转录
G87297AOPG109-
G91728ANA-
G95034AOPG115-
A100261GOPG118K606E早期转录因子（VETF）小亚基，ATP酶，预测解旋酶
C118161TOPG136R476Q主要核心蛋白 4a 的前体（p4a），形态发生
C119296TOPG136D98N主要核心蛋白 4a 的前体（p4a），形态发生
G120262AOPG137D221N新月膜和 Ⅳ 形成，VMAP
C121320TOPG139A17TMV膜磷蛋白，形态发生
G124130AOPG145E62KDNA 依赖性 ATP 酶、DNA 解旋酶
G124674AOPG145R243QDNA 依赖性 ATP 酶、DNA 解旋酶
), ArticleFig(id=1240633254575927842, tenantId=1146029695717560320, journalId=1227665162245664772, articleId=1240633243620406235, language=CN, label=表3, caption=

hMpxV/China/CQ-CQCDC-001/2022变异位点分析[2,11-12]

, figureFileSmall=null, figureFileBig=null, tableContent=
变异核苷酸位点编码基因氨基酸变异蛋白质结构域的结构、定位和功能
G1262AOPG001S105LPIE 结构域，趋化因子结合蛋白（Cop-C23L）
G2591AOPG002S54FT TNF-α 受体同源物和 PIE 结构域，趋化因子结合蛋白，CrmB
G3111AOPG003-
G3522AOPG003-
C3818TOPG003D264NANK 和 PRANC 结构域，与 NF-κB p105 亚基和 SCP1 结合
C7771TOPG019-
G14000TOPG025A423DANK 和 PRANC 结构域，针对 IFIT 进行降解，抗 IFN
G15428ANA-
A18769GOPG031-
G21723AOPG037-
C23105TOPG038E19KPIE 结构域，阻止 SD28 介导的 T 细胞活化，分泌型
C23564TOPG039-
G25661ANA-
T28175COPG044-
G30367AOPG047-
G31053AOPG047R48CBTB和Kelch结构域，有助于毒力，更大的病变大小
G34459AOPG053P78S成熟病毒粒子（MV）膜蛋白，进入融合复合体（EFC）成分
G37202AOPG056-
G38360AOPG056E125K包裹的病毒粒子（WV）成分与E2蛋白复合，促进WV运输
C38662TOPG056-
C39119TOPG057-
C39139TOPG057E353K棕榈脂化包膜病毒（EV）糖蛋白，P-脂肪酶同源物，WV 形成
G52885AOPG071-
G54117AOPG071L108FDNA 聚合酶
G54635AOPG072D56N巯基氧化酶，S-S 键形成途径，病毒蛋白
C63921TOPG083E266K丝氨酸蛋白酶，病毒粒子形态发生
G64297AOPG083-
C72362TOPG092D196N病毒磷蛋白，7蛋白复合体，早期形态发生
C73066TOPG093S30L病毒晚转录因子1，（VLTF-1），PCNA同源物
G73239AOPG093D88N病毒晚转录因子1，（VLTF-1），PCNA同源物
G74205AOPG094M142IMV膜，EFC组分，肉豆蔻酸酯
G77383AOPG098E162KDNA 结合核心转录蛋白（VP8），I7 依赖性裂解
G81275AOPG105-
A81977GOPG105-
C82373TOPG105-
G82451AOPG105-
C83326TOPG105S734LDNA 依赖性 RNA 聚合酶亚基 rop147
C84587TOPG105-
G87230AOPG109H740YRNA 聚合酶相关蛋白（RAP94），早期转录
G87297AOPG109-
G91728ANA-
G95034AOPG115-
A100261GOPG118K606E早期转录因子（VETF）小亚基，ATP酶，预测解旋酶
C118161TOPG136R476Q主要核心蛋白 4a 的前体（p4a），形态发生
C119296TOPG136D98N主要核心蛋白 4a 的前体（p4a），形态发生
G120262AOPG137D221N新月膜和 Ⅳ 形成，VMAP
C121320TOPG139A17TMV膜磷蛋白，形态发生
G124130AOPG145E62KDNA 依赖性 ATP 酶、DNA 解旋酶
G124674AOPG145R243QDNA 依赖性 ATP 酶、DNA 解旋酶
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中国大陆首例输入性猴痘病例的基因特征分析
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唐云 1, 2 , 文海燕 3 , 赵华 2 , 黄为 2 , 叶盛 2 , 裴晓方 1
现代预防医学 | 疾病预防控制 2024,51(10): 1878-1883
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现代预防医学 | 疾病预防控制 2024, 51(10): 1878-1883
中国大陆首例输入性猴痘病例的基因特征分析
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唐云1, 2, 文海燕3, 赵华2, 黄为2, 叶盛2, 裴晓方1
作者信息
  • 1.四川大学华西公共卫生学院/华西第四医院
  • 2.重庆市疾病预防控制中心
  • 3.重庆国际旅行卫生保健中心（重庆海关口岸门诊部

    • 唐云（1993—），女，硕士在读，主管技师，研究方向：微生物、公从健康与检验

通讯作者:

裴晓方，E-mail:
Genetic characteristics of the first imported monkeypox virus in the mainland, China
Yun TANG1, 2, Hai-yan WEN3, Hua ZHAO2, Wei HUANG2, Sheng YE2, Xiao-fang PEI1
Affiliations
  • West China School of Public Health / West China No4 Hospital, Sichuan University, Chengdu, Sichuan 610041, China
出版时间: 2024-05-25 doi: 10.20043/j.cnki.MPM.202403475
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摘要
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目的

通过不同文库构建方式，结合高通量测序方法，了解中国大陆首例输入性猴痘确诊病例病毒的基因特征，比较不同文库类型优缺点。

方法

以中国大陆首例输入性猴痘病例的疱疹液及鼻咽拭子为样本，提取核酸定量后直接进行文库构建或使用猴痘病毒全基因组捕获试剂盒进行扩增子文库构建，通过高通量测序，获得病毒全基因组序列。结合NCBI和GISAID数据库下载的34个猴痘病毒序列，以牛痘病毒、天花病毒和痘苗病毒序列为外群，构建系统发育进化树。

结果

两种文库构建方式均测序获得该病毒的全基因组序列，命名为hMpxV/China/CQ-CQCDC-001/2022，属于MPXV IIb分支，与属于IIb B.1分支的hMpxV/Germany/BE-ChVir28656/2022位于同一分支。宏基因组文库较扩增子文库所得序列覆盖度更均匀，但测序深度较低，有效数据量占比较少。

结论

IIb B.1分支猴痘病毒已经传入中国大陆境内，对后续发现的猴痘病毒测序时应根据样本量和测序时限、测序费效比等因素选择适合的文库构建方法。

猴痘病毒  /  高通量测序  /  基因特征
Objective

Different library construction methods, combined with high-throughput sequencing methods, were used to understand the genetic characteristics of the virus of the first confirmed imported monkeypox case in mainland China, and to compare the advantages and disadvantages of different library types.

Methods

The herpetic fluids and nasopharyngeal swabs of the first imported monkeypox case in the Chinese mainland were used as samples. After nucleic acid extraction and quantification, the library was directly constructed or the amplicon library was constructed using the monkeypox virus whole genome capture kit, and the whole genome sequence of the virus was obtained by high-throughput sequencing. Combined with 34 monkeypox virus sequences downloaded from the NCBI and GISAID databases, a phylogenetic evolutionary tree was constructed using vaccinia, variola, and cowpox sequences as out groups.

Results

The whole genome sequence of the virus was obtained by both library construction methods, named hMpxV/China/CQ-CQCDC-001/2022, which belongs to the monkeypox virus branch IIb, located in the same branch as hMpxV/Germany/BE-ChVir28656/2022 belonging to the IIb B.1 branch. Metagenomic libraries have more uniform sequence coverage than amplicon libraries, but the sequencing depth is lower and the effective data volume accounts for less.

Conclusion

Clade IIb B.1 monkeypox viruses have already been introduced into the mainland China, and the sequencing of subsequently discovered monkeypox viruses should be based on the sample size and sequencing timeframe, sequencing cost-effectiveness ratio, and other factors to choose the appropriate library construction method.

Monkeypox virus  /  High-throughput sequencing  /  Genetic characteristics
唐云, 文海燕, 赵华, 黄为, 叶盛, 裴晓方. 中国大陆首例输入性猴痘病例的基因特征分析. 现代预防医学, 2024 , 51 (10) : 1878 -1883 . DOI: 10.20043/j.cnki.MPM.202403475
Yun TANG, Hai-yan WEN, Hua ZHAO, Wei HUANG, Sheng YE, Xiao-fang PEI. Genetic characteristics of the first imported monkeypox virus in the mainland, China[J]. Modern Preventive Medicine, 2024 , 51 (10) : 1878 -1883 . DOI: 10.20043/j.cnki.MPM.202403475
正文
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猴痘（Monkeypox, Mpox）是一种通常由猴痘病毒（Monkeypox virus, MPXV）引起的自限性人兽共患疾病[1]。
猴痘病毒是痘病毒科正痘属的双链DNA病毒，曾分为“西非分支”和“中非”或“刚果盆地”分支两大主要分支。刚果盆地分支病死率较西非分支更高、传染性更强。猴痘病毒一般在一些非洲国家的人群中流行，且多是动物传播到人类，鲜有持续的人传人报道[2]。然而2022年西非分支在非猴痘流行国家如英国等多个国家引起了人群聚集性疫情[3-5]。截至2023年8月，全球已有112个国家和地区报告了超过8.8万例猴痘病例，其中包括149例死亡病例[6]。在如今全球疫情的流行情况下，为避免污名化病毒株，世卫组织不再使用地理位置命名病毒株，根据系统发育的相似性命名为I,IIa,IIb等分支。分支I对应于先前的“刚果盆地分支”，分支IIa和IIb对应于先前的“西非分支”。引起2022年疫情的病毒株属于IIb分支[7]。
继2022年9月14日，重庆市发现中国大陆首例输入性猴痘病例以来[8]，2023年我国大陆猴痘病例剧增，出现本土传播，截至2023年8月31日，中国内地（不含港澳台）25省（区、市）已报告千余例猴痘确诊病例[9]。为了解猴痘病例的基因特征，本研究使用了猴痘病毒高通量测序方法，对中国大陆首例输入病例的猴痘病毒进行了全基因组测序，分析其基因特征。
1　材料和方法
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1.1　样本采集与核酸提取
2022年9月，一名途经德国和西班牙的旅客进入重庆后，在新冠肺炎隔离点接受为期两周的隔离。隔离期间，该人员自述出现猴痘样临床表现，怀疑感染猴痘病毒[8]。立即采集患者2支鼻咽拭子和2支疱疹液拭子，分别置于含有胍盐灭活剂的病毒样本采集管中，低温（4~8℃）转运至重庆市疾病预防控制中心实验室。涡旋混匀后，每个样品取200 μl进行核酸提取（病毒核酸提取试剂，江苏硕世生物科技股份有限公司，中国泰州），60 μl洗脱液洗脱。
1.2　实时荧光定量PCR检测与文库构建
1.2.1　实时荧光定量PCR检测
按照猴痘实时荧光定量PCR检测试剂盒说明书（北京金豪药业有限公司，中国北京；上海伯杰医疗科技股份有限公司，中国上海）配制PCR反应体系，分别在ABI7500（Thermofisher，美国）和Bio-Rad CFX96（Bio-Rad，美国）实时荧光PCR仪上完成检测。根据Ct值（循环数阈值）选择较高病毒浓度的鼻咽拭子和疱疹液拭子核酸进行文库构建。
1.2.2　文库构建
本研究使用了两种文库构建方法：
方法1：使用Illumina DNA Prep（Illumina Inc，美国圣地亚哥）和NexteraTM DNA CD Indexes（Illumina Inc，美国圣地亚哥），从样本提取的核酸直接进行文库构建。构建的文库经Agilent 2100（Agilent Technologies，美国）测定文库片段长度后，将文库稀释至终浓度2 nM（2 nmol/L）；然后，吸取20 μl稀释好的文库至Illumina NextSeq 2000试剂盒上样孔中，装载试剂盒与测序芯片后，运行测序。
方法2：使用猴痘病毒全基因组捕获试剂盒（北京微未来）扩增核酸，扩增产物经AMpure Beads（Beckman Coulter Inc，美国）纯化产物并定量（Qubit 3.0,Thermofisher，美国），后续文库构建步骤同方法1。
1.2.3　基因组组装
下机FastQ数据导入CLC Genomics Workbench 22.0软件（Qiagen，德国）中。去除宿主基因组序列后，以猴痘病毒参考株（NC_063383.1）作为参考序列，将剩余reads进行有参拼接生成一致性序列。
1.2.4　系统发育分析
从NCBI和GISAID下载34个猴痘病毒序列，以及痘苗病毒、天花病毒和牛痘病毒序列各1条（表1）[10]，经Mafft 7.22对齐后，用MEGA vision 6.06构建系统发育进化树，建树方法选择最大似然法，Bootstrap值设定为1 000。痘苗病毒、天花病毒和牛痘病毒序列作为外群。
1.2.5　编码区域分析
基于序列相似性原理，利用NCBI参考序列（NC_063383.1）的注释和文献[2,11-12]对ORF功能的描述和预测，推断核苷酸突变对样品中病毒编码的氨基酸的影响。
2　结果
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2.1　实时荧光定量PCR检测
鼻咽拭子和疱疹液通过两个生产厂家的实时荧光定量PCR试剂检测，检测结果为猴痘病毒核酸阳性，且鼻咽拭子的Ct值低于疱疹液的Ct值（表2）。
2.2　NGS测序
由于两种样本类型的Ct值接近，因此从每种类型中选择一个样本进行文库构建。
方法1：经Qubit 3.0定量，鼻咽拭子和疱疹液样品中总DNA浓度分别为2.02 ng/μl和0.276 ng/μl。由于疱疹液中总DNA浓度太低，不满足Illumina DNA Prep试剂盒起始浓度的要求，因此仅使用30 μl鼻咽拭子样品核酸根据Illumina DNA Prep试剂盒说明书进行文库构建。文库终浓度为19.0 ng/μl，平均长度为583 bp。经测序总共获得了约2.43亿条reads。去除宿主序列reads后，将剩余reads以猴痘病毒序列（NC_063383.1）为参考基因组进行有参拼接，覆盖度100%（图1，图2），以30×为最小测序深度生成一致性序列。
方法2：鼻咽拭子样本使用猴痘病毒全基因组捕获试剂盒扩增。磁珠纯化后经Qubit 3.0定量，产物总DNA浓度为18.7 ng/μl。取10 μl PCR产物根据Illumina DNA Prep试剂盒说明书进行文库构建并上机测序，总共获得了约1.25亿条reads，与方法1以相同参数生成一致性序列。
两种方法得到的一致性序列相同，命名为hMpxV/China/CQ-CQCDC-001/2022，并上传至GISAID数据库，登录号为EPI_ISL_15005641。
系统发育分析显示，hMpxV/China/CQ-CQCDC-001/2022位于系统发育树中猴痘病毒大分支下的IIb分支中，与属于IIb B.1分支的另一株病毒hMpxV/Germany/BE-ChVir28656/2022位于同一分支，表明该病毒也属于IIb B.1分支（图3）。
与参考序列NC_063383.1相比，hMpxV/China/CQ-CQCDC-001/2022序列中的GC含量为33%，共有72个核苷酸变异位点（图4），其中10个位于序列5’和3’末端的ITR区域。64个变异位点分别位于38个编码区，其中有36个导致氨基酸变化（表3）。余下28个位点位于非编码区。
3　讨论
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由于样本为中国大陆首例猴痘病例，为在短时间内取得尽可能完整的病毒基因组数据进行后续研究，本研究选择两种文库构建方式互为补充验证。
方法1从临床样本中直接进行宏基因组文库构建。优点为无需耗时进行病毒培养，能够快速获得猴痘病毒的全基因组序列，但产出的数据中含有大量宿主序列，猴痘病毒序列仅占比0.19%（473 415条），因此需选用大通量测序试剂盒提升猴痘病毒序列的绝对量，测序成本更高。方法2使用猴痘病毒全基因组捕获试剂盒，靶向扩增病毒序列后再进行文库构建。产出的数据中猴痘病毒序列占比达到了74.76%（93 880 688条）。但由于进行了多轮PCR扩增，引入了PCR扩增偏倚，覆盖度和测序深度的差异较方法1大。且当时的猴痘病毒扩增子试剂盒的扩增效率和试剂盒引物覆盖情况不确定，因此使用两种构建方式互相比对，也互为补充。宏基因组文库通过高通量测序能获得较均匀的猴痘病毒序列覆盖度和较好的测序深度，但会产生大量的无关数据，浪费测序芯片通量；扩增子文库得到的猴痘病毒序列覆盖度分布均匀性稍差，但测序深度更高，且可能引入PCR扩增偏倚。对后续发现的猴痘病毒测序时需综合考量选择适合的文库构建方法。
系统发育树表明该病毒属于MPXV IIb B.1分支，与hMpxV/Germany/BE-ChVir 28656/2022位于同一分支，和患者曾到过德国的流行病学调查情况相符。
hMpxV/China/CQ-CQCDC-001/2022病毒编码区的核苷酸变异导致核苷酸编码的36个氨基酸发生变化。发生相关变化的基因中有14个与猴痘病毒-宿主相互作用有关；基因OPG001，OPG002，OPG003，OPG025，OPG176和OPG193被认为参与病毒的免疫逃逸[11,13]。免疫原性表面糖蛋白OPG210中发生的三种氨基酸变化（D209N、P722S和M1741I）与2022年一些国家猴痘疫情中的病毒特征一致[14]。最近的研究表明，2022年引起非流行地区猴痘暴发疫情的猴痘病毒较2018—2019年流行的病毒有着约50个单核苷酸多态性（SNP）的不同，远超过正痘病毒替代率（每个基因组每年产生1~2个替代变化），这表明当前广泛分布的病毒内的微进化很可能导致了病毒对人类和社区传播的适应性[14-15]。且IIb分支流行株的毒性较I分支低[16]，具有隐匿性，易被忽略，更应加强关注，因此及时完成病毒的序列测定及数据共享至关重要。
人员流动的频繁以及旅行和贸易的全球化，进一步促进了全球传播。2022年爆发的猴痘疫情涉及六大洲、100多个国家，感染人数和死亡病例较往年大大增加，造成极大的疾病负担。为应对猴痘疫情的变化，口岸和疾控部门应继续加强对猴痘疫情的防控监测，推进数据世界共享，有助于观察病毒进化轨迹、传播动态，有助于指导防控措施的制订及后续研究方向。
基金
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  • 四川省科技厅重大专项(2022ZDZX0017)
  • 重庆市技术创新与应用发展专项面上项目(CSTB2022TIAD-GPX0073)
  • 海关总署科研课题(2022HK68)
  • 重庆市卫生健康委医学科研项目(2024WSJK068)
文献
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参考文献 引证文献
排序方式：
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Bunge EM, Hoet B, Chen L, et al. The changing epidemiology of human monkeypox-A potential threat? A systematic review[J]. PLOS Neglected Tropical Diseases, 2022, 16(2): e0010141.
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Khare S, Gurry C, Freitas L, et al. GISAID’s role in pandemic response[J]. China CDC Weekly, 2021, 3(49): 1049-1051.
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Senkevich TG, Yutin N, Wolf YI, et al. Ancient gene capture and recent gene loss shape the evolution of Orthopoxvirus-Host interaction genes[J]. mBio, 2021, 12(4): e0149521.
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Chen NH, Li GY, Liszewski MK, et al. Virulence differences between monkeypox virus isolates from West Africa and the Congo basin[J]. Virology, 2005, 340(1): 46-63.
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Yu X, Shi HC, Cheng G. Mpox virus: its molecular evolution and potential impact on viral epidemiology[J]. Viruses, 2023, 15(4): 995.
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Isidro J, Borges V, Pinto M, et al. Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus[J]. Nature Medicine, 2022, 28(8): 1569-1572.
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2024年第51卷第10期
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文章信息
doi: 10.20043/j.cnki.MPM.202403475
  • 接收时间：2024-03-26
  • 首发时间：2026-03-17
  • 出版时间：2024-05-25
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出版历史
  • 收稿日期：2024-03-26
基金
四川省科技厅重大专项(2022ZDZX0017)
重庆市技术创新与应用发展专项面上项目(CSTB2022TIAD-GPX0073)
海关总署科研课题(2022HK68)
重庆市卫生健康委医学科研项目(2024WSJK068)
作者信息
    1.四川大学华西公共卫生学院/华西第四医院
    2.重庆市疾病预防控制中心
    3.重庆国际旅行卫生保健中心（重庆海关口岸门诊部

通讯作者:

裴晓方，E-mail:
参考文献
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2种不同金属材料的力学参数
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Number of
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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