Detection of echovirus 7 in effluent sewage treatment plant Ahvaz city, Iran
-
Golnaz Mokhtari Irani
,
Azarakhsh Azaran
,
Roya Pirmoradi
,
Alireza Tabibzadeh
,
Mohamad Rashno
,
Manoochehr Makvandi
Abstract
Background and Objectives: The enteroviruses may lead to conditions such as aseptic meningitis, encephalitis, acute flaccid myelitis, epidemic pleurodynia (Bornholm disease), hemorrhagic conjunctivitis, and myopericarditis among pediatric populations. The present study was undertaken to identify the presence of enteroviruses within sewage treatment systems.
Materials and Methods: 24 composite effluent sewage samples (500 ml each) were collected, centrifuged (1000 rpm for 30 minutes), and the first supernatant was saved. The precipitate was resuspended in 10 ml of supernatant, treated with 10% chloroform, and centrifuged again (1000 rpm for 5 minutes) to collect a second supernatant. The first and second supernatants were combined, treated with 2.2% sodium chloride and 7% polyethylene glycol 6000, and the mixture was agitated at 4°C overnight before being centrifuged for two hours at 2000 g. After discarding the supernatant, the pellet was resuspended at a 1:100 dilution. Each sample was then inoculated into RD and HeLa cells for virus isolation, followed by detection via RT-PCR. A phylogenetic tree was constructed to determine the genotypes of the isolated enteroviruses.
Results: Enterovirus was detected in 10 of 24 (41.7%) sewage effluent samples. Phylogenetic analysis of five randomly chosen positive samples identified echovirus 7.
Conclusion: The removal of enteroviruses during the sewage treatment process is of paramount importance, necessitating heightened attention to this critical phase of wastewater management.
1. Brouwer L, Moreni G, Wolthers KC, Pajkrt D. World-wide prevalence and genotype distribution of enteroviruses. Viruses 2021; 13: 434.
2. Kalam N, Balasubramaniam V. Changing Epidemiology of Hand, Foot, and Mouth Disease Causative Agents and Contributing Factors. Am J Trop Med Hyg 2024; 111: 740-755.
3. Messali S, Bertelli A, Dotta L, Giovanetti M, Sclavi L, Venneri G, et al. Outbreak of Enterovirus D68 in Young Children, Brescia, Italy, August to November 2024. J Med Virol 2025; 97(5): e70372.
4. Sousa Jr IP, Oliveira MD, Burlandy FM, Machado RS, Oliveira SS, Tavares FN, et al. Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005–2017). Emerg Microbes Infect 2020; 9: 2536-2546.
5. Grizer CS, Messacar K, Mattapallil JJ. Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children. Front Virol 2024; 4: 1328457.
6. Pietrasanta C, Ronchi A, Bassi L, De Carli A, Caschera L, Lo Russo FM, et al. Enterovirus and parechovirus meningoencephalitis in infants: A ten-year prospective observational study in a neonatal intensive care unit. J Clin Virol 2024; 173: 105664.
7. Brisca G, Bellini T, Pasquinucci M, Mariani M, Romanengo M, Buffoni I, et al. Clinical course and peculiarities of Parechovirus and Enterovirus central nervous system infections in newborns: a single-center experience. Eur J Pediatr 2024; 183: 2615-2623.
8. Xie Z, Khamrin P, Maneekarn N, Kumthip K. Epidemiology of Enterovirus Genotypes in Association with Human Diseases. Viruses 2024; 16: 1165.
9. Jartti M, Flodström-Tullberg M, Hankaniemi MM. Enteroviruses: epidemic potential, challenges and opportunities with vaccines. J Biomed Sci 2024; 31: 73.
10. Larivé O, Torii S, Derlon N, Kohn T. Selective elimination of enterovirus genotypes by activated sludge and chlorination. Environ Sci (Camb) 2023; 9: 1620-1633.
11. Gomes J, Domingues E, Frasson D, Martins RC, Matos AM. Virus Removal from Real Wastewater as an Environmental Management Approach. Molecules 2024; 29: 5601.
12. Reynaert E, Sylvestre É, Morgenroth E, Julian TR. Greywater recycling for diverse collection scales and appliances: Enteric pathogen log-removal targets and treatment trains. Water Res 2024; 264: 122216.
13. Cox SN, Casto AM, Franko NM, Chow EJ, Han PD, Gamboa L, et al. Clinical and Genomic Epidemiology of Coxsackievirus A21 and Enterovirus D68 in Homeless Shelters, King County, Washington, USA, 2019-2021. Emerg Infect Dis 2024; 30: 2250-2260.
14. Garcia-Pedemonte D, Carcereny A, Andrés C, Antón A, Pérez I, Blanco A, et al. Unveiling the enterovirus diversity in Barcelona, Spain (2020-2024) through wastewater and clinical surveillance. Emerg Microbes Infect 2025; 14: 2589547.
15. Moazeni M, Nikaeen M, Hadi M, Moghim S, Mouhebat L, Hatamzadeh M, et al. Estimation of health risks caused by exposure to enteroviruses from agricultural application of wastewater effluents. Water Res 2017; 125: 104-113.
16. Tiwari S, Dhole TN. Assessment of enteroviruses from sewage water and clinical samples during eradication phase of polio in North India. Virol J 2018; 15: 157.
17. Zhong Q, Carratalà A, Ossola R, Bachmann V, Kohn T. Cross-resistance of UV-or chlorine dioxide-resistant echovirus 11 to other disinfectants. Front Microbiol 2017; 8: 1928.
18. Ozawa H, Yoshida H, Usuku S. Environmental Surveillance Can Dynamically Track Ecological Changes in Enteroviruses. Appl Environ Microbiol 2019; 85(24): e01604-19.
19. El-Senousy WM, Abdel-Moneim A, Abdel-Latif M, El-Hefnawy MH, Khalil RG. Coxsackievirus B4 as a Causative Agent of Diabetes Mellitus Type 1: Is There a Role of Inefficiently Treated Drinking Water and Sewage in Virus Spreading? Food Environ Virol 2018; 10: 89-98.
20. Amdiouni H, Faouzi A, Fariat N, Hassar M, Soukri A, Nourlil J. Detection and molecular identification of human adenoviruses and enteroviruses in wastewater from Morocco. Lett Appl Microbiol 2012; 54: 359-366.
21. Mohamadpoor T, Nabavinia M, Gholoobi A, Alavi M, Meshkat Z. Enteroviruses in acute myocardial infarction. Iran J Public Health 2012; 41: 71-74.
22. Tang J, Yoshida H, Ding Z, Tao Z, Zhang J, Tian B, et al. Molecular epidemiology and recombination of human Enteroviruses from AFP surveillance in Yunnan, China from 2006 to 2010. Sci Rep 2014; 4: 6058.
23. Haghi Navand A, Jalilian S, Ahmadi Angali K, Karimi Babaahmadi M, Talaiezadeh A, Makvandi M. A new evaluation of the rearranged non-coding control region of JC virus in patients with colorectal cancer. BMC Cancer 2024; 24: 1001.
24. Szulc J, Okrasa M, Majchrzycka K, Sulyok M, Nowak A, Ruman T, et al. Microbiological and Toxicological Hazards in Sewage Treatment Plant Bioaerosol and Dust. Toxins (Basel) 2021; 13: 691.
25. Gwenzi W, Adelodun B, Kumar P, Ajibade FO, Silva LFO, Choi KS, et al. Human viral pathogens in the wastewater-source water-drinking water continuum: Evidence, health risks, and lessons for future outbreaks in low-income settings. Sci Total Environ 2024; 918: 170214.
26. Liang Z, Lin C, Huo D, Yang Y, Feng Z, Cui S, et al. First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022. J Med Virol 2024; 96(7): e29796.
27. Tao Z, Wang Z, Lin X, Wang S, Wang H, Yoshida H, et al. One-year Survey of human enteroviruses from sewage and the factors affecting virus adsorption to the suspended solids. Sci Rep 2016; 6: 31474.
28. Farías AA, Mojsiejczuk LN, Flores FS, Aguilar JJ, Prez VE, Masachessi G, et al. Environmental surveillance of human enteroviruses in Córdoba City, Argentina: prevalence and detection of serotypes from 2009 to 2014. Food and Environ Virol 2019;11:198-203.
29. Ndiaye N, Kébé O, Diarra M, Thiaw FD, Dia M, Dia N, et al. Non-polio enteroviruses circulation in acute flaccid paralysis cases and sewage in Senegal from 2013 to 2021. Int J Infect Dis 2024; 138: 54-62.
30. Moliner-Calderón E, Rabella-Garcia N, Turón-Viñas E, Ginovart-Galiana G, Figueras-Aloy J. Relevance of enteroviruses in neonatal meningitis. Enferm Infecc Microbiol Clin (Engl Ed) 2024; 42: 17-23.
31. Faleye TOC, Adewumi OM, Adeniji JA. Genomic Characterization of an Echovirus 7 Isolate of Southeast Asian Ancestry Recovered from a Child in Nigeria with Acute Flaccid Paralysis. Microbiol Resour Announc 2019; 8(33): e00830-19.
32. Nejati A, Zahraei SM, Mahmoudi S, Yousefi M, Mollaei-Kandelous Y, Tabatabaie H, et al. Molecular characterization of non-polio enteroviruses isolated from children with acute flaccid paralysis in IRAN, 2015-2018. Virus Genes 2020; 56: 531-536.
33. Lum LC, Chua KB, McMinn PC, Goh AY, Muridan R, Sarji SA, et al. Echovirus 7 associated encephalomyelitis. J Clin Virol 2002; 23:153-160.
34. Faleye TOC, Adewumi OM, Adeniji JA. Reference Echovirus 7 and 19 Genomes from Nigeria. Microbiol Resour Announc 2018; 7(22): e01465-18.
35. Kyriakopoulou Z, Dedepsidis E, Pliaka V, Mastorakos P, Stamati A, Pratti A, et al. Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece. Virus Genes 2010; 40: 183-192.
36. Rhodes ER, Huff EM, Hamilton DW, Jones JL. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices. J Virol Methods 2016; 228:31-38.
37. Karim MR, Rhodes ER, Brinkman N, Wymer L, Fout GS. New electropositive filter for concentrating enteroviruses and noroviruses from large volumes of water. Appl Environ Microbiol 2009; 75:2393-2399.
38. Amdiouni H, Maunula L, Hajjami K, Faouzi A, Soukri A, Nourlil J. Recovery comparison of two virus concentration methods from wastewater using cell culture and real-time PCR. Curr Microbiol 2012; 65: 432-437.
39. Chen W, Liu W, Jiao H, Zhang H, Cheng J, Wu Y. Development of a concentration method for detection of tobacco mosaic virus in irrigation water. Virol Sin 2014; 29: 155-161.
40. Farkas K, McDonald JE, Malham SK, Jones DL. Two-Step Concentration of Complex Water Samples for the Detection of Viruses. Methods Protoc 2018; 1: 35.
41. Rashid M, Khan MN, Jalbani N. Detection of Human Adenovirus, Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi, Pakistan. Food Environ Virol 2021; 13: 44-52.
42. Abd-Elshafy DN, Raslan HZ, Nadeem R, Awad MA, Shalaby S, Mohamed Bahgat M. Multiple infections with hepatitis A virus and development of rheumatoid arthritis among waste water treatment plant workers. J Infect Dev Ctries 2022; 16: 187-195.
43. Cuevas-Ferrando E, Randazzo W, Pérez-Cataluña A, Sánchez G. HEV occurrence in waste and drinking water treatment plants. Front Microbiol 2020; 10: 2937.
44. Sedji MI, Varbanov M, Meo M, Colin M, Mathieu L, Bertrand I. Quantification of human adenovirus and norovirus in river water in the north-east of France. Environ Sci Pollut Res Int 2018; 25: 30497-30507.
45. Di Bonito P, Iaconelli M, Gheit T, Tommasino M, Della Libera S, Bonadonna L, et al. Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents. Water Res 2017; 123: 549-555.
46. Jiang H, Zhang Z, Rao Q, Wang X, Wang M, Du T, et al. The epidemiological characteristics of enterovirus infection before and after the use of enterovirus 71 inactivated vaccine in Kunming, China. Emerg Microbes Infect 2021; 10: 619-628.
2. Kalam N, Balasubramaniam V. Changing Epidemiology of Hand, Foot, and Mouth Disease Causative Agents and Contributing Factors. Am J Trop Med Hyg 2024; 111: 740-755.
3. Messali S, Bertelli A, Dotta L, Giovanetti M, Sclavi L, Venneri G, et al. Outbreak of Enterovirus D68 in Young Children, Brescia, Italy, August to November 2024. J Med Virol 2025; 97(5): e70372.
4. Sousa Jr IP, Oliveira MD, Burlandy FM, Machado RS, Oliveira SS, Tavares FN, et al. Molecular characterization and epidemiological aspects of non-polio enteroviruses isolated from acute flaccid paralysis in Brazil: a historical series (2005–2017). Emerg Microbes Infect 2020; 9: 2536-2546.
5. Grizer CS, Messacar K, Mattapallil JJ. Enterovirus-D68 - A Reemerging Non-Polio Enterovirus that Causes Severe Respiratory and Neurological Disease in Children. Front Virol 2024; 4: 1328457.
6. Pietrasanta C, Ronchi A, Bassi L, De Carli A, Caschera L, Lo Russo FM, et al. Enterovirus and parechovirus meningoencephalitis in infants: A ten-year prospective observational study in a neonatal intensive care unit. J Clin Virol 2024; 173: 105664.
7. Brisca G, Bellini T, Pasquinucci M, Mariani M, Romanengo M, Buffoni I, et al. Clinical course and peculiarities of Parechovirus and Enterovirus central nervous system infections in newborns: a single-center experience. Eur J Pediatr 2024; 183: 2615-2623.
8. Xie Z, Khamrin P, Maneekarn N, Kumthip K. Epidemiology of Enterovirus Genotypes in Association with Human Diseases. Viruses 2024; 16: 1165.
9. Jartti M, Flodström-Tullberg M, Hankaniemi MM. Enteroviruses: epidemic potential, challenges and opportunities with vaccines. J Biomed Sci 2024; 31: 73.
10. Larivé O, Torii S, Derlon N, Kohn T. Selective elimination of enterovirus genotypes by activated sludge and chlorination. Environ Sci (Camb) 2023; 9: 1620-1633.
11. Gomes J, Domingues E, Frasson D, Martins RC, Matos AM. Virus Removal from Real Wastewater as an Environmental Management Approach. Molecules 2024; 29: 5601.
12. Reynaert E, Sylvestre É, Morgenroth E, Julian TR. Greywater recycling for diverse collection scales and appliances: Enteric pathogen log-removal targets and treatment trains. Water Res 2024; 264: 122216.
13. Cox SN, Casto AM, Franko NM, Chow EJ, Han PD, Gamboa L, et al. Clinical and Genomic Epidemiology of Coxsackievirus A21 and Enterovirus D68 in Homeless Shelters, King County, Washington, USA, 2019-2021. Emerg Infect Dis 2024; 30: 2250-2260.
14. Garcia-Pedemonte D, Carcereny A, Andrés C, Antón A, Pérez I, Blanco A, et al. Unveiling the enterovirus diversity in Barcelona, Spain (2020-2024) through wastewater and clinical surveillance. Emerg Microbes Infect 2025; 14: 2589547.
15. Moazeni M, Nikaeen M, Hadi M, Moghim S, Mouhebat L, Hatamzadeh M, et al. Estimation of health risks caused by exposure to enteroviruses from agricultural application of wastewater effluents. Water Res 2017; 125: 104-113.
16. Tiwari S, Dhole TN. Assessment of enteroviruses from sewage water and clinical samples during eradication phase of polio in North India. Virol J 2018; 15: 157.
17. Zhong Q, Carratalà A, Ossola R, Bachmann V, Kohn T. Cross-resistance of UV-or chlorine dioxide-resistant echovirus 11 to other disinfectants. Front Microbiol 2017; 8: 1928.
18. Ozawa H, Yoshida H, Usuku S. Environmental Surveillance Can Dynamically Track Ecological Changes in Enteroviruses. Appl Environ Microbiol 2019; 85(24): e01604-19.
19. El-Senousy WM, Abdel-Moneim A, Abdel-Latif M, El-Hefnawy MH, Khalil RG. Coxsackievirus B4 as a Causative Agent of Diabetes Mellitus Type 1: Is There a Role of Inefficiently Treated Drinking Water and Sewage in Virus Spreading? Food Environ Virol 2018; 10: 89-98.
20. Amdiouni H, Faouzi A, Fariat N, Hassar M, Soukri A, Nourlil J. Detection and molecular identification of human adenoviruses and enteroviruses in wastewater from Morocco. Lett Appl Microbiol 2012; 54: 359-366.
21. Mohamadpoor T, Nabavinia M, Gholoobi A, Alavi M, Meshkat Z. Enteroviruses in acute myocardial infarction. Iran J Public Health 2012; 41: 71-74.
22. Tang J, Yoshida H, Ding Z, Tao Z, Zhang J, Tian B, et al. Molecular epidemiology and recombination of human Enteroviruses from AFP surveillance in Yunnan, China from 2006 to 2010. Sci Rep 2014; 4: 6058.
23. Haghi Navand A, Jalilian S, Ahmadi Angali K, Karimi Babaahmadi M, Talaiezadeh A, Makvandi M. A new evaluation of the rearranged non-coding control region of JC virus in patients with colorectal cancer. BMC Cancer 2024; 24: 1001.
24. Szulc J, Okrasa M, Majchrzycka K, Sulyok M, Nowak A, Ruman T, et al. Microbiological and Toxicological Hazards in Sewage Treatment Plant Bioaerosol and Dust. Toxins (Basel) 2021; 13: 691.
25. Gwenzi W, Adelodun B, Kumar P, Ajibade FO, Silva LFO, Choi KS, et al. Human viral pathogens in the wastewater-source water-drinking water continuum: Evidence, health risks, and lessons for future outbreaks in low-income settings. Sci Total Environ 2024; 918: 170214.
26. Liang Z, Lin C, Huo D, Yang Y, Feng Z, Cui S, et al. First detection of multiple cases related to CV-A16 strain of B1c clade in Beijing in 2022. J Med Virol 2024; 96(7): e29796.
27. Tao Z, Wang Z, Lin X, Wang S, Wang H, Yoshida H, et al. One-year Survey of human enteroviruses from sewage and the factors affecting virus adsorption to the suspended solids. Sci Rep 2016; 6: 31474.
28. Farías AA, Mojsiejczuk LN, Flores FS, Aguilar JJ, Prez VE, Masachessi G, et al. Environmental surveillance of human enteroviruses in Córdoba City, Argentina: prevalence and detection of serotypes from 2009 to 2014. Food and Environ Virol 2019;11:198-203.
29. Ndiaye N, Kébé O, Diarra M, Thiaw FD, Dia M, Dia N, et al. Non-polio enteroviruses circulation in acute flaccid paralysis cases and sewage in Senegal from 2013 to 2021. Int J Infect Dis 2024; 138: 54-62.
30. Moliner-Calderón E, Rabella-Garcia N, Turón-Viñas E, Ginovart-Galiana G, Figueras-Aloy J. Relevance of enteroviruses in neonatal meningitis. Enferm Infecc Microbiol Clin (Engl Ed) 2024; 42: 17-23.
31. Faleye TOC, Adewumi OM, Adeniji JA. Genomic Characterization of an Echovirus 7 Isolate of Southeast Asian Ancestry Recovered from a Child in Nigeria with Acute Flaccid Paralysis. Microbiol Resour Announc 2019; 8(33): e00830-19.
32. Nejati A, Zahraei SM, Mahmoudi S, Yousefi M, Mollaei-Kandelous Y, Tabatabaie H, et al. Molecular characterization of non-polio enteroviruses isolated from children with acute flaccid paralysis in IRAN, 2015-2018. Virus Genes 2020; 56: 531-536.
33. Lum LC, Chua KB, McMinn PC, Goh AY, Muridan R, Sarji SA, et al. Echovirus 7 associated encephalomyelitis. J Clin Virol 2002; 23:153-160.
34. Faleye TOC, Adewumi OM, Adeniji JA. Reference Echovirus 7 and 19 Genomes from Nigeria. Microbiol Resour Announc 2018; 7(22): e01465-18.
35. Kyriakopoulou Z, Dedepsidis E, Pliaka V, Mastorakos P, Stamati A, Pratti A, et al. Molecular identification and full genome analysis of an echovirus 7 strain isolated from the environment in Greece. Virus Genes 2010; 40: 183-192.
36. Rhodes ER, Huff EM, Hamilton DW, Jones JL. The evaluation of hollow-fiber ultrafiltration and celite concentration of enteroviruses, adenoviruses and bacteriophage from different water matrices. J Virol Methods 2016; 228:31-38.
37. Karim MR, Rhodes ER, Brinkman N, Wymer L, Fout GS. New electropositive filter for concentrating enteroviruses and noroviruses from large volumes of water. Appl Environ Microbiol 2009; 75:2393-2399.
38. Amdiouni H, Maunula L, Hajjami K, Faouzi A, Soukri A, Nourlil J. Recovery comparison of two virus concentration methods from wastewater using cell culture and real-time PCR. Curr Microbiol 2012; 65: 432-437.
39. Chen W, Liu W, Jiao H, Zhang H, Cheng J, Wu Y. Development of a concentration method for detection of tobacco mosaic virus in irrigation water. Virol Sin 2014; 29: 155-161.
40. Farkas K, McDonald JE, Malham SK, Jones DL. Two-Step Concentration of Complex Water Samples for the Detection of Viruses. Methods Protoc 2018; 1: 35.
41. Rashid M, Khan MN, Jalbani N. Detection of Human Adenovirus, Rotavirus, and Enterovirus in Tap Water and Their Association with the Overall Quality of Water in Karachi, Pakistan. Food Environ Virol 2021; 13: 44-52.
42. Abd-Elshafy DN, Raslan HZ, Nadeem R, Awad MA, Shalaby S, Mohamed Bahgat M. Multiple infections with hepatitis A virus and development of rheumatoid arthritis among waste water treatment plant workers. J Infect Dev Ctries 2022; 16: 187-195.
43. Cuevas-Ferrando E, Randazzo W, Pérez-Cataluña A, Sánchez G. HEV occurrence in waste and drinking water treatment plants. Front Microbiol 2020; 10: 2937.
44. Sedji MI, Varbanov M, Meo M, Colin M, Mathieu L, Bertrand I. Quantification of human adenovirus and norovirus in river water in the north-east of France. Environ Sci Pollut Res Int 2018; 25: 30497-30507.
45. Di Bonito P, Iaconelli M, Gheit T, Tommasino M, Della Libera S, Bonadonna L, et al. Detection of oncogenic viruses in water environments by a Luminex-based multiplex platform for high throughput screening of infectious agents. Water Res 2017; 123: 549-555.
46. Jiang H, Zhang Z, Rao Q, Wang X, Wang M, Du T, et al. The epidemiological characteristics of enterovirus infection before and after the use of enterovirus 71 inactivated vaccine in Kunming, China. Emerg Microbes Infect 2021; 10: 619-628.
| Files | ||
| Issue | Vol 18 No 1 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i1.20938 | |
| Keywords | ||
| Wastewater Environment Enterovirus Polymerase chain reaction | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Mokhtari Irani G, Azaran A, Pirmoradi R, Tabibzadeh A, Rashno M, Makvandi M. Detection of echovirus 7 in effluent sewage treatment plant Ahvaz city, Iran. Iran J Microbiol. 2026;18(1):131-137.
