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1. Food Security: Microbiological and Chemical Risks

Author

Schlundt, Joergen, Tay, Moon Y. F., Chengcheng, Hu, Liwei, Chen, Masys, Anthony J., Series Editor, Bichler, Gisela, Advisory Editor, Bourlai, Thirimachos, Advisory Editor, Johnson, Chris, Advisory Editor, Karampelas, Panagiotis, Advisory Editor, Leuprecht, Christian, Advisory Editor, Morse, Edward C., Advisory Editor, Skillicorn, David, Advisory Editor, Yamagata, Yoshiki, Advisory Editor, Izurieta, Ricardo, editor, and Reina Ortiz, Miguel, editor

Published
2020
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3. Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance

Author

Munk, Patrick, Brinch, Christian, Møller, Frederik Duus, Petersen, Thomas N., Hendriksen, Rene S., Seyfarth, Anne Mette, Kjeldgaard, Jette S., Svendsen, Christina Aaby, van Bunnik, Bram, Berglund, Fanny, Bego, Artan, Power, Pablo, Rees, Catherine, Lambrinidis, Dionisia, Neilson, Elizabeth Heather Jakobsen, Gibb, Karen, Coventry, Kris, Collignon, Peter, Cassar, Susan, Allerberger, Franz, Begum, Anowara, Hossain, Zenat Zebin, Worrell, Carlon, Vandenberg, Olivier, Pieters, Ilse, Victorien, Dougnon Tamègnon, Gutierrez, Angela Daniela Salazar, Soria, Freddy, Grujić, Vesna Rudić, Mazalica, Nataša, Rahube, Teddie O., Tagliati, Carlos Alberto, Rodrigues, Dalia, Oliveira, Guilherme, de Souza, Larissa Camila Ribeiro, Ivanov, Ivan, Juste, Bonkoungou Isidore, Oumar, Traoré, Sopheak, Thet, Vuthy, Yith, Ngandijo, Antoinette, Nzouankeu, Ariane, Olivier, Ziem A. Abah Jacques, Yost, Christopher K., Kumar, Pratik, Brar, Satinder Kaur, Tabo, Djim-Adjim, Adell, Aiko D., Paredes-Osses, Esteban, Martinez, Maria Cristina, Cuadros-Orellana, Sara, Ke, Changwen, Zheng, Huanying, Baisheng, Li, Lau, Lok Ting, Chung, Teresa, Jiao, Xiaoyang, Yu, Yongjie, JiaYong, Zhao, Morales, Johan F. Bernal, Valencia, Maria Fernanda, Donado-Godoy, Pilar, Coulibaly, Kalpy Julien, Hrenovic, Jasna, Jergović, Matijana, Karpíšková, Renáta, Deogratias, Zozo Nyarukweba, Elsborg, Bodil, Hansen, Lisbeth Truelstrup, Jensen, Pernille Erland, Abouelnaga, Mohamed, Salem, Mohamed Fathy, Koolmeister, Marliin, Legesse, Mengistu, Eguale, Tadesse, Heikinheimo, Annamari, Le Guyader, Soizick, Schaeffer, Julien, Villacis, Jose Eduardo, Sanneh, Bakary, Malania, Lile, Nitsche, Andreas, Brinkmann, Annika, Schubert, Sara, Hesse, Sina, Berendonk, Thomas U., Saba, Courage Kosi Setsoafia, Mohammed, Jibril, Feglo, Patrick Kwame, Banu, Regina Ama, Kotzamanidis, Charalampos, Lytras, Efthymios, Lickes, Sergio A., Kocsis, Bela, Solymosi, Norbert, Thorsteinsdottir, Thorunn R., Hatha, Abdulla Mohamed, Ballal, Mamatha, Bangera, Sohan Rodney, Fani, Fereshteh, Alebouyeh, Masoud, Morris, Dearbhaile, O’Connor, Louise, Cormican, Martin, Moran-Gilad, Jacob, Battisti, Antonio, Diaconu, Elena Lavinia, Corno, Gianluca, Di Cesare, Andrea, Alba, Patricia, Hisatsune, Junzo, Yu, Liansheng, Kuroda, Makoto, Sugai, Motoyuki, Kayama, Shizuo, Shakenova, Zeinegul, Kiiyukia, Ciira, Ng’eno, Eric, Raka, Lul, Jamil, Kazi, Fakhraldeen, Saja Adel, Alaati, Tareq, Bērziņš, Aivars, Avsejenko, Jeļena, Kokina, Kristina, Streikisa, Madara, Bartkevics, Vadims, Matar, Ghassan M., Daoud, Ziad, Pereckienė, Asta, Butrimaite-Ambrozeviciene, Ceslova, Penny, Christian, Bastaraud, Alexandra, Rasolofoarison, Tiavina, Collard, Jean-Marc, Samison, Luc Hervé, Andrianarivelo, Mala Rakoto, Banda, Daniel Lawadi, Amin, Arshana, Rajandas, Heraa, Parimannan, Sivachandran, Spiteri, David, Haber, Malcolm Vella, Santchurn, Sunita J., Vujacic, Aleksandar, Djurovic, Dijana, Bouchrif, Brahim, Karraouan, Bouchra, Vubil, Delfino Carlos, Pal, Pushkar, Schmitt, Heike, van Passel, Mark, Jeunen, Gert-Jan, Gemmell, Neil, Chambers, Stephen T., Mendoza, Fania Perez, Huete-Pιrez, Jorge, Vilchez, Samuel, Ahmed, Akeem Olayiwola, Adisa, Ibrahim Raufu, Odetokun, Ismail Ayoade, Fashae, Kayode, Sørgaard, Anne-Marie, Wester, Astrid Louise, Ryrfors, Pia, Holmstad, Rune, Mohsin, Mashkoor, Hasan, Rumina, Shakoor, Sadia, Gustafson, Natalie Weiler, Schill, Claudia Huber, Rojas, Maria Luz Zamudio, Velasquez, Jorge Echevarria, Magtibay, Bonifacio B., Catangcatang, Kris, Sibulo, Ruby, Yauce, Felipe Campos, Wasyl, Dariusz, Manaia, Celia, Rocha, Jaqueline, Martins, Jose, Álvaro, Pedro, Di Yoong Wen, Doris, Shin, Hanseob, Hur, Hor-Gil, Yoon, Sukhwan, Bosevska, Golubinka, Kochubovski, Mihail, Cojocaru, Radu, Burduniuc, Olga, Hong, Pei-Ying, Perry, Meghan Rose, Gassama, Amy, Radosavljevic, Vladimir, Tay, Moon Y. F., Zuniga-Montanez, Rogelio, Wuertz, Stefan, Gavačová, Dagmar, Pastuchová, Katarína, Truska, Peter, Trkov, Marija, Keddy, Karen, Esterhuyse, Kerneels, Song, Min Joon, Quintela-Baluja, Marcos, Lopez, Mariano Gomez, Cerdà-Cuéllar, Marta, Perera, R.R.D.P., Bandara, N.K.B.K.R.G.W., Premasiri, H.I., Pathirage, Sujatha, Charlemagne, Kareem, Rutgersson, Carolin, Norrgren, Leif, Örn, Stefan, Boss, Renate, Van der Heijden, Tanja, Hong, Yu-Ping, Kumburu, Happiness Houka, Mdegela, Robinson Hammerthon, Hounmanou, Yaovi Mahuton Gildas, Chonsin, Kaknokrat, Suthienkul, Orasa, Thamlikitkul, Visanu, de Roda Husman, Ana Maria, Bidjada, Bawimodom, Njanpop-Lafourcade, Berthe-Marie, Nikiema-Pessinaba, Somtinda Christelle, Levent, Belkis, Kurekci, Cemil, Ejobi, Francis, Kalule, John Bosco, Thomsen, Jens, Obaidi, Ouidiane, Jassim, Laila Mohamed, Moore, Andrew, Leonard, Anne, Graham, David W., Bunce, Joshua T., Zhang, Lihong, Gaze, William H., Lefor, Brett, Capone, Drew, Sozzi, Emanuele, Brown, Joe, Meschke, John Scott, Sobsey, Mark D., Davis, Michael, Beck, Nicola Koren, Sukapanpatharam, Pardi, Truong, Phuong, Lilienthal, Ronald, Kang, Sanghoon, Wittum, Thomas E., Rigamonti, Natalia, Baklayan, Patricia, Van, Chinh Dang, Tran, Doan Minh Nguyen, Do Phuc, Nguyen, Kwenda, Geoffrey, Larsson, D. G. Joakim, Koopmans, Marion, Woolhouse, Mark, Aarestrup, Frank M., Virology, Producció Animal, and Sanitat Animal

Subjects
genomic analysis, sewage, antimicrobial resistance, Multidisciplinary, Sewage, Drug Resistance, Bacterial, Metagenome, General Physics and Astronomy, Drug Resistance, Bacterial/genetics, Genomics, General Chemistry, General Biochemistry, Genetics and Molecular Biology, Anti-Bacterial Agents/pharmacology, Anti-Bacterial Agents
Abstract

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention. Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.

Published
2022
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4. Food Security: Microbiological and Chemical Risks

Author

Schlundt, Joergen, Tay, Moon Y. F., Chengcheng, Hu, and Liwei, Chen

Subjects
Sustainability, education, Microbiological risks, Food security, humanities, health care economics and organizations, Article
Abstract

Food Security within a health security context relates to systems dealing with the prevention and control of not only acute but also sporadic and chronic foodborne diseases. The description of food security and safety systems in this chapter will hence include oversight of both microbiological and chemical hazards, which both can cause acute as well as chronic disease events. The Chapter includes a description of existing national and international surveillance systems for foodborne diseases and regulatory systems enabling risk mitigation action for both chemical and microbiological hazards, with specific inclusion of the concerning increase in antimicrobial resistance (AMR) of foodborne microorganisms also related to the animal production systems. The Chapter also includes descriptions of methodology for the quantitative assessment of sustainability of food production systems.

Published
2019

7. Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community

Author

Sala, Claudia, primary, Mordhorst, Hanne, additional, Grützke, Josephine, additional, Brinkmann, Annika, additional, Petersen, Thomas N., additional, Poulsen, Casper, additional, Cotter, Paul D., additional, Crispie, Fiona, additional, Ellis, Richard J., additional, Castellani, Gastone, additional, Amid, Clara, additional, Hakhverdyan, Mikhayil, additional, Guyader, Soizick Le, additional, Manfreda, Gerardo, additional, Mossong, Joël, additional, Nitsche, Andreas, additional, Ragimbeau, Catherine, additional, Schaeffer, Julien, additional, Schlundt, Joergen, additional, Tay, Moon Y. F., additional, Aarestrup, Frank M., additional, Hendriksen, Rene S., additional, Pamp, Sünje Johanna, additional, and De Cesare, Alessandra, additional

Published
2020
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8. Proficiency Testing of Metagenomics-Based Detection of Food-Borne Pathogens Using a Complex Artificial Sequencing Dataset

Author

Höper, Dirk, primary, Grützke, Josephine, additional, Brinkmann, Annika, additional, Mossong, Joël, additional, Matamoros, Sébastien, additional, Ellis, Richard J., additional, Deneke, Carlus, additional, Tausch, Simon H., additional, Cuesta, Isabel, additional, Monzón, Sara, additional, Juliá, Miguel, additional, Petersen, Thomas Nordahl, additional, Hendriksen, Rene S., additional, Pamp, Sünje J., additional, Leijon, Mikael, additional, Hakhverdyan, Mikhayil, additional, Walsh, Aaron M., additional, Cotter, Paul D., additional, Chandrasekaran, Lakshmi, additional, Tay, Moon Y. F., additional, Schlundt, Joergen, additional, Sala, Claudia, additional, De Cesare, Alessandra, additional, Nitsche, Andreas, additional, Beer, Martin, additional, and Wylezich, Claudia, additional

Published
2020
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9. Occurrence and Antimicrobial Resistance Traits of Escherichia coli from Wild Birds and Rodents in Singapore

Author

Ong, Kar Hui, primary, Khor, Wei Ching, additional, Quek, Jing Yi, additional, Low, Zi Xi, additional, Arivalan, Sathish, additional, Humaidi, Mahathir, additional, Chua, Cliff, additional, Seow, Kelyn L. G., additional, Guo, Siyao, additional, Tay, Moon Y. F., additional, Schlundt, Joergen, additional, Ng, Lee Ching, additional, and Aung, Kyaw Thu, additional

Published
2020
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10. A Glycosylated Cationic Block Poly(β‐peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram‐Negative Bacteria

Author

Si, Zhangyong, primary, Lim, Hui Wen, additional, Tay, Moon Y. F., additional, Du, Yu, additional, Ruan, Lin, additional, Qiu, Haofeng, additional, Zamudio‐Vazquez, Rubí, additional, Reghu, Sheethal, additional, Chen, Yahua, additional, Tiong, Wen Shuo, additional, Marimuthu, Kalisvar, additional, De, Partha Pratim, additional, Ng, Oon Tek, additional, Zhu, Yabin, additional, Gan, Yunn‐Hwen, additional, Chi, Yonggui Robin, additional, Duan, Hongwei, additional, Bazan, Guillermo C., additional, Greenberg, E. Peter, additional, Chan‐Park, Mary B., additional, and Pethe, Kevin, additional

Published
2020
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11. Metagenomics-Based Proficiency Test of Smoked Salmon Spiked with a Mock Community

Author

Sala, Claudia, Mordhorst, Hanne, Grützke, Joesphine, Brinkmann, Annika, Petersen, Thomas N, Poulsen, Casper, Cotter, Paul D, Crispie, Fiona, Ellis, Richard J, Castellani, Gastone, Amid, Clara, Hakhverdyan, Mikhayil, Le Guyader, Soizick, Manfreda, Gerardo, Mossong, Joël, Nitsche, Andreas, Ragimbeau, Catherine, Schaeffer, Julien, Schlundt, Joergen, Tay, Moon Y. F., Aarestrup, Frank M., Hendriksen, Rene S., Pamp, Sünje Johanna, De Cesare, Alessandra, Sala, Claudia, Mordhorst, Hanne, Grützke, Joesphine, Brinkmann, Annika, Petersen, Thomas N, Poulsen, Casper, Cotter, Paul D, Crispie, Fiona, Ellis, Richard J, Castellani, Gastone, Amid, Clara, Hakhverdyan, Mikhayil, Le Guyader, Soizick, Manfreda, Gerardo, Mossong, Joël, Nitsche, Andreas, Ragimbeau, Catherine, Schaeffer, Julien, Schlundt, Joergen, Tay, Moon Y. F., Aarestrup, Frank M., Hendriksen, Rene S., Pamp, Sünje Johanna, and De Cesare, Alessandra

Abstract

An inter-laboratory proficiency test was organized to assess the ability of participants to perform shotgun metagenomic sequencing of cold smoked salmon, experimentally spiked with a mock community composed of six bacteria, one parasite, one yeast, one DNA, and two RNA viruses. Each participant applied its in-house wet-lab workflow(s) to obtain the metagenomic dataset(s), which were then collected and analyzed using MG-RAST. A total of 27 datasets were analyzed. Sample pre-processing, DNA extraction protocol, library preparation kit, and sequencing platform, influenced the abundance of specific microorganisms of the mock community. Our results highlight that despite differences in wet-lab protocols, the reads corresponding to the mock community members spiked in the cold smoked salmon, were both detected and quantified in terms of relative abundance, in the metagenomic datasets, proving the suitability of shotgun metagenomic sequencing as a genomic tool to detect microorganisms belonging to different domains in the same food matrix. The implementation of standardized wet-lab protocols would highly facilitate the comparability of shotgun metagenomic sequencing dataset across laboratories and sectors. Moreover, there is a need for clearly defining a sequencing reads threshold, to consider pathogens as detected or undetected in a food sample.

Published
2020
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12. Setting a baseline for global urban virome surveillance in sewage

Author

Nieuwenhuijse, David, Munnink, Bas B. Oude, Phan, My V. T., Munk, Patrick, Venkatakrishnan, Shweta, Aarestrup, Frank M., Cotten, Matthew, Koopmans, Marion P., Hendriksen, Rene S., Bego, Artan, Rees, Catherine, Neilson, Elizabeth Heather, Coventry, Kris, Collignon, Peter, Allerberger, Franz, Rahube, Teddie O., Oliveira, Guilherme, Ivanov, Ivan, Sopheak, Thet, Vuthy, Yith, Yost, Christopher K., Tabo, Djim-adjim, Cuadros-Orellana, Sara, Ke, Changwen, Zheng, Huanying, Li, Baisheng, Jiao, Xiaoyang, Donado-Godoy, Pilar, Coulibaly, Kalpy Julien, Hrenović, Jasna, Jergović, Matijana, Karpiskova, Renata, Elsborg, Bodil, Legesse, Mengistu, Eguale, Tadesse, Heikinheimo, Annamari, Eduardo Villacis, Jose, Sanneh, Bakary, Malania, Lile, Nitsche, Andreas, Brinkmann, Annika, Saba, Courage Kosi Setsoafa, Kocsis, Bela, Solymosi, Norbert, Thorsteinsdottir, Thorunn R., Hatha, Abdulla Mohamed, Alebouyeh, Masoud, Morris, Dearbhaile, O'Connor, Louise, Cormican, Martin, Moran-Gilad, Jacob, Battisti, Antonio, Alba, Patricia, Shakenova, Zeinegul, Kiiyukia, Ciira, Ng'eno, Eric, Raka, Lul, Berzins, Aivars, Avsejenko, Jelena, Bartkevics, Vadims, Penny, Christian, Rajandas, Heraa, Parimannan, Sivachandran, Haber, Malcolm Vella, Pal, Pushkar, Schmitt, Heike, van Passel, Mark, van de Schans, Milou G. M., Zuidema, Tina, Jeunen, Gert-Jan, Gemmell, Neil, Fashae, Kayode, Wester, Astrid Louise, Holmstad, Rune, Hasan, Rumina, Shakoor, Sadia, Zamudio Rojas, Maria Luz, Wasyl, Dariusz, Bosevska, Golubinka, Kochubovski, Mihail, Radu, Cojocaru, Gassama, Amy, Radosavljević, Vladimir, Tay, Moon Y. F., Zuniga-Montanez, Rogelio, Wuertz, Stefan, Gavacova, Dagmar, Trkov, Marija, Keddy, Karen, Esterhuyse, Kerneels, Cerda-Cuellar, Marta, Pathirage, Sujatha, Larsson, D. G. Joakim, Norrgren, Leif, Orn, Stefan, Van der Heijden, Tanja, Kumburu, Happiness Houka, de RodaHusman, Ana Maria, Njanpop-Lafourcade, Berthe-Marie, Bidjada, Pawou, Nikiema-Pessinaba, Somtinda Christelle, Levent, Belkis, Meschke, John Scott, Beck, Nicola Koren, Chinh, Van Dang, Doan Minh Nguyen, Tran, Nguyen, Do Phuc, Kwenda, Geofrey, Nieuwenhuijse, David, Munnink, Bas B. Oude, Phan, My V. T., Munk, Patrick, Venkatakrishnan, Shweta, Aarestrup, Frank M., Cotten, Matthew, Koopmans, Marion P., Hendriksen, Rene S., Bego, Artan, Rees, Catherine, Neilson, Elizabeth Heather, Coventry, Kris, Collignon, Peter, Allerberger, Franz, Rahube, Teddie O., Oliveira, Guilherme, Ivanov, Ivan, Sopheak, Thet, Vuthy, Yith, Yost, Christopher K., Tabo, Djim-adjim, Cuadros-Orellana, Sara, Ke, Changwen, Zheng, Huanying, Li, Baisheng, Jiao, Xiaoyang, Donado-Godoy, Pilar, Coulibaly, Kalpy Julien, Hrenović, Jasna, Jergović, Matijana, Karpiskova, Renata, Elsborg, Bodil, Legesse, Mengistu, Eguale, Tadesse, Heikinheimo, Annamari, Eduardo Villacis, Jose, Sanneh, Bakary, Malania, Lile, Nitsche, Andreas, Brinkmann, Annika, Saba, Courage Kosi Setsoafa, Kocsis, Bela, Solymosi, Norbert, Thorsteinsdottir, Thorunn R., Hatha, Abdulla Mohamed, Alebouyeh, Masoud, Morris, Dearbhaile, O'Connor, Louise, Cormican, Martin, Moran-Gilad, Jacob, Battisti, Antonio, Alba, Patricia, Shakenova, Zeinegul, Kiiyukia, Ciira, Ng'eno, Eric, Raka, Lul, Berzins, Aivars, Avsejenko, Jelena, Bartkevics, Vadims, Penny, Christian, Rajandas, Heraa, Parimannan, Sivachandran, Haber, Malcolm Vella, Pal, Pushkar, Schmitt, Heike, van Passel, Mark, van de Schans, Milou G. M., Zuidema, Tina, Jeunen, Gert-Jan, Gemmell, Neil, Fashae, Kayode, Wester, Astrid Louise, Holmstad, Rune, Hasan, Rumina, Shakoor, Sadia, Zamudio Rojas, Maria Luz, Wasyl, Dariusz, Bosevska, Golubinka, Kochubovski, Mihail, Radu, Cojocaru, Gassama, Amy, Radosavljević, Vladimir, Tay, Moon Y. F., Zuniga-Montanez, Rogelio, Wuertz, Stefan, Gavacova, Dagmar, Trkov, Marija, Keddy, Karen, Esterhuyse, Kerneels, Cerda-Cuellar, Marta, Pathirage, Sujatha, Larsson, D. G. Joakim, Norrgren, Leif, Orn, Stefan, Van der Heijden, Tanja, Kumburu, Happiness Houka, de RodaHusman, Ana Maria, Njanpop-Lafourcade, Berthe-Marie, Bidjada, Pawou, Nikiema-Pessinaba, Somtinda Christelle, Levent, Belkis, Meschke, John Scott, Beck, Nicola Koren, Chinh, Van Dang, Doan Minh Nguyen, Tran, Nguyen, Do Phuc, and Kwenda, Geofrey

Abstract

The rapid development of megacities, and their growing connectedness across the world is becoming a distinct driver for emerging disease outbreaks. Early detection of unusual disease emergence and spread should therefore include such cities as part of risk-based surveillance. A catch-all metagenomic sequencing approach of urban sewage could potentially provide an unbiased insight into the dynamics of viral pathogens circulating in a community irrespective of access to care, a potential which already has been proven for the surveillance of poliovirus. Here, we present a detailed characterization of sewage viromes from a snapshot of 81 high density urban areas across the globe, including in-depth assessment of potential biases, as a proof of concept for catch-all viral pathogen surveillance. We show the ability to detect a wide range of viruses and geographical and seasonal differences for specific viral groups. Our findings offer a cross-sectional baseline for further research in viral surveillance from urban sewage samples and place previous studies in a global perspective.

Published
2020

15. Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage

Author

Hendriksen, Rene S., Munk, Patrick, Njage, Patrick, van Bunnik, Bram, McNally, Luke, Lukjancenko, Oksana, Roder, Timo, Nieuwenhuijse, David, Pedersen, Susanne Karlsmose, Kjeldgaard, Jette, Kaas, Rolf S., Clausen, Philip, Vogt, Josef Korbinian, Leekitcharoenphon, Pimlapas, van de Schans, Milou G. M., Zuidema, Tina, Husman, Ana Maria de Roda, Rasmussen, Simon, Petersen, Bent, Amid, Clara, Cochrane, Guy, Sicheritz-Ponten, Thomas, Schmitt, Heike, Alvarez, Jorge Raul Matheu, Aidara-Kane, Awa, Pamp, Sunje J., Lund, Ole, Hald, Tine, Woolhouse, Mark, Koopmans, Marion P., Vigre, Hakan, Petersen, Thomas Nordahl, Aarestrup, Frank M., Bego, Artan, Rees, Catherine, Cassar, Susan, Coventry, Kris, Collignon, Peter, Allerberger, Franz, Rahube, Teddie O., Oliveira, Guilherme, Ivanov, Ivan, Vuthy, Yith, Sopheak, Thet, Yost, Christopher K., Ke, Changwen, Zheng, Huanying, Li, Baisheng, Jiao, Xiaoyang, Donado-Godoy, Pilar, Coulibaly, Kalpy Julien, Jergović, Matijana, Hrenović, Jasna, Karpiskova, Renata, Eduardo Villacis, Jose, Legesse, Mengistu, Eguale, Tadesse, Heikinheimo, Annamari, Malania, Lile, Nitsche, Andreas, Brinkmann, Annika, Saba, Courage Kosi Setsoafia, Kocsis, Bela, Solymosi, Norbert, Thorsteinsdottir, Thorunn R., Hatha, Abdulla Mohamed, Alebouyeh, Masoud, Morris, Dearbhaile, Cormican, Martin, O'Connor, Louise, Moran-Gilad, Jacob, Alba, Patricia, Battisti, Antonio, Shakenova, Zeinegul, Kiiyukia, Ciira, Ng'eno, Eric, Raka, Lul, Avsejenko, Jelena, Berzins, Aivars, Bartkevics, Vadims, Penny, Christian, Rajandas, Heraa, Parimannan, Sivachandran, Haber, Malcolm Vella, Pal, Pushkar, Jeunen, Gert-Jan, Gemmell, Neil, Fashae, Kayode, Holmstad, Rune, Hasan, Rumina, Shakoor, Sadia, Rojas, Maria Luz Zamudio, Wasyl, Dariusz, Bosevska, Golubinka, Kochubovski, Mihail, Radu, Cojocaru, Gassama, Amy, Radosavljević, Vladimir, Wuertz, Stefan, Zuniga-Montanez, Rogelio, Tay, Moon Y. F., Gavacova, Dagmar, Pastuchova, Katarina, Truska, Peter, Trkov, Marija, Esterhuyse, Kerneels, Keddy, Karen, Cerda-Cuellar, Marta, Pathirage, Sujatha, Norrgren, Leif, Orn, Stefan, Larsson, D. G. Joakim, Van der Heijden, Tanja, Kumburu, Happiness Houka, Sanneh, Bakary, Bidjada, Pawou, Njanpop-Lafourcade, Berthe-Marie, Nikiema-Pessinaba, Somtinda Christelle, Levent, Belkis, Meschke, John Scott, Beck, Nicola Koren, Chinh, Dang Van, Nguyen, Do Phuc, Doan Minh Nguyen, Tran, Kwenda, Geoffrey, Tabo, Djim-Adjim, Wester, Astrid Louise, Cuadros-Orellana, Sara, Hendriksen, Rene S., Munk, Patrick, Njage, Patrick, van Bunnik, Bram, McNally, Luke, Lukjancenko, Oksana, Roder, Timo, Nieuwenhuijse, David, Pedersen, Susanne Karlsmose, Kjeldgaard, Jette, Kaas, Rolf S., Clausen, Philip, Vogt, Josef Korbinian, Leekitcharoenphon, Pimlapas, van de Schans, Milou G. M., Zuidema, Tina, Husman, Ana Maria de Roda, Rasmussen, Simon, Petersen, Bent, Amid, Clara, Cochrane, Guy, Sicheritz-Ponten, Thomas, Schmitt, Heike, Alvarez, Jorge Raul Matheu, Aidara-Kane, Awa, Pamp, Sunje J., Lund, Ole, Hald, Tine, Woolhouse, Mark, Koopmans, Marion P., Vigre, Hakan, Petersen, Thomas Nordahl, Aarestrup, Frank M., Bego, Artan, Rees, Catherine, Cassar, Susan, Coventry, Kris, Collignon, Peter, Allerberger, Franz, Rahube, Teddie O., Oliveira, Guilherme, Ivanov, Ivan, Vuthy, Yith, Sopheak, Thet, Yost, Christopher K., Ke, Changwen, Zheng, Huanying, Li, Baisheng, Jiao, Xiaoyang, Donado-Godoy, Pilar, Coulibaly, Kalpy Julien, Jergović, Matijana, Hrenović, Jasna, Karpiskova, Renata, Eduardo Villacis, Jose, Legesse, Mengistu, Eguale, Tadesse, Heikinheimo, Annamari, Malania, Lile, Nitsche, Andreas, Brinkmann, Annika, Saba, Courage Kosi Setsoafia, Kocsis, Bela, Solymosi, Norbert, Thorsteinsdottir, Thorunn R., Hatha, Abdulla Mohamed, Alebouyeh, Masoud, Morris, Dearbhaile, Cormican, Martin, O'Connor, Louise, Moran-Gilad, Jacob, Alba, Patricia, Battisti, Antonio, Shakenova, Zeinegul, Kiiyukia, Ciira, Ng'eno, Eric, Raka, Lul, Avsejenko, Jelena, Berzins, Aivars, Bartkevics, Vadims, Penny, Christian, Rajandas, Heraa, Parimannan, Sivachandran, Haber, Malcolm Vella, Pal, Pushkar, Jeunen, Gert-Jan, Gemmell, Neil, Fashae, Kayode, Holmstad, Rune, Hasan, Rumina, Shakoor, Sadia, Rojas, Maria Luz Zamudio, Wasyl, Dariusz, Bosevska, Golubinka, Kochubovski, Mihail, Radu, Cojocaru, Gassama, Amy, Radosavljević, Vladimir, Wuertz, Stefan, Zuniga-Montanez, Rogelio, Tay, Moon Y. F., Gavacova, Dagmar, Pastuchova, Katarina, Truska, Peter, Trkov, Marija, Esterhuyse, Kerneels, Keddy, Karen, Cerda-Cuellar, Marta, Pathirage, Sujatha, Norrgren, Leif, Orn, Stefan, Larsson, D. G. Joakim, Van der Heijden, Tanja, Kumburu, Happiness Houka, Sanneh, Bakary, Bidjada, Pawou, Njanpop-Lafourcade, Berthe-Marie, Nikiema-Pessinaba, Somtinda Christelle, Levent, Belkis, Meschke, John Scott, Beck, Nicola Koren, Chinh, Dang Van, Nguyen, Do Phuc, Doan Minh Nguyen, Tran, Kwenda, Geoffrey, Tabo, Djim-Adjim, Wester, Astrid Louise, and Cuadros-Orellana, Sara

Abstract

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use meta-genomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Published
2019

16. Whole Genome Sequencing Analysis of Nontyphoidal Salmonella enterica of Chicken Meat and Human Origin Under Surveillance in Sri Lanka

Author

Tay, Moon Y F, Pathirage, Sujatha, Chandrasekaran, Lakshmi, Wickramasuriya, Uddami, Sadeepanie, Nirasha, Waidyarathna, Kaushalya D K, Liyanage, Liyanaralalage Dilini Chathurika, Seow, Kelyn L G, Hendriksen, Rene S., Takeuchi, Masami T, Schlundt, Jørgen, Tay, Moon Y F, Pathirage, Sujatha, Chandrasekaran, Lakshmi, Wickramasuriya, Uddami, Sadeepanie, Nirasha, Waidyarathna, Kaushalya D K, Liyanage, Liyanaralalage Dilini Chathurika, Seow, Kelyn L G, Hendriksen, Rene S., Takeuchi, Masami T, and Schlundt, Jørgen

Abstract

A total of 73 nontyphoidal Salmonella enterica isolates, 33 from raw chicken meat and 40 from routine clinical specimens, were collected between 2015 and 2017 from eight cities in Sri Lanka for a pilot study of whole genome sequencing for Salmonella surveillance. The isolates were characterized by conventional serotyping and whole genome sequencing. The raw sequenced data were assembled and analyzed to predict Salmonella serotypes, determine sequence type (ST) profiles of genome and plasmid, and identify plasmid replicon sequences and antimicrobial resistance (AMR) genes. The most common serovar isolated from chicken meat was Salmonella enterica serovar Agona of ST13 (n = 16), in contrast to Salmonella enterica serovar Enteritidis of ST11 (n = 21) in human. Salmonella enterica serovar Corvallis is the only serovar that was overlapping between human and chicken meat. The level of agreement between serotyping and serotype prediction results was 100%. Among the 33 chicken isolates, multidrug resistance (MDR) was observed in five isolates, including two Salmonella enterica serovar Kentucky ST314, which harbored six different classes of AMR determinants. Among the 40 human isolates, MDR was detected in two Salmonella enterica serovar Chester (ST2063) isolates containing five different antibiotic classes of AMR determinants. Out of 73 isolates, the only human Salmonella enterica serovar Typhimurium strain of ST36 was found to possess extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15, and it was positive for ESBL production. In summary, this study identified S. enterica serovars that were dominating in chicken meat and human and showed the genomic differences among the chicken meat and human strains. It should be noted that the limited number of isolates and sampling at a different time period means that thorough source attribution is not possible. To the best of our knowledge, this is the first report on the use of whole genome sequencing analysis of nontyphoidal

Published
2019

17. Prevalence and genomic analysis of ESBL-producing Escherichia coli in retail raw meats in Singapore.

Author

Guo, Siyao, Aung, Kyaw Thu, Leekitcharoenphon, Pimlapas, Tay, Moon Y F, Seow, Kelyn L G, Zhong, Yang, Ng, Lee Ching, Aarestrup, Frank Møller, and Schlundt, Joergen

Abstract

Objectives: To determine the prevalence and genetic characteristics of ESBL-producing Escherichia coli in retail raw meats from Singapore markets.Methods: A total of 634 raw meat (chicken, pork and beef) samples were collected from markets in Singapore during June 2017-October 2018. The samples were enriched overnight and then incubated on Brilliance™ ESBL Agar. Presumptive ESBL isolates were confirmed using the double-disc synergy test. Confirmed ESBL-producing E. coli were sent for WGS and bioinformatic analysis was performed.Results: The prevalence of ESBL-producing E. coli in chicken, pork and beef meats was 51.2% (109/213), 26.9% (58/216) and 7.3% (15/205), respectively. A total of 225 ESBL-producing E. coli were isolated from 184 samples. β-Lactam resistance genes were detected in all isolates. After β-lactam resistance genes, the most common antimicrobial resistance genes detected were aminoglycoside resistance genes (92.4%). One hundred and seventy-two (76.4%), 102 (45.3%) and 52 (23.1%) isolates carried blaCTX-M genes, blaTEM genes and blaSHV genes, respectively. blaCTX-M-55 (57/225, 25.3%) and blaCTX-M-65 (40/225, 17.8%) were the most frequent ESBL genes. Colistin resistance genes (including mcr-1, mcr-3 and mcr-5) were found in 15.6% of all isolates.Conclusions: This study indicates that ESBL-producing E. coli are widely found in retail raw meats, especially chicken, in Singapore. Occurrence of MDR (resistance to at least three classes of antimicrobial) and colistin resistance genes in retail raw meat suggests potential food safety and public health risks. [ABSTRACT FROM AUTHOR]

Published
2021
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18. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production

Author

Tay, Moon Y. F., primary, Smith, Kate, additional, Ng, Ivan H. W., additional, Chan, Kitti W. K., additional, Zhao, Yongqian, additional, Ooi, Eng Eong, additional, Lescar, Julien, additional, Luo, Dahai, additional, Jans, David A., additional, Forwood, Jade K., additional, and Vasudevan, Subhash G., additional

Published
2016
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19. A Crystal Structure of the Dengue Virus NS5 Protein Reveals a Novel Inter-domain Interface Essential for Protein Flexibility and Virus Replication

Author

Zhao, Yongqian, primary, Soh, Tingjin Sherryl, additional, Zheng, Jie, additional, Chan, Kitti Wing Ki, additional, Phoo, Wint Wint, additional, Lee, Chin Chin, additional, Tay, Moon Y. F., additional, Swaminathan, Kunchithapadam, additional, Cornvik, Tobias C., additional, Lim, Siew Pheng, additional, Shi, Pei-Yong, additional, Lescar, Julien, additional, Vasudevan, Subhash G., additional, and Luo, Dahai, additional

Published
2015
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24. Extended-spectrum β-lactamase-producing Proteus mirabilis with multidrug resistance isolated from raw chicken in Singapore: Genotypic and phenotypic analysis.

Author

Guo S, Aung KT, Tay MYF, Seow KLG, Ng LC, and Schlundt J

Subjects
Animals, Chickens microbiology, Disease Reservoirs microbiology, Genome, Bacterial, Genotype, Microbial Sensitivity Tests, Phenotype, Proteus Infections microbiology, Proteus mirabilis enzymology, Proteus mirabilis genetics, Singapore, Whole Genome Sequencing, beta-Lactamases genetics, Anti-Bacterial Agents pharmacology, Disease Reservoirs veterinary, Drug Resistance, Multiple, Bacterial, Proteus Infections veterinary, Proteus mirabilis drug effects
Abstract

Objectives: Proteus mirabilis is ubiquitous in soil and water. It is an important catheter-associated urinary tract pathogen and has reportedly been associated with antimicrobial-resistant infections. This study reports the draft genome of a multidrug-resistant P. mirabilis isolated from raw retail chicken meat in Singapore., Methods: The P. mirabilis strain was isolated on Brilliance TM ESBL Agar and was screened for antimicrobial susceptibility against 29 antimicrobial agents using a MicroScan® Neg MIC Panel Type 44. The double-disk synergy test (DDST) was used for confirmation of extended-spectrum β-lactamase (ESBL) production. Genomic DNA from the pure culture isolate was extracted and was sent for sequencing based on Illumina HiSeq 2500 technology. Further bioinformatics analysis was performed using online tools available at the Center for Genomic Epidemiology., Results: Species identification of the isolate was performed by KmerFinder. Antimicrobial susceptibility testing of the isolate showed multidrug resistance to broad-spectrum β-lactams, fluoroquinolones and aminoglycosides, among others. ESBL production was confirmed by the DDST. A total of 29 antimicrobial resistance genes were detected by ResFinder., Conclusion: To the best of our knowledge, this is the first report of the whole-genome sequence of a multidrug-resistant P. mirabilis producing an ESBL from raw chicken meat in Singapore. This indicates that raw meat in Singapore can be a reservoir for drug-resistant pathogens., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

Published
2019
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25. Whole-Genome Sequencing Analysis of Nontyphoidal Salmonella enterica of Chicken Meat and Human Origin Under Surveillance in Sri Lanka.

Author

Tay MYF, Pathirage S, Chandrasekaran L, Wickramasuriya U, Sadeepanie N, Waidyarathna KDK, Liyanage LDC, Seow KLG, Hendriksen RS, Takeuchi MT, and Schlundt J

Subjects
Animals, Chickens, Humans, Microbial Sensitivity Tests, Pilot Projects, Retrospective Studies, Salmonella enterica drug effects, Salmonella enteritidis isolation & purification, Serotyping, Sri Lanka, Whole Genome Sequencing, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial, Meat Products microbiology, Salmonella enterica classification, Salmonella enterica isolation & purification
Abstract

A total of 73 nontyphoidal Salmonella enterica isolates, 33 from raw chicken meat and 40 from routine clinical specimens, were collected between 2015 and 2017 from eight cities in Sri Lanka for a pilot study of whole-genome sequencing for Salmonella surveillance. The isolates were characterized by conventional serotyping and whole-genome sequencing. The raw sequenced data were assembled and analyzed to predict Salmonella serotypes, determine sequence type (ST) profiles of genome and plasmid, and identify plasmid replicon sequences and antimicrobial resistance (AMR) genes. The most common serovar isolated from chicken meat was Salmonella enterica serovar Agona of ST13 ( n  = 16), in contrast to Salmonella enterica serovar Enteritidis of ST11 ( n  = 21) in human. Salmonella enterica serovar Corvallis is the only serovar that was overlapping between human and chicken meat. The level of agreement between serotyping and serotype prediction results was 100%. Among the 33 chicken isolates, multidrug resistance (MDR) was observed in five isolates, including two Salmonella enterica serovar Kentucky ST314, which harbored six different classes of AMR determinants. Among the 40 human isolates, MDR was detected in two Salmonella enterica serovar Chester (ST2063) isolates containing five different antibiotic classes of AMR determinants. Out of 73 isolates, the only human Salmonella enterica serovar Typhimurium strain of ST36 was found to possess extended-spectrum beta-lactamase (ESBL) gene, bla CTX-M-15 , and it was positive for ESBL production. In summary, this study identified S . enterica serovars that were dominating in chicken meat and human and showed the genomic differences among the chicken meat and human strains. It should be noted that the limited number of isolates and sampling at a different time period means that thorough source attribution is not possible. To the best of our knowledge, this is the first report on the use of whole-genome sequencing analysis of nontyphoidal S . enterica isolated from chicken meat and human in Sri Lanka.

Published
2019
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26. The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

Author

Tay MY, Saw WG, Zhao Y, Chan KW, Singh D, Chong Y, Forwood JK, Ooi EE, Grüber G, Lescar J, Luo D, and Vasudevan SG

Subjects
Animals, Antiviral Agents chemistry, Binding Sites, Cell Line, Cell Line, Tumor, Cricetinae, DNA, Complementary metabolism, Enzyme-Linked Immunosorbent Assay, Humans, Lysine chemistry, Mutagenesis, Site-Directed, Mutation, Plasmids metabolism, Protein Binding, Protein Interaction Mapping, Protein Structure, Tertiary, RNA-Dependent RNA Polymerase, Scattering, Radiation, Dengue Virus physiology, RNA, Viral chemistry, Serine Endopeptidases chemistry, Viral Nonstructural Proteins chemistry, Virus Replication
Abstract

Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Published
2015
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27. Identification and molecular characterization of human antibody fragments specific for dengue NS5 protein.

Author

Zhao Y, Moreland NJ, Tay MY, Lee CC, Swaminathan K, and Vasudevan SG

Subjects
Amino Acid Sequence, Cross Reactions, Dengue immunology, Dengue Virus classification, Dengue Virus genetics, Dengue Virus immunology, Epitope Mapping, Humans, Immunoglobulin Fab Fragments analysis, Immunoglobulin Fab Fragments genetics, Immunoglobulin Fab Fragments immunology, Methyltransferases genetics, Methyltransferases immunology, Molecular Sequence Data, RNA-Dependent RNA Polymerase genetics, RNA-Dependent RNA Polymerase immunology, Sequence Alignment, Species Specificity, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins immunology, Virus Replication, Dengue virology, Dengue Virus enzymology, Methyltransferases chemistry, RNA-Dependent RNA Polymerase chemistry, Viral Nonstructural Proteins chemistry
Abstract

The multifunctional dengue nonstructural (NS) protein 5 from the four serotypes of dengue virus (DENV1-4) is essential for viral replication and harbors a methyl transferase (MTase) and a RNA-dependent RNA-polymerase domain (RdRp). There are limited comparative studies of NS5 from the four DENV serotypes and this is further hampered by a lack of cross-reactive NS5 antibodies. In this study, recombinant NS5 proteins were expressed, purified, enzymatically characterized, and used strategically as bait in biopanning experiments with a naïve human Fab phage-display library to identify serotype specific or cross-reactive Fab fragments. Using a combination of peptide competition ELISA and peptide phage display the epitopes of the cross-reactive Fabs were mapped to the first alpha helix of the MTase domain (5M1) and the priming loop of the RdRp domain (5R3). The epitope of a third, serotype-specific Fab (5M3) was mapped to aa19-30 of the DENV3 MTase domain. Together the recombinant proteins and specific antibodies will facilitate further mechanistic studies of the DENV replication complex., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Published
2014
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28. Monoclonal antibodies against dengue NS2B and NS3 proteins for the study of protein interactions in the flaviviral replication complex.

Author

Moreland NJ, Tay MY, Lim E, Rathore AP, Lim AP, Hanson BJ, and Vasudevan SG

Subjects
Animals, Antibodies, Monoclonal immunology, Antibodies, Viral immunology, Enzyme-Linked Immunosorbent Assay methods, Humans, Immunoglobulin G immunology, Immunoglobulin G isolation & purification, Immunohistochemistry methods, Immunoprecipitation methods, Mice, Protein Binding, RNA Helicases immunology, RNA Helicases metabolism, Reproducibility of Results, Sensitivity and Specificity, Serine Endopeptidases immunology, Serine Endopeptidases metabolism, Viral Nonstructural Proteins immunology, Antibodies, Monoclonal isolation & purification, Antibodies, Viral isolation & purification, Dengue Virus physiology, Protein Interaction Mapping, Viral Nonstructural Proteins metabolism
Abstract

The replication of dengue virus (DENV) RNA requires at least two viral non-structural (NS) proteins, NS3 and NS5. To facilitate the study of the DENV replication complex, human monoclonal IgG that are specific for NS proteins have been generated and characterised. The anti-NS3 IgG, 3F8, binds a conserved epitope (aa526-531) in the NS3 helicase domain, and cross-reacts with NS3 from all four DENV serotypes and the related yellow fever virus. The anti-NS2B IgG, 3F10, binds aa49-66 of NS2B (CF18), which forms part of the 47 aa hydrophilic cofactor region required for NS3 protease activity. The specificity of the IgG for their respective non-structural proteins has been demonstrated by immunofluorescence of cells infected with DENV and Western blotting. 3F8 is able to co-immunoprecipitate NS3 and NS5 from BHK-21 cells infected with DENV2, and 3F10 is able to detect an interaction between recombinant NS2B(CF18)NS3 full-length protein and the NS5 RNA-dependent RNA polymerase (RdRp) domain in an ELISA-based binding assay. The assay is specific and highly reproducible, with a clear binding curve seen when RdRp is incubated with increasing amounts of full-length NS3, but not the NS3 protease domain. The NS3 helicase domain competes with NS3 full-length for NS5 RdRp binding, with a K(d.) of 2.5μM. Since NS3 and NS5 are required for DENV replication, this fascile assay could be used to screen for non-nucleoside, allosteric inhibitors that disrupt the interaction between the two proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Published
2012
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