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Metagenomic shotgun sequencing reveals host species as an important driver of virome composition in mosquitoes

  • 1.

    Cadwell, K. The virome in host health and disease. Immunity 42, 805–813 (2015).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 2.

    Paez-Espino, D. et al. Uncovering earth’s virome. Nature https://doi.org/10.1038/nature19094 (2016).

    Article 
    PubMed 

    Google Scholar 

  • 3.

    Shi, M. et al. The evolutionary history of vertebrate RNA viruses. Nature 556, 197–202 (2018).

    ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 4.

    Dolja, V. V. & Koonin, E. V. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res. 244, 36–52 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 5.

    Li, C.-X. et al. Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. Elife 4, e05378 (2015).

    PubMed Central 
    Article 
    CAS 
    PubMed 

    Google Scholar 

  • 6.

    Shi, M. et al. Redefining the invertebrate RNA virosphere. Nature 540, 539–543 (2016).

    ADS 
    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 7.

    Atoni, E. et al. Metagenomic Virome Analysis of Culex Mosquitoes from Kenya and China. Viruses 10, 30 (2018).

    PubMed Central 
    Article 
    CAS 
    PubMed 

    Google Scholar 

  • 8.

    Sadeghi, M. et al. Virome of > 12 thousand Culex mosquitoes from throughout California. Virology 523, 74–88 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 9.

    Zakrzewski, M. et al. Mapping the virome in wild-caught Aedes aegypti from Cairns and Bangkok. Nat. Publ. Group https://doi.org/10.1038/s41598-018-22945-y (2018).

    Article 

    Google Scholar 

  • 10.

    Xia, H. et al. Comparative metagenomic profiling of viromes associated with four common mosquito species in China. Virol. Sin. 33, 59–66 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 11.

    Frey, K. G. et al. Bioinformatic characterization of mosquito viromes within the eastern United States and Puerto Rico: ciscovery of novel viruses. Evolut. Bioinform. 12s2, EBO.S38518 (2016).

    Article 

    Google Scholar 

  • 12.

    Chandler, J. A., Liu, R. M. & Bennett, S. N. RNA shotgun metagenomic sequencing of northern California (USA) mosquitoes uncovers viruses, bacteria, and fungi. Front. Microbiol. 06, 403 (2015).

    Article 

    Google Scholar 

  • 13.

    Chandler, J. A. et al. Metagenomic shotgun sequencing of a Bunyavirus in wild-caught Aedes aegypti from Thailand informs the evolutionary and genomic history of the Phleboviruses. Virology 464–465, 312–319 (2014).

    PubMed 
    Article 
    CAS 

    Google Scholar 

  • 14.

    Cholleti, H. et al. Discovery of novel viruses in mosquitoes from the Zambezi valley of Mozambique. PLoS ONE 11, e0162751 (2016).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • 15.

    Scarpassa, V. M. et al. An insight into the sialotranscriptome and virome of Amazonian anophelines. BMC Genom. https://doi.org/10.1186/s12864-019-5545-0 (2019).

    Article 

    Google Scholar 

  • 16.

    Hameed, M. et al. A viral metagenomic analysis reveals rich viral abundance and diversity in mosquitoes from pig farms. Transbound. Emerg. Dis. 67, 328–343 (2019).

    PubMed 
    Article 

    Google Scholar 

  • 17.

    Fauver, J. R. et al. West African Anopheles gambiae mosquitoes harbor a taxonomically diverse virome including new insect-speci. Virology 498, 288–299 (2016).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 18.

    Xiao, P. et al. Metagenomic sequencing from mosquitoes in China reveals a variety of insect and human viruses. Front. Cell. Infect. Microbiol. 8, 131–211 (2018).

    Article 
    CAS 

    Google Scholar 

  • 19.

    Shi, C. et al. Stable distinct core eukaryotic viromes in different mosquito species from Guadeloupe, using single mosquito viral metagenomics. Microbiome https://doi.org/10.1186/s40168-019-0734-2 (2019).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 20.

    World Health Organization. A global brief on vector-borne diseases. (2014).

  • 21.

    Vasilakis, N. & Tesh, R. B. Insect-specific viruses and their potential impact on arbovirus transmission. Curr. Opin. Virol. 15, 69–74 (2015).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 22.

    Goenaga, S. et al. Potential for co-infection of a mosquito-specific flavivirus, Nhumirim virus, to block West Nile virus transmission in mosquitoes. Viruses 7, 5801–5812 (2015).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 23.

    Hall-Mendelin, S. et al. The insect-specific Palm Creek virus modulates West Nile virus infection in and transmission by Australian mosquitoes. Parasit. Vectors 9, 414 (2016).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 24.

    Colmant, A. M. G. et al. The recently identified flavivirus Bamaga virus is transmitted horizontally by Culex mosquitoes and interferes with West Nile virus replication in vitro and transmission in vivo. PLoS Negl. Trop. Dis. 12, e0006886 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 25.

    Romo, H., Kenney, J. L., Blitvich, B. J. & Brault, A. C. Restriction of Zika virus infection and transmission in Aedes aegypti mediated by an insect-specific flavivirus. Emerg. Microbes Infect 7, 181 (2018).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 26.

    Schultz, M. J., Frydman, H. M. & Connor, J. H. Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells. Virology 518, 406–413 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 27.

    Thongsripong, P. et al. Mosquito vector diversity across habitats in central Thailand endemic for dengue and other arthropod-borne diseases. PLoS Negl. Trop. Dis. 7, e2507 (2013).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 28.

    Kukutla, P., Steritz, M. & Xu, J. Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq. JoVE https://doi.org/10.3791/50093 (2013).

    Article 
    PubMed 

    Google Scholar 

  • 29.

    Rattanarithikul, R., Harrison, B. A. & Panthusiri, P. Coleman RE (2005) Illustrated keys to the mosquitoes of Thailand I. Background; geographic distribution; lists of genera, subgenera, and species; and a key to the genera. Southeast Asian J. Trop. Med. Public Health 36 Suppl 1, 1–80 (2005).

    PubMed 

    Google Scholar 

  • 30.

    Rattanarithikul, R. et al. Illustrated keys to the mosquitoes of Thailand. II. Genera Culex and Lutzia. Southeast Asian J. Trop. Med. Public Health 36 Suppl 2, 1–97 (2005).

    PubMed 

    Google Scholar 

  • 31.

    Rattanarithikul, R., Harrison, B. A., Panthusiri, P., Peyton, E. L. & Coleman, R. E. Illustrated keys to the mosquitoes of Thailand III. Genera Aedeomyia, Ficalbia, Mimomyia, Hodgesia, Coquillettidia, Mansonia, and Uranotaenia. Southeast Asian J. Trop. Med. Public Health 37 Suppl 1, 1–85 (2006).

    PubMed 

    Google Scholar 

  • 32.

    Rattanarithikul, R., Harrison, B. A., Harbach, R. E., Panthusiri, P. & Coleman, R. E. Illustrated keys to the mosquitoes of Thailand. IV. Anopheles. Southeast Asian J. Trop. Med. Public Health 37 Suppl 2, 1–128 (2006).

    PubMed 

    Google Scholar 

  • 33.

    Rattanarithikul, R., Harbach, R. E., Harrison, B. A., Panthusiri, P. & Coleman, R. E. Illustrated keys to the mosquitoes of Thailand V. Genera Orthopodomyia, Kimia, Malaya, Topomyia, Tripteroides, and Toxorhynchites. Southeast Asian J. Trop. Med. Public Health 38, 1–65 (2007).

    PubMed 

    Google Scholar 

  • 34.

    Rattanarithikul, R. et al. Illustrated keys to the mosquitoes of Thailand. VI. Tribe Aedini. Southeast Asian J. Trop. Med. Public Health 41 Suppl 1, 1–225 (2010).

    PubMed 

    Google Scholar 

  • 35.

    Grabherr, M. G. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 36.

    Haas, B. J. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494–1512 (2013).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 37.

    Buchfink, B., Xie, C. & Huson, D. H. Fast and sensitive protein alignment using DIAMOND. Nat. Methods 12, 59–60 (2015).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 38.

    Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. https://doi.org/10.1093/bib/bbx108 (2017).

    Article 
    PubMed Central 
    PubMed 

    Google Scholar 

  • 39.

    Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 40.

    Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165 (2011).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 41.

    Kozlov, A. M., Darriba, D., Flouri, T., Morel, B. & Stamatakis, A. RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference. bioRxiv 447110 (2018).

  • 42.

    Miller, M. A., Pfeiffer, W. & Schwartz, T. Creating the CIPRES science gateway for interface of large phylogenetic trees. 1–8 (2010).

  • 43.

    Letunic, I. & Bork, P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. https://doi.org/10.1093/nar/gkz239 (2019).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 44.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat Methods 9, 357–359 (2012).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 45.

    Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • 46.

    Ryan, F. P. Human endogenous retroviruses in multiple sclerosis: potential for novel neuro-pharmacological research. Curr. Neuropharmacol. 9, 360–369 (2011).

  • 47.

    Wood, D. E. & Salzberg, S. L. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol 15, R46 (2014).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 48.

    Kopylova, E., Noe, L. & Touzet, H. SortMeRNA: fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28, 3211–3217 (2012).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 49.

    Simmonds, P. et al. ICTV virus taxonomy profile: Flaviviridae. J. Gen. Virol. 98, 2–3 (2017).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 50.

    Kyaw, A. K. et al. Virus research. Virus Res. 247, 120–124 (2018).

    Article 
    CAS 

    Google Scholar 

  • 51.

    Valles, S. M. et al. ICTV virus taxonomy profile: Iflaviridae. J. Gen. Virol. 98, 527–528 (2017).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 52.

    Kobayashi, D. et al. Isolation and characterization of a new iflavirus from Armigeres spp. mosquitoes in the Philippines. J. Gen. Virol. 98, 2876–2881 (2017).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 53.

    Viruses, I. C. O. T. O., King, A. M. Q., Adams, M. J., Lefkowitz, E. & Carstens, E. B. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses (Elsevier, Amsterdam, 2011).

    Google Scholar 

  • 54.

    Hillman, B. I. & Cai, G. The family narnaviridae: Simplest of RNA viruses. Adv. Virus Res. 86, 149–176 (2013).

    PubMed 
    Article 

    Google Scholar 

  • 55.

    Turina, M. et al. ICTV virus taxonomy profile: Ourmiavirus. J. Gen. Virol. 98, 129–130 (2017).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 56.

    Yong, C. Y., Yeap, S. K., Omar, A. R. & Tan, W. S. Advances in the study of nodavirus. PeerJ 5, e3841 (2017).

    PubMed 
    PubMed Central 
    Article 
    CAS 

    Google Scholar 

  • 57.

    Sahul Hameed, A. S. et al. ICTV virus taxonomy profile: Nodaviridae. J. Gen. Virol. 100, 3–4 (2019).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 58.

    Sanborn, M. et al. Metagenomic analysis reveals three novel and prevalent mosquito biruses from a single pool of Aedes vexans nipponii collected in the Republic of Korea. Viruses 11, 222 (2019).

    CAS 
    PubMed Central 
    Article 
    PubMed 

    Google Scholar 

  • 59.

    Olendraite, I. et al. ICTV virus taxonomy profile: Polycipiviridae. J. Gen. Virol. 100, 554–555 (2019).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 60.

    Wichgers Schreur, P. J., Kormelink, R. & Kortekaas, J. Genome packaging of the Bunyavirales. Curr. Opin. Virol. 33, 151–155 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 61.

    Marklewitz, M., Zirkel, F., Kurth, A., Drosten, C. & Junglen, S. Evolutionary and phenotypic analysis of live virus isolates suggests arthropod origin of a pathogenic RNA virus family. Proc. Natl. Acad. Sci. U.S.A. 112, 7536–7541 (2015).

    ADS 
    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 62.

    Walker, P. J. et al. ICTV virus taxonomy profile: Rhabdoviridae. J. Gen. Virol. 99, 447–448 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 63.

    Sun, Q. et al. Complete genome sequence of Menghai rhabdovirus, a novel mosquito-borne rhabdovirus from China. Adv. Virol. 162, 1103–1106 (2017).

    CAS 

    Google Scholar 

  • 64.

    Hilgenboecker, K., Hammerstein, P., Schlattmann, P., Telschow, A. & Werren, J. H. How many species are infected with Wolbachia? A statistical analysis of current data. FEMS Microbiol Lett 281, 215–220 (2008).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 65.

    Flegontov, P. et al. Paratrypanosoma is a novel early-branching trypanosomatid. Curr Biol 23, 1787–1793 (2013).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 66.

    Kaur, D. et al. Occurrence of Setaria digitata in a cow. J Parasit Dis 39, 477–478 (2015).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 67.

    Heneberg, P. et al. Intermediate hosts of the trematode Collyriclum faba (Plagiochiida: Collyriclidae) identified by an integrated morphological and genetic approach. Parasit. Vectors 8, 85 (2015).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 68.

    Enabulele, E. E., Lawton, S. P., Walker, A. J. & Kirk, R. S. Molecular and morphological characterization of the cercariae of Lecithodendrium linstowi (Dollfus, 1931), a trematode of bats, and incrimination of the first intermediate snail host Radix balthica. Parasitology 145, 307–312 (2018).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 69.

    Greiman, S. E. et al. Real-time PCR detection and phylogenetic relationships of Neorickettsia spp. in digeneans from Egypt, Philippines, Thailand, Vietnam and the United States. Parasitol. Int. 66, 1003–1007 (2017).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 70.

    Lantova, L. & Volf, P. Mosquito and sand fly gregarines of the genus Ascogregarina and Psychodiella (Apicomplexa: Eugregarinorida, Aseptatorina)—Overview of their taxonomy, life cycle, host specificity and pathogenicity. Infect. Genet. Evol. 28, 616–627 (2014).

    PubMed 
    Article 

    Google Scholar 

  • 71.

    Roychoudhury, S. et al. Comparison of the morphology of oocysts and the phylogenetic analysis of four Ascogregarina species (Eugregarinidae: Lecudinidae) as inferred from small subunit ribosomal DNA sequences. Parasitol. Int. 56, 113–118 (2007).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 72.

    Muslim, A., Fong, M.-Y., Mahmud, R., Lau, Y.-L. & Sivanandam, S. Armigeres subalbatus incriminated as a vector of zoonotic Brugia pahangi filariasis in suburban Kuala Lumpur Peninsular Malaysia. Parasites Vectors 6, 219 (2013).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 73.

    Hiscox, A. et al. Armigeres subalbatus colonization of damaged pit latrines: A nuisance and potential health risk to residents of resettlement villages in Laos. Med. Vet. Entomol. 30, 95–100 (2016).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 74.

    Chaves, L. F., Imanishi, N. & Hoshi, T. Population dynamics of Armigeres subalbatus (Diptera: Culicidae) across a temperate altitudinal gradient. Bull. Entomol. Res. 105, 589–597 (2015).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 75.

    Ohba, S.-Y., Van Soai, N., Van Anh, D. T., Nguyen, Y. T. & Takagi, M. Study of mosquito fauna in rice ecosystems around Hanoi, northern Vietnam. Acta Trop. 142, 89–95 (2015).

    PubMed 
    Article 

    Google Scholar 

  • 76.

    Tsuda, Y., Takagi, M., Suwonkerd, W., Sugiyama, A. & Wada, Y. Comparisons of rice field mosquito (Diptera: Culicidae) abundance among areas with different agricultural practices in northern Thailand. J. Med. Entom. 35, 845–848 (1998).

    CAS 
    Article 

    Google Scholar 

  • 77.

    Ohba, S.-Y. et al. Mosquitoes and their potential predators in rice agroecosystems of the Mekong Delta, southern Vietnam. J. Am. Mosq. Control Assoc. 27, 384–392 (2011).

    PubMed 
    Article 

    Google Scholar 

  • 78.

    Su, C.-L. et al. Molecular epidemiology of Japanese encephalitis virus in mosquitoes in Taiwan during 2005–2012. PLoS Negl. Trop. Dis. 8, e3122 (2014).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 79.

    Keiser, J. et al. Effect of irrigated rice agriculture on Japanese encephalitis, including challenges and opportunities for integrated vector management. Acta Trop. 95, 40–57 (2005).

    PubMed 
    Article 

    Google Scholar 

  • 80.

    Apiwathnasorn, C., Samung, Y., Prummongkol, S., Asavanich, A. & Komalamisra, N. Surveys for natural host plants of Mansonia mosquitoes inhabiting Toh Daeng peat swamp forest, Narathiwat Province, Thailand. Southeast Asian J. Trop. Med. Public Health 37, 279–282 (2006).

    PubMed 

    Google Scholar 

  • 81.

    Surtees, G., Simpson, D. I. H., Bowen, E. T. W. & Grainger, W. E. Ricefield development and arbovirus epidemiology, Kano Plain, Kenya. Trans. R. Soc. Trop. Med. Hyg. 64, 511–518 (1970).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 82.

    Kwa, B. H. Environmental change, development and vector-borne disease: Malaysia’s experience with filariasis, scrub typhus and dengue. Environ. Dev. Sustain. 10, 209–217 (2008).

    Article 

    Google Scholar 

  • 83.

    Cook, S. et al. Molecular evolution of the insect-specific flaviviruses. J. Gen. Virol. 93, 223–234 (2012).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 84.

    Parry, R. & Asgari, S. Aedes anphevirus: an insect-specific virus distributed worldwide in Aedes aegypti mosquitoes that has complex interplays with Wolbachia and Dengue Virus Infection in Cells. J. Virol. 92, e00224–18 (2018).

    CAS 
    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 85.

    Shi, M. et al. High-resolution metatranscriptomics reveals the ecological dynamics of mosquito-associated RNA viruses in western Australia. J. Virol. 91, e00680–17 (2017).

    PubMed 
    PubMed Central 

    Google Scholar 

  • 86.

    Thongsripong, P. et al. Mosquito vector-associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod-borne diseases. Ecol. Evol. 8, 1352–1368 (2018).

    PubMed 
    Article 

    Google Scholar 

  • 87.

    Eisenhofer, R. et al. Contamination in low microbial biomass microbiome studies: Issues and recommendations. Trends Microbiol. 27, 105–117 (2019).

    CAS 
    PubMed 
    Article 

    Google Scholar 

  • 88.

    Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 1–12 (2014).

    MathSciNet 
    Article 
    CAS 

    Google Scholar 

  • 89.

    Pollock, J., Glendinning, L., Wisedchanwet, T. & Watson, M. The madness of microbiome: attempting to find consensus ‘best practice’ for 16S microbiome studies. Appl. Environ. Microbiol. 84, e02627–17 (2018).

    PubMed 
    PubMed Central 
    Article 

    Google Scholar 

  • 90.

    Blair, C. D., Olson, K. E. & Bonizzoni M. The widespread occurrence and potential biological roles of endogenous viral elements in insect genomes. Curr. Issues Mol. Biol. 34, 13–30 (2020).

    PubMed 
    Article 

    Google Scholar 


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