Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

Changcheng Wu; Ruhan A; Sheng Ye; Fei Ye; Weibang Huo; Roujian Lu; Yue Tang; Jianwei Yang; Xuehong Meng; Yun Tang; Shuang Chen; Li Zhao; Baoying Huang; Zhongxian Zhang; Yuda Chen; Dongfang Li; Wenling Wang; Ke-jia Shan; Jian Lu; Wenjie Tan

doi:10.1016/j.virs.2023.12.002

February 2024

. doi: 10.1016/j.virs.2023.12.002

Citation: Changcheng Wu, Ruhan A, Sheng Ye, Fei Ye, Weibang Huo, Roujian Lu, Yue Tang, Jianwei Yang, Xuehong Meng, Yun Tang, Shuang Chen, Li Zhao, Baoying Huang, Zhongxian Zhang, Yuda Chen, Dongfang Li, Wenling Wang, Ke-jia Shan, Jian Lu, Wenjie Tan. Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing .VIROLOGICA SINICA, 2024, 39(1) : 134-143. http://dx.doi.org/10.1016/j.virs.2023.12.002

基于宏基因和扩增子测序快速测定临床样品中猴痘病毒完整基因组并进行分子溯源

吴长城 ^a ,
阿茹罕 ^a ,
叶盛 ^b ,
叶飞 ^a ,
霍威邦 ^a ,
陆柔剑 ^a ,
唐岳 ^c,h ,
杨健威 ^c ,
孟雪红 ^d ,
唐云 ^b ,
陈爽 ^b ,
赵莉 ^a ,
黄保英 ^a ,
张钟贤 ^a,e ,
陈昱达 ^a,e ,
栗东方 ^f ,
王文玲 ^a ,
单科家 ^g ,
陆剑 ^g,, ,
谭文杰 ^a,e,,

a. 中国疾病预防控制中心病毒病预防控制所;
b. 重庆市疾病预防控制中心;
c. 北京大学生命科学学院;
d. 深圳华大智造科技股份有限公司;
e. 赛默飞世尔科技公司;
f. 包头医学院;
g. 深圳华大因源医药科技有限公司

通讯作者： 陆剑, luj@pku.edu.cn; 谭文杰, tanwj@ivdc.chinacdc.cn
收稿日期： 2023-09-23
录用日期： 2023-12-04

摘要

猴痘病毒（MPXV）当前在全球范围内流行。基因组测序和分子溯源有助于诊断和控制猴痘病毒。猴痘病毒基因组较大、AT碱基偏倚严重且含有大量串联重复序列，从低病毒载量的临床样本中快速鉴定猴痘病毒完整基因组的策略需要不断地优化，以有效地追踪病毒遗传变异。针对来自中国五个猴痘病例的多个临床样本，本研究快速地测定了多个临床样本中猴痘病毒的完整基因组并准确组装了复杂串联重复序列。我们评估了mNGS、扩增子测序技术以及三种测序平台在猴痘病毒基因组测序方面的综合表现。扩增子测序可高效地对临床样本进行测序且成本较低，利于获得高质量的猴痘病毒基因组。三代测序策略可显著提升猴痘病毒基因组中末端串联重复区域的组装质量并纠正了已发表猴痘病毒基因组序列中一处常见的组装错误。此外，我们还在首个输入型猴痘病例感染的病毒中鉴定出了多个单核苷酸变异，提示猴痘病毒可在宿主体内快速演化。本研究成果对制定临床样品中猴痘病毒完整基因组的快速测定策略具有重要意义，将促进猴痘病毒变异及流行趋势监测工作。
- 猴痘病毒（MPXV）
- , 宏基因组
- , 第二代测序
- , 扩增子
- , 第三代测序

Rapid identification of full-length genome and tracing variations of monkeypox virus in clinical specimens based on mNGS and amplicon sequencing

Changcheng Wu ^a ,
Ruhan A ^a ,
Sheng Ye ^b ,
Fei Ye ^a ,
Weibang Huo ^a ,
Roujian Lu ^a ,
Yue Tang ^c,h ,
Jianwei Yang ^c ,
Xuehong Meng ^d ,
Yun Tang ^b ,
Shuang Chen ^b ,
Li Zhao ^a ,
Baoying Huang ^a ,
Zhongxian Zhang ^a,e ,
Yuda Chen ^a,e ,
Dongfang Li ^f ,
Wenling Wang ^a ,
Ke-jia Shan ^g ,
Jian Lu ^g,, ,
Wenjie Tan ^a,e,,

a. NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 100052, China;
b. Chongqing Municipal Key Laboratory for High Pathogenic Microbes, Chongqing Center for Disease Control and Prevention, Chongqing, 400042, China;
c. MGI, BGI-Shenzhen, Shenzhen, 518083, China;
d. Thermo Fisher Scientific, Beijing, 100013, China;
e. School of Public Health, Baotou Medical College, Baotou, 014030, China;
f. BGI PathoGenesis Pharmaceutical Technology, Shenzhen, 518000, China;
g. State Key Laboratory of Protein and Plant Gene Research, Center for Bioinformatics, School of Life Sciences, Peking University, Beijing, 100871, China;
h. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China

Corresponding author: Jian Lu, luj@pku.edu.cn Wenjie Tan, tanwj@ivdc.chinacdc.cn
Received Date: 23 September 2023
Accepted Date: 04 December 2023

Abstract

The monkeypox virus (MPXV) has triggered a current outbreak globally. Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control. It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads, as it is one of the DNA viruses with the largest genome and the most AT-biased, and has a significant number of tandem repeats. Here we evaluated the performance of metagenomic and amplicon sequencing techniques, and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland. We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens. Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes. Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences. Besides, several intra-host single nucleotide variations were identified in the first imported mpox case. This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens. The findings of this study will significantly enhance the surveillance of MPXV.
- Monkeypox virus (MPXV)
- , Metagenomic
- , Next generation sequencing
- , Amplicon
- , Third-generation sequencing

References
1. Aksamentov, I., Roemer, C., Hodcroft, E.B.,Neher, R.A., 2021. Nextclade: clade assignment, mutation calling and quality control for viral genomes. Journal of Open Source Software, 6, 3773.
2. Aljabali, A.A., Obeid, M.A., Nusair, M.B., Hmedat, A.,Tambuwala, M.M., 2022. Monkeypox virus: An emerging epidemic. Microb Pathog, 173, 105794.
3. Anwar, F.,Waris, A., 2022. Monkeypox virus outbreak: a brief timeline. New Microbes New Infect, 48, 101004.
4. Benson, G., 1999. Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res, 27, 573-580.
5. Bunge, E.M., Hoet, B., Chen, L., Lienert, F., Weidenthaler, H., Baer, L.R.,Steffen, R., 2022. The changing epidemiology of human monkeypox-A potential threat? A systematic review. PLoS Negl Trop Dis, 16, e0010141.
6. Charre, C., Ginevra, C., Sabatier, M., Regue, H., Destras, G., Brun, S., Burfin, G., Scholtes, C., Morfin, F., Valette, M., Lina, B., Bal, A.,Josset, L., 2020. Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation. Virus Evol, 6, veaa075.
7. Chen, N.F.G., Chaguza, C., Gagne, L., Doucette, M., Smole, S., Buzby, E., Hall, J., Ash, S., Harrington, R., Cofsky, S., Clancy, S., Kapsak, C.J., Sevinsky, J., Libuit, K., Park, D.J., Hemarajata, P., Garrigues, J.M., Green, N.M., Sierra-Patev, S., Carpenter-Azevedo, K., Huard, R.C., Pearson, C., Incekara, K., Nishimura, C., Huang, J.P., Gagnon, E., Reever, E., Razeq, J., Muyombwe, A., Borges, V., Ferreira, R., Sobral, D., Duarte, S., Santos, D., Vieira, L., Gomes, J.P., Aquino, C., Savino, I.M., Felton, K., Bajwa, M., Hayward, N., Miller, H., Naumann, A., Allman, R., Greer, N., Fall, A., Mostafa, H.H., Mchugh, M.P., Maloney, D.M., Dewar, R., Kenicer, J., Parker, A., Mathers, K., Wild, J., Cotton, S., Templeton, K.E., Churchwell, G., Lee, P.A., Pedrosa, M., Mcgruder, B., Schmedes, S., Plumb, M.R., Wang, X., Barcellos, R.B., Godinho, F.M.S., Salvato, R.S., Ceniseros, A., Breban, M.I., Grubaugh, N.D., Gallagher, G.R.,Vogels, C.B.F., 2023. Development of an amplicon-based sequencing approach in response to the global emergence of mpox. PLoS Biol, 21, e3002151.
8. Chen, S., Zhou, Y., Chen, Y.,Gu, J., 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 34, i884-i890.
9. Chen, Y., Wu, C., A, R., Zhao, L., Zhang, Z.,Tan, W., 2023. Perspective on the application of genome sequencing for monkeypox virus surveillance. Virol Sin, 38, 327-333.
10. Cingolani, P., Platts, A., Wang Le, L., Coon, M., Nguyen, T., Wang, L., Land, S.J., Lu, X.,Ruden, D.M., 2012. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin), 6, 80-92.
11. Danecek, P., Bonfield, J.K., Liddle, J., Marshall, J., Ohan, V., Pollard, M.O., Whitwham, A., Keane, T., Mccarthy, S.A., Davies, R.M.,Li, H., 2021. Twelve years of SAMtools and BCFtools. Gigascience, 10, giab008.
12. Fuchs, J., Nekrutenko, A., Kohl, A.-K., Technau-Hafsi, K., Hornuß, D., Rieg, S., Jaki, L., Huzly, D., Maier, W., Grüning, B., Gutbrod, L., Falcone, V., Hengel, H.,Panning, M. 2022. Travel-associated Monkeypox virus genomes from two German patients and of a derived virus isolate all closely related to a US sequence, 2022 [Online]. Available: https://virological.org/t/travel-associated-monkeypox-virus-genomes-from-two-german-patients-and-of-a-derived-virus-isolate-all-closely-related-to-a-us-sequence-2022/844 [Accessed 03 June 2022].
13. Gigante, C.M., Korber, B., Seabolt, M.H., Wilkins, K., Davidson, W., Rao, A.K., Zhao, H., Smith, T.G., Hughes, C.M., Minhaj, F., Waltenburg, M.A., Theiler, J., Smole, S., Gallagher, G.R., Blythe, D., Myers, R., Schulte, J., Stringer, J., Lee, P., Mendoza, R.M., Griffin-Thomas, L.A., Crain, J., Murray, J., Atkinson, A., Gonzalez, A.H., Nash, J., Batra, D., Damon, I., Mcquiston, J., Hutson, C.L., Mccollum, A.M.,Li, Y., 2022. Multiple lineages of monkeypox virus detected in the United States, 2021-2022. Science, 378, 560-565.
14. Gohl, D.M., Garbe, J., Grady, P., Daniel, J., Watson, R.H.B., Auch, B., Nelson, A., Yohe, S.,Beckman, K.B., 2020. A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2. BMC Genomics, 21, 863.
15. Gorgé, O., Jarjaval, F., Nolent, F., Criqui, A., Lourenco, J., Badaoui, A., Khoury, R., Burrel, S.L., O., Chapus, C., Simon-Loriere, E., Tournier, J.N.,Ferraris, O. 2022. MinIon-produced Illumina-confirmed genomes of the two first isolated cases from France [Online]. Available: https://virological.org/t/minion-produced-illumina-confirmed-genomes-of-the-two-first-isolated-cases-from-france/868 [Accessed 17 June 2022].
16. Hraib, M., Jouni, S., Albitar, M.M., Alaidi, S.,Alshehabi, Z., 2022. The outbreak of monkeypox 2022: An overview. Ann Med Surg (Lond), 79, 104069.
17. Huo, S., Chen, Y., Lu, R., Zhang, Z., Zhang, G., Zhao, L., Deng, Y., Wu, C.,Tan, W., 2022. Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage. Biosaf Health, 4, 392-398.
18. Isidro, J., Borges, V., Pinto, M., Sobral, D., Santos, J.D., Nunes, A., Mixao, V., Ferreira, R., Santos, D., Duarte, S., Vieira, L., Borrego, M.J., Nuncio, S., De Carvalho, I.L., Pelerito, A., Cordeiro, R.,Gomes, J.P., 2022. Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med, 28, 1569-1572.
19. Ladnyj, I.D., Ziegler, P.,Kima, E., 1972. A human infection caused by monkeypox virus in Basankusu Territory, Democratic Republic of the Congo. Bull World Health Organ, 46, 593-597.
20. Li, H.,Durbin, R., 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754-1760.
21. Li, J., Du, P., Yang, L., Zhang, J., Song, C., Chen, D., Song, Y., Ding, N., Hua, M., Han, K., Song, R., Xie, W., Chen, Z., Wang, X., Liu, J., Xu, Y., Gao, G., Wang, Q., Pu, L., Di, L., Li, J., Yue, J., Han, J., Zhao, X., Yan, Y., Yu, F., Wu, A.R., Zhang, F., Gao, Y.Q., Huang, Y., Wang, J., Zeng, H.,Chen, C., 2022. Two-step fitness selection for intra-host variations in SARS-CoV-2. Cell Rep, 38, 110205.
22. Liu, Q., Zhao, S., Shi, C.M., Song, S., Zhu, S., Su, Y., Zhao, W., Li, M., Bao, Y., Xue, Y.,Chen, H., 2020. Population Genetics of SARS-CoV-2: Disentangling Effects of Sampling Bias and Infection Clusters. Genomics Proteomics Bioinformatics, 18, 640-647.
23. Lythgoe, K.A., Hall, M., Ferretti, L., De Cesare, M., Macintyre-Cockett, G., Trebes, A., Andersson, M., Otecko, N., Wise, E.L., Moore, N., Lynch, J., Kidd, S., Cortes, N., Mori, M., Williams, R., Vernet, G., Justice, A., Green, A., Nicholls, S.M., Ansari, M.A., Abeler-Dorner, L., Moore, C.E., Peto, T.E.A., Eyre, D.W., Shaw, R., Simmonds, P., Buck, D., Todd, J.A., Oxford Virus Sequencing Analysis, G., Connor, T.R., Ashraf, S., Da Silva Filipe, A., Shepherd, J., Thomson, E.C., Consortium, C.-G.U., Bonsall, D., Fraser, C.,Golubchik, T., 2021. SARS-CoV-2 within-host diversity and transmission. Science, 372, eabg0821
24. Martínez-Puchol, S., Coello, A., Bordoy, A.E., Soler, L., Panisello, D., González-Gómez, S., Clarà, G., León, A.P.D., Not, A., Hernández, Á., Bofill-Mas, S., Saludes, V., Martró, E.,Cardona, P.J. 2022. Spanish draft genome sequence of Monkeypox virus related to multi-country outbreak (May 2022) [Online]. Available: https://virological.org/t/spanish-draft-genome-sequence-of-monkeypox-virus-related-to-multi-country-outbreak-may-2022/825 [Accessed 27 May 2022].
25. Mckenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M.,Depristo, M.A., 2010. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res, 20, 1297-1303.
26. Monzón, S., Varona, S., Negredo, A., Patiño-Galindo, J.A., Vidal-Freire, S., Zaballos, A., Orviz, E., Ayerdi, O., Muñoz-García, A., Delgado-Iribarren, A., Estrada, V., García, C., Molero, F., Sánchez, P., Torres, M., Vázquez, A., Galán, J.-C., Torres, I., Causse Del Río, M., Merino, L., López, M., Galar, A., Cardeñoso, L., Gutiérrez, A., Loras, C., Escribano, I., Alvarez-Argüelles, M.E., Del Río, L., Simón, M., Meléndez, M.A., Camacho, J., Herrero, L., Sancho, P.J., Navarro-Rico, M.L., Kuhn, J.H., Sanchez-Lockhart, M., Paola, N.D., Kugelman, J.R., Giannetti, E., Guerra, S., García-Sastre, A., Palacios, G., Cuesta, I.,Sánchez-Seco, M.P., 2022. Changes in a new type of genomic accordion may open the pallets to increased monkeypox transmissibility. bioRxiv, 10.1101/2022.09.30.510261, 2022.2009.2030.510261.
27. Nguyen, L.T., Schmidt, H.A., Von Haeseler, A.,Minh, B.Q., 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol, 32, 268-274.
28. Nurk, S., Koren, S., Rhie, A., Rautiainen, M., Bzikadze, A.V., Mikheenko, A., Vollger, M.R., Altemose, N., Uralsky, L., Gershman, A., Aganezov, S., Hoyt, S.J., Diekhans, M., Logsdon, G.A., Alonge, M., Antonarakis, S.E., Borchers, M., Bouffard, G.G., Brooks, S.Y., Caldas, G.V., Chen, N.C., Cheng, H., Chin, C.S., Chow, W., De Lima, L.G., Dishuck, P.C., Durbin, R., Dvorkina, T., Fiddes, I.T., Formenti, G., Fulton, R.S., Fungtammasan, A., Garrison, E., Grady, P.G.S., Graves-Lindsay, T.A., Hall, I.M., Hansen, N.F., Hartley, G.A., Haukness, M., Howe, K., Hunkapiller, M.W., Jain, C., Jain, M., Jarvis, E.D., Kerpedjiev, P., Kirsche, M., Kolmogorov, M., Korlach, J., Kremitzki, M., Li, H., Maduro, V.V., Marschall, T., Mccartney, A.M., Mcdaniel, J., Miller, D.E., Mullikin, J.C., Myers, E.W., Olson, N.D., Paten, B., Peluso, P., Pevzner, P.A., Porubsky, D., Potapova, T., Rogaev, E.I., Rosenfeld, J.A., Salzberg, S.L., Schneider, V.A., Sedlazeck, F.J., Shafin, K., Shew, C.J., Shumate, A., Sims, Y., Smit, A.F.A., Soto, D.C., Sovic, I., Storer, J.M., Streets, A., Sullivan, B.A., Thibaud-Nissen, F., Torrance, J., Wagner, J., Walenz, B.P., Wenger, A., Wood, J.M.D., Xiao, C., Yan, S.M., Young, A.C., Zarate, S., Surti, U., Mccoy, R.C., Dennis, M.Y., Alexandrov, I.A., Gerton, J.L., O'neill, R.J., Timp, W., Zook, J.M., Schatz, M.C., Eichler, E.E., Miga, K.H.,Phillippy, A.M., 2022. The complete sequence of a human genome. Science, 376, 44-53.
29. Pathak, A.K., Mishra, G.P., Uppili, B., Walia, S., Fatihi, S., Abbas, T., Banu, S., Ghosh, A., Kanampalliwar, A., Jha, A., Fatma, S., Aggarwal, S., Dhar, M.S., Marwal, R., Radhakrishnan, V.S., Ponnusamy, K., Kabra, S., Rakshit, P., Bhoyar, R.C., Jain, A., Divakar, M.K., Imran, M., Faruq, M., Sowpati, D.T., Thukral, L., Raghav, S.K.,Mukerji, M., 2022. Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes. Nucleic Acids Res, 50, 1551-1561.
30. Robinson, J.T., Thorvaldsdóttir, H., Winckler, W., Guttman, M., Lander, E.S., Getz, G.,Mesirov, J.P., 2011. Integrative genomics viewer. Nat Biotechnol, 29, 24-26.
31. Sayers, E.W., Bolton, E.E., Brister, J.R., Canese, K., Chan, J., Comeau, D.C., Connor, R., Funk, K., Kelly, C., Kim, S., Madej, T., Marchler-Bauer, A., Lanczycki, C., Lathrop, S., Lu, Z., Thibaud-Nissen, F., Murphy, T., Phan, L., Skripchenko, Y., Tse, T., Wang, J., Williams, R., Trawick, B.W., Pruitt, K.D.,Sherry, S.T., 2021. Database resources of the national center for biotechnology information. Nucleic Acids Res., 50 , D10-D17.
32. Shchelkunov, S.N., Totmenin, A.V., Safronov, P.F., Mikheev, M.V., Gutorov, V.V., Ryazankina, O.I., Petrov, N.A., Babkin, I.V., Uvarova, E.A., Sandakhchiev, L.S., Sisler, J.R., Esposito, J.J., Damon, I.K., Jahrling, P.B.,Moss, B., 2002. Analysis of the monkeypox virus genome. Virology, 297, 172-194.
33. Shu, Y.L.,Mccauley, J., 2017. GISAID: Global initiative on sharing all influenza data - from vision to reality. Euro Surveill., 22, 2-4.
34. Tan, W.,Gao, F.G., 2022. Perspectives: Neglected Zoonotic Monkeypox in Africa but Now Back in the Spotlight Worldwide. China CDC Weekly, 4, 2.
35. Wang, Y., Wang, D., Zhang, L., Sun, W., Zhang, Z., Chen, W., Zhu, A., Huang, Y., Xiao, F., Yao, J., Gan, M., Li, F., Luo, L., Huang, X., Zhang, Y., Wong, S.S., Cheng, X., Ji, J., Ou, Z., Xiao, M., Li, M., Li, J., Ren, P., Deng, Z., Zhong, H., Xu, X., Song, T., Mok, C.K.P., Peiris, M., Zhong, N., Zhao, J., Li, Y., Li, J.,Zhao, J., 2021. Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients. Genome Med, 13, 30.
36. Wu, C., Cui, L., Pan, Y., Lv, Z., Yao, M., Wang, W., Ye, F., Huo, W., Zhao, L., Huang, B., Zhu, F., Lu, R., Deng, Y., Wang, W.,Tan, W., 2023. Characterization of whole genomes from recently emerging Mpox cases in several regions of China, 2023. Sci China Life Sci, https://doi.org/10.1007/s11427-023-2485-8.
37. Wu, F., Oghuan, J., Gitter, A., Mena, K.D.,Brown, E.L., 2023. Wide mismatches in the sequences of primers and probes for monkeypox virus diagnostic assays. J Med Virol, 95, e28395.
38. Yang, Z., 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol, 24, 1586-1591.
39. Yeh, T.-Y., Hsieh, Z.-Y., Feehley, M.C., Feehley, P.J., Contreras, G.P., Su, Y.-C., Hsieh, S.-L.,Lewis, D.A., 2022. Recombination shapes 2022 monkeypox outbreak. medRxiv, 10.1101/2022.08.09.22278589, 2022.2008.2009.22278589.
40. Yu, G., Smith, D.K., Zhu, H., Guan, Y.,Lam, T.T.-Y., 2017. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution, 8, 28-36.
41. Zhao, H., Wang, W., Zhao, L., Ye, S., Song, J., Lu, R., Zong, H., Wu, C., Huang, W., Huang, B., Deng, Y., A, R., Xie, W., Qi, L., Xu, W., Lin, H.,Tan, W., 2022. The First Imported Case of Monkeypox in the Mainland of China — Chongqing Municipality, China, September 16, 2022. China CDC Weekly, 4, 853-854.
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