Skip to main content
SpringerLink
Log in

Genetic Characteristics and Phylogeographic Dynamics of Echovirus

  • Virology
  • Published:
Journal of Microbiology Aims and scope Submit manuscript

Abstract

Echoviruses belong to the genus Enterovirus in the Picornaviridae family, forming a large group of Enterovirus B (EV-B) within the Enteroviruses. Previously, Echoviruses were classified based on the coding sequence of VP1. In this study, we performed a reliable phylogenetic classification of 277 sequences isolated from 1992 to 2019 based on the full-length genomes of Echovirus. In this report, phylogenetic, phylogeographic, recombination, and amino acid variability landscape analyses were performed to reveal the evolutional characteristics of Echovirus worldwide. Echoviruses were clustered into nine major clades, e.g., G1–G9. Phylogeographic analysis showed that branches G2–G9 were linked to common strains, while the branch G1 was only linked to G5. In contrast, strains E12, E14, and E16 clustered separately from their G3 and G7 clades respectively, and became a separate branch. In addition, we identified a total of 93 recombination events, where most of the events occurred within the VP1-VP4 coding regions. Analysis of amino acid variation showed high variability in the a positions of VP2, VP1, and VP3. This study updates the phylogenetic and phylogeographic information of Echovirus and indicates that extensive recombination and significant amino acid variation in the capsid proteins drove the emergence of new strains.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

The nucleotide and protein sequence data used in this study are available on the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/nuccore/).

References

  • Abzug, M. J. (2004). Presentation, diagnosis, and management of enterovirus infections in neonates. Pediatric Drugs, 6, 1–10.

    Article  PubMed  Google Scholar 

  • Angez, M., Shaukat, S., Zahra, R., Sharif, S., Alam, M. M., Khurshid, A., Rana, M. S., & Zaidi, S. S. (2015). Identification of new genotype of Echovirus 19 from children with Acute Flaccid Paralysis in Pakistan. Scientific Reports, 5, 17456.

    Article  PubMed  PubMed Central  Google Scholar 

  • Benschop, K. S. M., Broberg, E. K., Hodcroft, E., Schmitz, D., Albert, J., Baicus, A., Bailly, J. L., Baldvinsdottir, G., Berginc, N., Blomqvist, S., Böttcher, S., Brytting, M., Bujaki, E., Cabrerizo, M., Celma, C., Cinek, O., Claas, E. C. J., Cremer, J., Dean, J., … Demchyshyna, I. (2021). Molecular epidemiology and evolutionary trajectory of emerging Echovirus 30, Europe. Emerging Infectious Diseases, 27, 1616–1626.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brouwer, L., Moreni, G., Wolthers, K. C., & Pajkrt, D. (2021). World-wide prevalence and genotype distribution of Enteroviruses. Viruses, 13, 434.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Castro, C. M., Oliveira, D. S., Macedo, O., Lima, M. J., Santana, M. B., Wanzeller, A. L., Silveira, E., & Gomes, M. L. (2009). Echovirus 30 associated with cases of aseptic meningitis in state of Pará, Northern Brazil. Memorias Do Instituto Oswaldo Cruz, 104, 444–450.

    Article  PubMed  Google Scholar 

  • Chen, J. H., Chiu, N. C., Chang, J. H., Huang, F. Y., Wu, K. B., & Lin, T. L. (2005). A neonatal echovirus 11 outbreak in an obstetric clinic. Journal of Microbiology, Immunology, and Infection, 38, 332–337.

    PubMed  Google Scholar 

  • Chen, P., Li, Y., Tao, Z., Wang, H., Lin, X., Liu, Y., Wang, S., Zhou, N., Wang, P., & Xu, A. (2017a). Evolutionary phylogeography and transmission pattern of echovirus 14: An exploration of spatiotemporal dynamics based on the 26-year acute flaccid paralysis surveillance in Shandong, China. BMC Genomics, 18, 48.

    Article  PubMed  PubMed Central  Google Scholar 

  • Chen, X., Li, J., Guo, J., Xu, W., Sun, S., & Xie, Z. (2017b). An outbreak of echovirus 18 encephalitis/meningitis in children in Hebei Province, China, 2015. Emerging Microbes & Infections, 6, e54.

    Article  Google Scholar 

  • Chen, X., Qu, X., Liu, C., Zhang, Y., Zhang, G., Han, P., Duan, Y., Li, Q., Wang, L., Ruan, W., Wang, P., Wei, W., Gao, G. F., Zhao, X., & Xie, Z. (2022). Human FcRn is a two-in-one attachment-uncoating receptor for Echovirus 18. mBio, 13, e0116622.

    Article  PubMed  Google Scholar 

  • Diedrich, S., & Schreier, E. (2001). Aseptic meningitis in Germany associated with echovirus type 13. BMC Infectious Diseases, 1, 14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dilcher, M., Howard, J. C., Dalton, S. C., Anderson, T., Clinghan, R. T., & Werno, A. M. (2022). Clinical, laboratory, and molecular epidemiology of an outbreak of aseptic meningitis due to a triple-recombinant Echovirus in Ashburton, New Zealand. Viruses, 14, 658.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Faleye, T. O. C., Adewumi, O. M., Klapsa, D., Majumdar, M., Martin, J., & Adeniji, J. A. (2019). Genome Sequences of two dual-serotype-specific Echovirus 20 Strains from Nigeria. Microbiology Resource Announcements, 8, e00894-e919.

    Article  PubMed  PubMed Central  Google Scholar 

  • Fields, B. N., Knipe, D. M., & Howley, P. M. (2007). Fields virology (5th ed.). Wolters Kluwer Health/Lippincott Williams & Wilkins.

    Google Scholar 

  • Fourgeaud, J., Mirand, A., Demortier, J., Kamus, L., Collet, L., Olivier, S., Henquell, C., & Vauloup-Fellous, C. (2022). Enterovirus meningitis in Mayotte French Comoros Island, March–June 2019. Journal of Clinical Virology, 150–151, 105154.

    Article  PubMed  Google Scholar 

  • Fratty, I. S., Kriger, O., Weiss, L., Vasserman, R., Erster, O., Mendelson, E., Sofer, D., & Weil, M. (2023). Increased detection of Echovirus 6-associated meningitis in patients hospitalized during the COVID-19 pandemic, Israel 2021–2022. Journal of Clinical Virology, 162, 105425.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Garcia-Boronat, M., Diez-Rivero, C. M., Reinherz, E. L., & Reche, P. A. (2008). PVS: A web server for protein sequence variability analysis tuned to facilitate conserved epitope discovery. Nucleic Acids Research, 36, W35–W41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grädel, C., Ireddy, N. R., Koch, M. C., Baumann, C., Terrazos Miani, M. A., Barbani, M. T., Steinlin-Schopfer, J., Suter-Riniker, F., Leib, S. L., & Ramette, A. (2022). Genome sequences of rare human enterovirus genotypes recovered from clinical respiratory samples in Bern. Switzerland. Microbiology Resource Announcements, 11, e0027622.

    Article  PubMed  Google Scholar 

  • Grenfell, B. T., Pybus, O. G., Gog, J. R., Wood, J. L., Daly, J. M., Mumford, J. A., & Holmes, E. C. (2004). Unifying the epidemiological and evolutionary dynamics of pathogens. Science, 303, 327–332.

    Article  CAS  PubMed  Google Scholar 

  • Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.

    CAS  Google Scholar 

  • Handsher, R., Shulman, L. M., Abramovitz, B., Silberstein, I., Neuman, M., Tepperberg-Oikawa, M., Fisher, T., & Mendelson, E. (1999). A new variant of echovirus 4 associated with a large outbreak of aseptic meningitis. Journal of Clinical Virology, 13, 29–36.

    Article  CAS  PubMed  Google Scholar 

  • Hirade, T., Abe, Y., Ito, S., Suzuki, T., Katano, H., Takahashi, N., Koike, D., Nariai, A., & Kato, F. (2023). Congenital Echovirus 11 infection in a neonate. The Pediatric Infectious Disease Journal. https://doi.org/10.1097/inf.0000000000004052

    Article  PubMed  Google Scholar 

  • Home of Picornaviruses. (2023). Enterovirus B. The Pirbright Institute. Retrieved from https://www.picornaviridae.com/ensavirinae/enterovirus/ev-b/ev-b.htm.

  • Huang, Q. S., Carr, J. M., Nix, W. A., Oberste, M. S., Kilpatrick, D. R., Pallansch, M. A., Croxson, M. C., Lindeman, J. A., Baker, M. G., & Grimwood, K. (2003). An echovirus type 33 winter outbreak in New Zealand. Clinical Infectious Diseases, 37, 650–657.

    Article  PubMed  Google Scholar 

  • ICTV, International Committee on Taxonomy of Viruses. (2023). Taxon Details. Enterovirus B. Retrieved from https://ictv.global/taxonomy/taxondetails?taxnode_id=202101984 (20 Feb 2023).

  • Jiang, C., Xu, Z., Li, J., Zhang, J., Xue, X., Jiang, J., Jiang, G., Wang, X., Peng, Y., Chen, T., et al. (2022). Case report: Clinical and virological characteristics of aseptic meningitis caused by a recombinant echovirus 18 in an immunocompetent adult. Frontiers in Medicine, 9, 1094347.

    Article  PubMed  Google Scholar 

  • Kabat, E. A., Wu, T. T., & Bilofsky, H. (1977). Unusual distributions of amino acids in complementarity determining (hypervariable) segments of heavy and light chains of immunoglobulins and their possible roles in specificity of antibody-combining sites. Journal of Biological Chemistry, 252, 6609–6616.

    Article  CAS  PubMed  Google Scholar 

  • Khetsuriani, N., Lamonte-Fowlkes, A., Oberst, S., Pallansch, M. A., Centers for Disease Control and Prevention. (2006). Enterovirus surveillance—United States, 1970–2005. Morbidity and Mortality Weekly Report. Surveillance Summaries, 55, 1–20.

    PubMed  Google Scholar 

  • Kyriakopoulou, Z., Bletsa, M., Tsakogiannis, D., Dimitriou, T. G., Amoutzias, G. D., Gartzonika, C., Levidiotou-Stefanou, S., & Markoulatos, P. (2015a). Molecular epidemiology and evolutionary dynamics of Echovirus 3 serotype. Infection, Genetics and Evolution, 32, 305–312.

    Article  PubMed  Google Scholar 

  • Kyriakopoulou, Z., Pliaka, V., Amoutzias, G. D., & Markoulatos, P. (2015b). Recombination among human non-polio enteroviruses: Implications for epidemiology and evolution. Virus Genes, 50, 177–188.

    Article  CAS  PubMed  Google Scholar 

  • Lavania, M., Viswanathan, R., Bhardwaj, S. D., Oswal, J. S., Chavan, N., Shinde, M., & Katendra, S. (2022). Detection of Echovirus-18 in children suspected With SARS-CoV-2 infection with multisystem inflammatory syndrome: A case report from India. Frontiers in Public Health, 10, 897662.

    Article  PubMed  PubMed Central  Google Scholar 

  • Leigh, J. W., & Bryant, D. (2015). POPART: Full-feature software for haplotype network construction. Methods in Ecology and Evolution, 6, 1110–1116.

    Article  Google Scholar 

  • Lema, C., Torres, C., Van der Sanden, S., Cisterna, D., Freire, M. C., & Gómez, R. M. (2019). Global phylodynamics of Echovirus 30 revealed differential behavior among viral lineages. Virology, 531, 79–92.

    Article  CAS  PubMed  Google Scholar 

  • Li, J., Yan, D., Chen, L., Zhang, Y., Song, Y., Zhu, S., Ji, T., Zhou, W., Gan, F., Wang, X., Hong, M., Guan, L., Shi, Y., Wu, G., & Xu, W. (2019). Multiple genotypes of Echovirus 11 circulated in mainland China between 1994 and 2017. Scientific Reports, 9, 10583.

    Article  PubMed  PubMed Central  Google Scholar 

  • Liu, H., Zhang, J., Zhao, Y., Zhang, H., Lin, K., Sun, H., Huang, X., Yang, Z., & Ma, S. (2018). Molecular characterization of echovirus 12 strains isolated from healthy children in China. Scientific Reports, 8, 11716.

    Article  PubMed  PubMed Central  Google Scholar 

  • Lizasoain, A., Mir, D., Victoria, M., Barrios, M. E., Blanco-Fernández, M. D., Rodríguez-Osorio, N., Nates, S., Cisterna, D., Mbayed, V. A., & Colina, R. (2021). Human Enterovirus diversity by next-generation sequencing analysis in urban sewage samples from Buenos Aires Metropolitan Area, Argentina: A retrospective study. Food and Environmental Virology, 13, 259–269.

    Article  CAS  PubMed  Google Scholar 

  • Lukashev, A. N., Lashkevich, V. A., Ivanova, O. E., Koroleva, G. A., Hinkkanen, A. E., & Ilonen, J. (2005). Recombination in circulating Human enterovirus B: Independent evolution of structural and non-structural genome regions. The Journal of General Virology, 86, 3281–3290.

    Article  CAS  PubMed  Google Scholar 

  • Martin, D. P., Murrell, B., Golden, M., Khoosal, A., & Muhire, B. (2015). RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evolution, 1, vev003.

    Article  PubMed  PubMed Central  Google Scholar 

  • Miyata, I., Hanaoka, N., Okabe, N., Fujimoto, T., Sakamoto, S., Kasahara, M., & Saitoh, A. (2014). Echovirus 3 as another enterovirus causing life-threatening neonatal fulminant hepatitis. Journal of Clinical Virology, 59, 132–134.

    Article  PubMed  Google Scholar 

  • Morsli, M., Vincent, J. J., Milliere, L., Colson, P., & Drancourt, M. (2021). Direct next-generation sequencing diagnosis of echovirus 9 meningitis, France. European Journal of Clinical Microbiology & Infectious Diseases, 40, 2037–2039.

    Article  Google Scholar 

  • Oberste, M. S., Maher, K., & Pallansch, M. A. (2004). Evidence for frequent recombination within species Human Enterovirus B based on complete genomic sequences of all thirty-seven serotypes. Journal of Virology, 78, 855–867.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oberste, M. S., Nix, W. A., Kilpatrick, D. R., Flemister, M. R., & Pallansch, M. A. (2003). Molecular epidemiology and type-specific detection of echovirus 11 isolates from the Americas, Europe, Africa, Australia, southern Asia and the Middle East. Virus Research, 91, 241–248.

    Article  CAS  PubMed  Google Scholar 

  • Ogbole, O. O., Akinleye, T. E., Segun, P. A., Faleye, T. C., & Adeniji, A. J. (2018). In vitro antiviral activity of twenty-seven medicinal plant extracts from Southwest Nigeria against three serotypes of echoviruses. Virology Journal, 15, 110.

    Article  PubMed  PubMed Central  Google Scholar 

  • Paul, A. V., & Wimmer, E. (2015). Initiation of protein-primed picornavirus RNA synthesis. Virus Research, 206, 12–26.

    Article  CAS  PubMed  Google Scholar 

  • Pedrosa, C., Lage, M. J., & Virella, D. (2013). Congenital echovirus 21 infection causing fulminant hepatitis in a neonate. BMJ Case Reports. https://doi.org/10.1136/bcr-2012-008394

    Article  PubMed  PubMed Central  Google Scholar 

  • Racaniello, V. R. (2007). Picornaviridae: The viruses and their replication. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman, & S. E. Strauss (Eds.), Fields virology (5th ed., pp. 795–838). Lippincott Williams & Wilkins.

    Google Scholar 

  • Ramos-Alvarez, M., & Sabin, A. B. (1954). Characteristics of poliomyelitis and other enteric viruses recovered in tissue culture from healthy American children. Proceedings of the Society for Experimental Biology and Medicine, 87, 655–661.

    Article  CAS  PubMed  Google Scholar 

  • Rao, C. D. (2015). Non-polio enteroviruses, the neglected and emerging human pathogens: Are we waiting for the sizzling enterovirus volcano to erupt. Proceedings of the Indian National Science Academy, 81, 447–462.

    Google Scholar 

  • Roberts, J. A., Hobday, L. K., Ibrahim, A., & Thorley, B. R. (2020). Australian National Enterovirus Reference laboratory annual report 2018. Communicable Diseases Intelligence. https://doi.org/10.33321/cdi.2020.44.26

    Article  PubMed  Google Scholar 

  • Rueca, M., Lanini, S., Giombini, E., Messina, F., Castilletti, C., Ippolito, G., Capobianchi, M. R., & Valli, M. B. (2022). Detection of recombinant breakpoint in the genome of human enterovirus E11 strain associated with a fatal nosocomial outbreak. Virol Journal, 19, 97.

    Article  CAS  Google Scholar 

  • Shaukat, S., Angez, M., Mahmood, T., Alam, M. M., Sharif, S., Khurshid, A., Rana, M. S., & Zaidi, S. S. (2017). Molecular characterization of echovirus 13 uncovering high genetic diversity and identification of new genotypes in Pakistan. Infection, Genetics and Evolution, 48, 102–108.

    Article  CAS  PubMed  Google Scholar 

  • Sousa, I. P., Jr., Burlandy, F. M., Lima, S. T. S., Maximo, A. C. B., Figueiredo, M. A. A., Maia, Z., & da Silva, E. E. (2019). Echovirus 30 detection in an outbreak of acute myalgia and rhabdomyolysis, Brazil 2016–2017. Clinical Microbiology and Infection, 25, 252.e2-252.e8.

    Article  Google Scholar 

  • Sun, Y., Miao, Z., Yan, J., Gong, L., Chen, Y., Chen, Y., Mao, H., & Zhang, Y. (2019). Sero-molecular epidemiology of enterovirus-associated encephalitis in Zhejiang Province, China, from 2014 to 2017. International Journal of Infectious Diseases, 79, 58–64.

    Article  PubMed  Google Scholar 

  • Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38, 3022–3027.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trifinopoulos, J., Nguyen, L. T., von Haeseler, A., & Minh, B. Q. (2016). W-IQ-TREE: A fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Research, 44, W232–W235.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wieczorek, M., Krzysztoszek, A., Ciąćka, A., & Figas, A. (2017). Molecular characterization of environmental and clinical echovirus 6 isolates from Poland, 2006–2014. Journal of Medical Virology, 89, 936–940.

    Article  PubMed  Google Scholar 

  • Xi, H., Tian, Y., Shao, H., Yin, X., Ma, L., Yang, P., & Li, X. (2023). An outbreak of nosocomial infection of neonatal aseptic meningitis caused by echovirus 18. Epidemiology and Infection, 151, e107.

    Article  PubMed  PubMed Central  Google Scholar 

  • Xiao, J., Zhang, Y., Hong, M., Han, Z., Zhang, M., Song, Y., Yan, D., Zhu, S., & Xu, W. (2020). Phylogenetic characteristics and molecular epidemiological analysis of novel enterovirus EV-B83 isolated from Tibet, China. Scientific Reports, 10, 6630.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yao, X., Bian, L. L., Mao, Q. Y., Zhu, F. C., Ye, Q., & Liang, Z. L. (2015). Echovirus 7 associated with hand, foot, and mouth disease in mainland China has undergone a recombination event. Archives of Virology, 160, 1291–1295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang, D., Lu, J., & Lu, J. (2010). Enterovirus 71 vaccine: Close but still far. International Journal of Infectious Diseases, 14, e739–e743.

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang, M., Guo, W., Xu, D., Feng, C., Bao, G., Sun, H., Yang, Z., & Ma, S. (2022). Molecular characterization of echovirus 9 strains isolated from hand-foot-and-mouth disease in Kunming, Yunnan Province. China. Scientific Reports, 12, 2293.

    Article  CAS  PubMed  Google Scholar 

  • Zhu, Y., Zhou, X., Liu, J., Xia, L., Pan, Y., Chen, J., Luo, N., Yin, J., & Ma, S. (2016). Molecular identification of human enteroviruses associated with aseptic meningitis in Yunnan province, Southwest China. Springerplus, 5, 1515.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work is supported by Four “batches” innovation project of invigorating medical through science and technology of Shanxi province for Dr. Li Xing (2023XM015), the Fundamental Research Program of Shanxi Province, China (20210302124187) for Dr. Yue Liu, and the Programme of Introducing Talents of Discipline to Universities (D21004). We thank all the persons who contributed to the collection and production of Echovirus sequences in NCBI GenBank.

Author information

Author notes

  1. Yan Wang, Pir Tariq Shah and Yue Liu have contributed equally to this work.

Authors and Affiliations

  1. Institutes of Biomedical Sciences, Shanxi University, Taiyuan, 030006, Shanxi, People’s Republic of China

    Yan Wang, Pir Tariq Shah, Yue Liu, Amina Nawal Bahoussi & Li Xing

  2. Shanxi Provincial Key Laboratory of Medical Molecular Cell Biology, Shanxi University, Taiyuan, 030006, People’s Republic of China

    Li Xing

  3. Shanxi Provincial Key Laboratory for Prevention and Treatment of Major Infectious Diseases, Taiyuan, 030006, People’s Republic of China

    Li Xing

  4. The Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Shanxi University, Taiyuan, 030006, People’s Republic of China

    Li Xing

Authors
  1. Yan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pir Tariq Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Amina Nawal Bahoussi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Li Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LX conceived the study. YW, PTS, and YL performed analysis. YW, PTS, and YL wrote the manuscript. PTS, YW, ANB, and LX revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Li Xing.

Ethics declarations

Conflict of Interest

The authors declare no conflict of interest.

Institutional Review Board Statement and Informed Consent Statement

Not applicable.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 38062 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Y., Shah, P.T., Liu, Y. et al. Genetic Characteristics and Phylogeographic Dynamics of Echovirus. J Microbiol. 61, 865–877 (2023). https://doi.org/10.1007/s12275-023-00078-w

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12275-023-00078-w

Keywords

Search

Navigation