Sequencing of monkeypox virus from infected patients reveals viral genomes with APOBEC3-like editing, gene inactivation, and bacterial agents of skin superinfection.


Journal

Journal of medical virology
ISSN: 1096-9071
Titre abrégé: J Med Virol
Pays: United States
ID NLM: 7705876

Informations de publication

Date de publication:
06 2023
Historique:
revised: 12 04 2023
received: 27 09 2022
accepted: 08 05 2023
medline: 23 6 2023
pubmed: 21 6 2023
entrez: 21 6 2023
Statut: ppublish

Résumé

A large outbreak of Monkeypox virus (MPXV) infections has arisen in May 2022 in nonendemic countries. Here, we performed DNA metagenomics using next-generation sequencing with Illumina or Nanopore technologies for clinical samples from MPXV-infected patients diagnosed between June and July 2022. Classification of the MPXV genomes and determination of their mutational patterns were performed using Nextclade. Twenty-five samples from 25 patients were studied. A MPXV genome was obtained for 18 patients, essentially from skin lesions and rectal swabbing. All 18 genomes were classified in clade IIb, lineage B.1, and we identified four B.1 sublineages (B.1.1, B.1.10, B.1.12, B.1.14). We detected a high number of mutations (range, 64-73) relatively to a 2018 Nigerian genome (genome GenBank Accession no. NC_063383.1), which were harbored by a large part of a set of 3184 MPXV genomes of lineage B.1 recovered from GenBank and Nextstrain; and we detected 35 mutations relatively to genome ON563414.3 (a B.1 lineage reference genome). Nonsynonymous mutations occurred in genes encoding central proteins, among which transcription factors and core and envelope proteins, and included two mutations that would truncate a RNA polymerase subunit and a phospholipase d-like protein, suggesting an alternative start codon and gene inactivation, respectively. A large majority (94%) of nucleotide substitutions were G > A or C > U, suggesting the action of human APOBEC3 enzymes. Finally, >1000 reads were identified as from Staphylococcus aureus and Streptococcus pyogenes for 3 and 6 samples, respectively. These findings warrant a close genomic monitoring of MPXV to get a better picture of the genetic micro-evolution and mutational patterns of this virus, and a close clinical monitoring of skin bacterial superinfection in monkeypox patients.

Identifiants

pubmed: 37342884
doi: 10.1002/jmv.28799
doi:

Substances chimiques

APOBEC3 proteins, human EC 3.5.4.5
APOBEC Deaminases EC 3.5.4.5

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

e28799

Informations de copyright

© 2023 Wiley Periodicals LLC.

Références

Senkevitch TG, Yutin N, Wolf YI, Koonin EV, Moss B. Ancient gene capture and recent gene loss shape the evolution of Or-thopoxvirus-Host interaction genes. mBio. 2021;12:e0149521. doi:10.1128/mBio.01495-21
Alakunle EF, Okeke MI. Monkeypox virus: a neglected zoonotic pathogen spreads globally. Nat Rev Microbiol. 2022;20:507-508. doi:10.1038/s41579-022-00776-z
Mauldin MR, McCollum AM, Nakazawa YJ, et al. Exportation of monkeypox virus from the African continent. J Infect Dis. 2022;225:1367-1376. doi:10.1093/infdis/jiaa559
Berthet N, Descorps-Declère S, Besombes C, et al. Genomic history of human monkey pox infections in the Central African Republic between 2001 and 2018. Sci Rep. 2021;11:13085. doi:10.1038/s41598-021-92315-8
Isidro J, Borges V, Pinto M, et al. Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med. 2022b;28:1569-1572. doi:10.1038/s41591-022-01907-y
Luna N, Ramírez AL, Muñoz M, et al. Phylogenomic analysis of the monkeypox virus (MPXV) 2022 outbreak: emergence of a novel viral lineage? Travel Med Infect Dis. 2022;49:102402. doi:10.1016/j.tmaid.2022.102402
Wang L, Shang J, Weng S, et al. Genomic annotation and molecular evolution of monkeypox virus outbreak in 2022. J Med Virol. Published online July 29, 2022. doi:10.1002/jmv.28036
Otu A, Ebenso B, Walley J, Barceló JM, Ochu CL. Global human monkeypox outbreak: atypical presentation demanding urgent public health action. The Lancet Microbe. 2022;3:e554-e555. doi:10.1016/S2666-5247(22)00153-7
Vaughan AM, Cenciarelli O, Colombe S, et al. A large multi-country outbreak of monkeypox across 41 countries in the WHO European Region, 7 March to 23 August 2022. Euro Surveill. 2022;27:2200620. doi:10.2807/1560-7917.ES.2022.27.36.2200620
Bragazzi NL, Kong JD, Wu J. Is monkeypox a new, emerging sexually transmitted disease? A rapid review of the literature. J Med Virol. 2023;95:e28145. doi:10.1002/jmv.28145
Isidro J, Borges V, Pinto M, et al. First draft genome sequence of Monkeypox virus associated with the suspected multi-country outbreak, May 2022 (confirmed case in Portugal). Virological.org. 2022. https://virological.org/t/first-draft-genome-sequence-of-monkeypox-virus-associated-with-the-suspected-multi-country-outbreak-may-2022-confirmed-case-in-portugal/799
Croville G, Mathilda W, Jean-Luc G, Jean-Michel M, Christophe P, Jacques I. First French draft genome sequence of Monkeypox virus, may 2022. Virological.org. 2022. https://virological.org/t/first-french-draft-genome-sequence-of-monkeypox-virus-may-2022/819
Jones TC, Schneider J, Mühlemann B, et al. Genetic variability, including gene duplication and deletion, in early sequences from the 2022 European monkeypox outbreak. bioRxiv. 2022:501239. doi:10.1101/2022.07.23.501239
Wolf JM, Wolf LM, Bello GL, Maccari JG, Nasi LA. Molecular evolution of SARS-CoV-2 from December 2019 to August 2022. J Med Virol. 2023;95(1):e28366. doi:10.1002/jmv.28366
Firth C, Kitchen A, Shapiro B, Suchard MA, Holmes EC, Rambaut A. Using time- structured data to estimate evolutionary rates of double-stranded DNA viruses. Mol Biol Evol. 2010;27:2038-2051. doi:10.1093/molbev/msq088
Cassir N, Cardona F, Tissot-Dupont H, et al. Observational cohort study of evolving epi-demiologic, clinical, and virologic features of monkeypox in Southern France. Emerging Infect Dis. 2022;28(12):2409-2415. doi:10.3201/eid2812.221440
Scaramozzino N, Ferrier-Rembert A, Favier A, et al. Real-time PCR to identify variola virus or other human pathogenic orthopox viruses. Clin Chem. 2007;53:606-613. doi:10.1373/clinchem.2006.068635
Ninove L, Nougairede A, Gazin C, et al. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests. PLoS One. 2011;6:e16142. doi:10.1371/journal.pone.0016142
Colson P, Fournier PE, Chaudet H, et al. Analysis of SARS- CoV-2 variants from 24,181 patients exemplifies the role of globali-zation and zoonosis in pandemics. Front Microbiol. 2022;12:786233. doi:10.3389/fmicb.2021.786233
Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094-3100. doi:10.1093/bioinformatics/bty191
Aksamentov I, Roemer C, Hodcroft E, Neher R. Nextclade: clade assignment, mutation calling and quality control for viral genomes. J Open Source Soft. 2021;6:3773. doi:10.21105/joss.03773
Garrison E, Marth Gabor. Haplotype-based variant detection from short-read sequencing. arXiv. 2012:1207.3907v2. doi:10.48550/arXiv.1207.3907
Edge P, Bansal V. Longshot enables accurate variant calling in diploid genomes from single-molecule long read sequencing. Nat Commun. 2019;10:4660. doi:10.1038/s41467-019-12493-y
Elbe S, Buckland-Merrett G. Data, disease and diplomacy: GISAID's innovative contribution to global health: data, disease and diplomacy. Global Challenges. 2017;1:33-46. doi:10.1002/gch2.1018
Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772-780. doi:10.1093/molbev/mst010
Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maxi-mum-likelihood phylogenies. Mol Biol Evol. 2015;32:268-274. doi:10.1093/molbev/msu300
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547-1549. doi:10.1093/molbev/msy096
Hadfield J, Megill C, Bell SM, et al. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics. 2018;34(23):4121-4123. doi:10.1093/bioinformatics/bty407
Sayers EW, Cavanaugh M, Clark K, et al. GenBank. Nucleic Acids Res. 2022;50:D161-D164. doi:10.1093/nar/gkab1135
Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 2014;15:R46. doi:10.1186/gb-2014-15-3-r46
Sereewit J, Lieberman NAP, Xie H, et al. ORF-Interrupting mutations in monkeypox virus genomes from Washington and ohio, 2022. Viruses. 2022;14(11):2393. doi:10.3390/v14112393
McDaniel YZ, Wang D, Love RP, et al. Deamination hotspots among APOBEC3 family members are defined by both target site sequence context and ssDNA secondary structure. Nucleic Acids Res. 2020;48:1353-1371. doi:10.1093/nar/gkz1164
Upton C, Slack S, Hunter AL, Ehlers A, Roper RL. Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome. J Virol. 2003;77:7590-7600. doi:10.1128/jvi.77.13.7590-7600.2003
Ojeda S, Domi A, Moss B. Vaccinia virus G9 protein is an essential component of the poxvirus entry-fusion complex. J Virol. 2006;80(19):9822-9830. doi:10.1128/JVI.00987-06
Smithson C, Imbery J, Upton C. Re-assembly and analysis of an ancient variola virus genome. Viruses. 2017;9(9):253. doi:10.3390/v9090253
Peng C, Moss B. Repair of a previously uncharacterized second host-range gene contributes to full replication of modified vaccinia virus Ankara (MVA) in human cells. Proc Nat Acad Sci. 2020;117(7):3759-3767. doi:10.1073/pnas.1921098117
Vliegen I, Yang G, Hruby D, Jordan R, Neyts J. Deletion of the vaccinia virus F13L gene results in a highly attenuated virus that mounts a protective immune response against subsequent vaccinia virus challenge. Antiviral Res. 2012;93(1):160-166. doi:10.1016/j.antiviral.2011.11.010
DeHaven BC, Gupta K, Isaacs SN. The vaccinia virus A56 protein: a multifunctional transmembrane glycoprotein that anchors two secreted viral proteins. J Gen Virol. 2011;92(Pt 9):1971-1980. doi:10.1099/vir.0.030460-0
Wassenaar TM, Wanchai V, Ussery DW. Comparison of monkeypox virus genomes from the 2017 Nigeria outbreak and the 2022 outbreak. J Appl Microbiol. 2022;133(6):3690-3698. doi:10.1111/jam.15806
Forni D, Moltrasio C, Sironi M, et al. Whole-Genome sequencing of hMPXV1 in five Italian cases confirms the occurrence of the predominant epidemic lineage. J Med Virol. 2023;95:e28493. doi:10.1002/jmv.28493
Suspène R, Raymond KA, Boutin L, et al. APOBEC3F is the main source of editing identified during the 2022 outbreak of human monkeypox virus. bioRxiv. 2023:522979. doi:10.1101/2023.01.06.522979
O'Toole A, Rambaut A. Initial observations about putative APOBEC3 deaminase editing driving short-term evolution of MPXV since 2017. Virological. org. 2022. https://virological.org/t/initial-observations-about-putative-apobec3-deaminase-editing-driving-short-term-evolution-of-mpxv-since-2017/830
Hammarlund E, Lewis MW, Carter SV, et al. Multiple diagnostic techniques identify previously vaccinated individuals with protective immunity against monkeypox. Nature Med. 2005;11:1005-1011. doi:10.1038/nm1273
Al-Khodor S, Price CT, Kalia A, Abu Kwaik Y. Functional diversity of ankyrin repeats in microbial proteins. TIM. 2010;18:132-139. doi:10.1016/j.tim.2009.11.004
Pagnier I, Yutin N, Croce O, et al. Babela massiliensis, a repre-sentative of a widespread bacterial phylum with unusual adaptations to parasitism in amoebae. Biol Direct. 2015;10:13. doi:10.1186/s13062-015-0043-z
Iyer LM, Aravind L, Koonin EV. Common origin of four diverse families of large eukaryotic DNA viruses. J Virol. 2001;75:11720-11734. doi:10.1128/JVI.75.23.11720-11734.2001
Barry M, van Buuren N, Burles K, Mottet K, Wang Q, Teale A. Poxvirus exploitation of the ubiquitin-proteasome system. Viruses. 2010;2:2356-2380. doi:10.3390/v2102356
Forni D, Cagliani R, Pozzoli U, Sironi M. An APOBEC3 mutational signature in the genomes of human-infecting orthopoxviruses. mSphere. 2023;8:e0006223. doi:10.1128/msphere.00062-23
Gigante CM, Plumb M, Ruprecht A, et al. Genomic deletions and rearrangements in monkeypox virus from the 2022 outbreak, USA. bioRxiv. 2022:508251. doi:10.1101/2022.09.16.508251
Yeh TY, Hsieh ZY, Feehley MC, et al. Recombination shapes the 2022 monkeypox (mpox) outbreak. Med (New York, N.Y.). 2022;3(12):824-826. doi:10.1016/j.medj.2022.11.003
McLysaght A, Baldi PF, Gaut BS. Extensive gene gain associated with adaptive evolution of poxviruses. Proc Nat Acad Sci. 2003;100(26):15655-15660. doi:10.1073/pnas.2136653100
Gigante CM, Korber B, Seabolt MH, et al. Multiple lineages of monkeypox virus detected in the United States, 2021-2022. Science. 2022;378(6619):560-565. doi:10.1126/science.add4153
Bryk P, Brewer MG, Ward BM. Vaccinia virus phospholipase protein F13 promotes rapid entry of extracellular virions into cells. J Virol. 2018;92(11):e02154-17. doi:10.1128/JVI.02154-17
Selvy PE, Lavieri RR, Lindsley CW, Brown HA. Phospholipase D: enzymology, functionality, and chemical modulation. Chem Rev. 2011;111:6064-6119. doi:10.1021/cr200296t
Mutsafi Y, Zauberman N, Sabanay I, Minsky A. Vaccinia-like cytoplasmic replication of the giant mimivirus. Proc Nat Acad Scie. 2010;107(13):5978-5982. doi:10.1073/pnas.0912737107
Witt ASA, Trindade GS, Souza FG, et al. Ultrastructural analysis of monkeypox virus replication in vero cells. J Med Virol. 2023;95:e28536. doi:10.1002/jmv.28536
Hendrickson RC, Wang C, Hatcher EL, Lefkowitz EJ. Orthopoxvirus genome evolution: the role of gene loss. Viruses. 2010;2:1933-1967. doi:10.3390/v2091933
Brennan G, Stoian AMM, Yu H, et al. Molecular mechanisms of poxvirus evolution. mBio. 2023;14(1):e0152622. doi:10.1128/mbio.01526-22
Americo JL, Earl PL, Moss B. Virulence differences of mpox (monkeypox) virus clades I, IIa, and IIb.1 in a small animal model. Proc Nat Acad Sci. 2023;120(8):e2220415120. doi:10.1073/pnas.2220415120
Kugelman JR, Johnston SC, Mulembakani PM, et al. Genomic variability of monkeypox virus among humans, democratic republic of the Congo. Emerging Infect Dis. 2014;20:232-239. doi:10.3201/eid2002.130118
Colson P, Raoult D. Lamarckian evolution of the giant mimivirus in allopatric laboratory culture on amoebae. Front Cell Infect Microbiol. 2012;2:91. doi:10.3389/fcimb.2012.00091
Moran NA. Microbial minimalism. Cell. 2002;108(5):583-586. doi:10.1016/s0092-8674(02)00665-7
Rubins KH, Hensley LE, Bell GW, et al. Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes. PLoS One. 2008;3(7):e2628. doi:10.1371/journal.pone.0002628
Yang Z, Bruno DP, Martens CA, Porcella SF, Moss B. Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing. Proc Nat Acad Sci. 2010;107(25):11513-11518. doi:10.1073/pnas.1006594107
Kerr PJ, Ghedin E, DePasse JV, et al. Evolutionary history and attenuation of myxoma virus on two continents. PLoS Pathog. 2012;8(10):e1002950. doi:10.1371/journal.ppat.1002950
Babkin IV, Babkina IN. Molecular dating in the evolution of vertebrate poxviruses. Intervirology. 2011;54:253-260. doi:10.1159/000320964
Chen Y, Li M, Fan H. The monkeypox outbreak in 2022: adaptive evolution associated with APOBEC3 may account for. Signal Transduct Target Ther. 2022;7(1):323. doi:10.1038/s41392-022-01181-x
Harris RS, Liddament MT. Retroviral restriction by APOBEC proteins. Nat Rev Immunol. 2004;4:868-877. doi:10.1038/nri1489
Armitage AE, Katzourakis A, de Oliveira T, et al. Conserved footprints of APOBEC3G on hypermutated human immunodeficiency virus type 1 and human endogenous ret-rovirus HERV-K(HML2) sequences. J Virol. 2008;82:8743-8761. doi:10.1128/JVI.00584-08
Dobrovolná M, Brázda V, Warner EF, Bidula S. Inverted repeats in the monkeypox virus genome are hot spots for mutation. J Med Virol. 2023;95(1):e28322. doi:10.1002/jmv.28322
Vartanian JP, Guétard D, Henry M, Wain-Hobson S. Evidence for editing of human papillomavirus DNA by APOBEC3 in benign and precancerous lesions. Science. 2008;320:230-233. doi:10.1126/science.1153201
Freidl GS, Sonder GJ, Bovée LP, et al. Hepatitis A outbreak among men who have sex with men (MSM) predominantly linked with the EuroPride, the Netherlands, July 2016 to February 2017. Euro Surveill. 2017;22:30468. doi:10.2807/1560-7917.ES.2017.22.8.30468
Moschese D, Giacomelli A, Beltrami M, et al. Hospital-isation for monkeypox in milan, Italy. Travel Med Infect Dis. 2022;49:102417. doi:10.1016/j.tmaid.2022.102417
Ogoina D, Iroezindu M, James HI, et al. Clinical course and outcome of human monkeypox in Nigeria. Clin Infect Dis. 2020;71:e210-e214. doi:10.1093/cid/ciaa143
Girometti N, Byrne R, Bracchi M, et al. Demographic and clinical characteristics of confirmed human monkeypox virus cases in individuals attending a sexual health centre in London, UK: an observational analysis. Lancet Infect Dis. 2022;22:1321-1328. doi:10.1016/S1473-3099(22)00411-X
Patel A, Bilinska J, Tam JCH, et al. Clinical features and novel presentations of human monkeypox in a central London centre during the 2022 outbreak: descriptive case series. BMJ. 2022;378:e072410. doi:10.1136/bmj-2022-072410
World Health Organization (WHO). Clinical management and infection prevention and control for monkeypox: Interim rapid response guidance, 10 June 2022. Accessed September 12, 2022. https://www.who.int/publications/i/item/WHO-MPX-Clinical-and-IPC-2022.1
Centers for Disease Control and Prevention (CDC). Monkeypox-treatment information for healthcare professionals. 2022. Accessed September 12, 2022. https://www.cdc.gov/poxvirus/monkeypox/clinicians/treatment.html
Webb E, Rigby I, Michelen M, et al. Availability, scope and quality of monkeypox clinical management guidelines globally: a systematic review. BMJ Glob Health. 2022;7:e009838. doi:10.1136/bmjgh-2022-009838

Auteurs

Philippe Colson (P)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Gwilherm Penant (G)

IHU Méditerranée Infection, Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Jeremy Delerce (J)

IHU Méditerranée Infection, Marseille, France.

Céline Boschi (C)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Nathalie Wurtz (N)

IHU Méditerranée Infection, Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Marielle Bedotto (M)

IHU Méditerranée Infection, Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Stéphanie Branger (S)

Service de Médecine Interne Infectiologie Aïgue Polyvalente, Centre hospitalier d'Avignon, Avignon, France.

Philippe Brouqui (P)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Philippe Parola (P)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Institut de Recherche pour le Développement (IRD), Vecteurs - Infections Tropicales et Méditerranéennes (VITROME), Aix-Marseille Univ., Marseille, France.

Jean-Christophe Lagier (JC)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Nadim Cassir (N)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Hervé Tissot-Dupont (H)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Matthieu Million (M)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Sarah Aherfi (S)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

Bernard La Scola (B)

IHU Méditerranée Infection, Marseille, France.
Institut de Recherche pour le Développement (IRD), Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille Univ., Marseille, France.
Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France.

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