Genomic surveillance of avian influenza viruses in Lao PDR
Collaboration
• National Animal Health Laboratory (NAHL), Ministry of Agriculture and Forestry, Lao PDR.
Funding
• World Health Organization.
Objectives
The objective is to support NAHL by providing genomic information on avian influenza viruses (AIVs) detected within the framework of their national surveillance network.
Background
Avian Influenza Viruses (AIVs), particularly the H5N1 subtype, are known to cause severe respiratory illnesses, pneumonia, and fatalities in humans. First identified in 1996 in China and subsequently in humans in Hong Kong in 1997, H5N1 has resulted in approximately 900 reported human cases worldwide since 2003, with a case fatality rate of 50% (1). Despite only two officially reported cases of H5N1 in Lao PDR (in 2014 and 2020), the true disease burden is likely much higher due to the country’s limited surveillance capabilities for respiratory infections. In fact, thousands of H5N1 strains have been detected regularly by the National Animal Health Laboratory (NAHL) through active surveillance of poultry (2).
Given the ongoing threat posed by AIVs worldwide, particularly in light of recent human H5N1 cases reported in Cambodia and Vietnam, IPL is assisting NAHL for the sequencing and the genome analysis of specific strains collected through national surveillance efforts.
Methodology
Samples.
Samples were collected from ducks, chickens, or geese (oropharyngeal swabs and tissues) or from the environment (water or fecal swabs) by NAHL, in 2023 and 2024, in the framework of their national surveillance in Lao PDR.
Avian influenza virus detection and subtype identification.
Virus nucleic acids are extracted from collected swabs by Nucleo Spin RNA virus kit (Macherey-Nagel) according to the manufacturer’s instructions. Extracted RNAs are then screened for the presence of Avian Influenza virus (AIVs) by RT-qPCR assay targeting the Matrix (M) gene.
Samples testing positive for AIVs are subsequently subjected to additional molecular assays, including conventional and/or real-time RT-PCR, to identify AIV subtypes in accordance with FAO recommendations.
NGS sequencing.
Whole genomes were amplified using custom Uni12, and Uni13 integrated barcoded primers and Superscript II One-step RT–PCR with Platinum Taq High Fidelity kit (Thermo Fisher, Massachusetts, USA), as previously described (3). Sequencing libraries were prepared using the ligation sequencing kit SQK-LSK109 (Oxford Nanopore Technologies, Oxford, UK) and sequenced on the MinION Mk1B platform (Oxford Nanopore Technologies, Oxford, UK). Sequencing reads were de-multiplexed, quality-trimmed, and filtered using Porechop software (https://github.com/rrwick/ Porechop). Consensus sequences were generated using IRMA v1.02 using default settings.
Phylogenetic analyses.
A selection of sequences of H5N1 and H9N2 viruses, with a special focus on viruses from Lao PDR, Vietnam, and Cambodia, were obtained from GISAID. Only sequences with full-length HA genes were selected. A selection of sequences representative of diverse clades circulating worldwide were also downloaded. Sequences were aligned using MAFFT. Each alignment was checked manually. Maximum likelihood (ML) trees were constructed using IQ Tree web server (http://iqtree.cibiv. univie.ac.at) with automatic substitution model selection using ModelFinder and then edited with FigTree v1.4.4 software (http://tree.bio.ed.ac.uk).
Results
Subtype identification.
The results of the serotype identification have been shared with NAHL and will be reported in a future publication.
NGS sequencing results.
Based on the Ct values, 28 samples were selected for genomic sequencing using NGS. Genomic sequences were successfully generated for 22 samples with Ct values below 30. Among these 22 samples, full-length genomes (8 segments) were obtained for 10 strains.
Molecular and phylogenetic analyses.
Comprehensive molecular and phylogenetic analyses, including the identification of virulence markers, have been provided to NAHL and local authorities and will be reported in a future publication.
Discussion
Avian Influenza Viruses (AIVs) were detected in all samples processed at IPL, with Ct values ranging from 20 to 42, reflecting a wide range of viral loads. Hemagglutinin (HA) subtypes were successfully identified in all samples, while the neuraminidase (NA) subtypes could not be determined in four samples, likely due to limitations of the molecular assays as these samples have high Ct values, reflecting a low viral load.
Complete full-length genomes, encompassing all eight segments were obtained for three strains. The ability to sequence full-length genomes from original samples using multi-segment RT-PCR with a universal set of primers appears to be limited to samples with high viral loads, specifically those with Ct values below 30, underscoring the need to explore other techniques to amplify or enrich the viral RNA to allow for more efficient full-genome sequencing, especially in samples with higher Ct values. One potential approach is to utilize conventional PCR targeting specific influenza subtypes and subsequently sequence the resulting amplicons using the Sanger method, which can provide further insights into known critical mutation.
Conclusion & perspectives
Full-length genome sequences of AIVs could be reasonably efficiently generated by NGS for samples with Ct values below 30. For samples with higher Ct values, an additional amplification step could be performed by inoculating specified pathogen-free (SPF) embryonated eggs in a Biosafety Level 3 (BSL-3) laboratory. The allantoic fluid from dead eggs is then harvested, and the amplified isolates, with a higher viral load, can be sequenced by NGS to generate whole-genome sequences. This capacity will be set up at IPL in the framework of a project initiated in October 2024 on tracking AIVs in live bird markets of Laos, funded by the International Pathogen Surveillance Network (IPSN).
The recent re-emergence of human H5N1 cases in Cambodia and Vietnam underscores the importance of enhancing AIV sequencing capabilities. Strengthening these capabilities will better equip Lao health authorities and international stakeholders to respond more effectively to AIV outbreaks. Through this initiative, IPL aims to bolster intersectoral collaboration with the Ministry of Agriculture and Forestry and the Ministry of Health, thereby enhancing both animal and public health efforts in Laos.
References
1. Cumulative number of confirmed human cases for avian influenza A(H5N1) reported to WHO, 2003-2024, 26 February 2024 [Internet]. Available from: https://www. who.int/publications/m/item/cumulative-number-ofconfirmed- human-cases-for-avian-influenza-a(h5n1)- reported-to-who–2003-2024-26-february-2024.
2. Horwood PF, Horm SV, Suttie A, Thet S, Y P, Rith S, et al. Co-circulation of Influenza A H5, H7, and H9 Viruses and Co-infected Poultry in Live Bird Markets, Cambodia. Emerg Infect Dis. 2018 Feb;24(2):352–5.
3. Ratcliff JD, Merritt B, Gooden H, Siegers JY, Srikanth A, Yann S, et al. Improved Resolution of Highly Pathogenic Avian Influenza Virus Haemagglutinin Cleavage Site Using Oxford Nanopore R10 Sequencing Chemistry [Internet]. bioRxiv; 2023 [cited 2024 Mar 20]. p. 2023.09.30.560331. Available from: https://www. biorxiv.org/content/10.1101/2023.09.30.560331v1.
Publications in 2024
1. Viral etiology of measles-like rash in Guinean children during the COVID epidemic in 2022. Anguinze RS, Touré A, Cissé F, Grayo S, Troupin C, Tordo N, Kouamou E, Roques P. J Med Virol. 2024 Feb;96(2):e29437. doi: 10.1002/jmv.29437.
2. Geographic Disparities in Domestic Pig Population Exposure to Ebola Viruses, Guinea, 2017-2019. Grayo S, Camara A, Doukouré B, Ellis I, Troupin C, Fischer K, Vanhomwegen J, White M, Groschup MH, Diederich S, Tordo N. Emerg Infect Dis. 2024 Apr;30(4):681-690. doi: 10.3201/eid3004.231034.
3. Seroprevalence and Phylogenetic Characterization of Hepatitis E Virus (Paslahepevirus balayani) in Guinean Pig Population. Doukouré B, Le Pennec Y, Troupin C, Grayo S, Eiden M, Groschup MH, Tordo N, Roques P. Vector Borne Zoonotic Dis. 2024 Aug;24(8):540-545. doi: 10.1089/ vbz.2023.0104. Epub 2024 Apr 23.
4. Spatial analysis of dengue fever incidence and serotype distribution in Vientiane Capital, Laos: A multi-year study. Phanhkongsy S, Suwannatrai A, Thinkhamrop K, Somlor S, Sorsavanh T, Tavinyan V, Sentian V, Khamphilavong S, Samountry B, Phanthanawiboon S. Acta Trop. 2024 Aug;256:107229. doi: 10.1016/j.actatropica.2024.107229. Epub 2024 May 18.
5. Viromes of arthropod parasites and their hosts: The case of bats and bat ectoparasites. Tendu A, Li R, Kane Y, Nalikka B, Omondi V, Bienes KM, Berthet N, Wong G. Acta Trop. 2024 Nov;259:107375. doi: 10.1016/j.actatropica.2024.107375. Epub 2024 Sep 1.
Congress
Oral presentations:
1. Troupin C. Surveillance of arboviruses and their vectors in Vientiane capital (and in Laos), One Urban Health workshop in Southeast Asia, 30-31 January 2024, Singapore.
2. Troupin C. Dengue surveillance at IPL (in the framework of Arboshield Plus project), Arboshield Plus project closing meeting, 26-27 March 2024, Vientiane.
Posters:
1. Vanhnollat C. “Joint collaboration on capacity building at Institut Pasteur in Laos”, Global Health Security Conference 2024, Sydney, Australia, 18-21 June 2024. Congress attendance: 1. Troupin C. Arbovirus Summit, 22-23 April 2024, Bali, Indonesia, invited panelist on “Need and Opportunities for Integrated Arbovirus Surveillance”.
2. Troupin C. Regional Technical Consultation on Reassortant Avian Influenza in Southeast Asia, 23-25 July 2025, Bangkok, Thailand, online attendance.
Teaching activities
(lectures given in universities, during seminars, etc.)
1. Wong G. Novel pathogen discovery in the Greater Mekong Subregion. 1st A*IDL-Institut Pasteur Joint Symposium. Paris, France. October 2023.
2. Wong G. Novel pathogen discovery in Southeast Asia. Institut Pasteur de Lille. Lille, France. December 2023.
3. Wong G. Viral diversity in wild and urban rodents of Yunnan Province, China. Pasteur English Immersion Club (PEIC). Online. May 2024.
4. Troupin C. Principles of virological diagnosis, Arboviruses surveillance activities at IPL, Diagnosis of COVID-19 at IPL. Arboshield Plus intern, IPL, Vientiane, Laos. March-April 2024.
Training activities
Training given by the team:
1. “Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024.
2. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024.
3. Provided an internship for a PhD student from the University of Hokkaido on using IFA (immunofluorescence assay) to detect bat- and shrewborne hantaviruses, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 02 April to 14 May 2024.
Training undertaken by the team:
Troupin C.
1. “Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024.
Vanhnollat C.
1. “Adeno-Associated Viral Vector in Gene Therapy and Genome Editing”, Mahidol University, Thailand. 23-25 April 2024.
2. “Sample inactivation/extraction, protein expression and ELISA”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 01-12 July 2024.
3. “Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024. 4. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024.
Somlor S.
1. “Training in Cell Culture Techniques”. Institut Pasteur du Cambodge, Phnom Penh, Cambodia. 11-24 February 2024. 2. “Training on maintenance BSL3’’. Institut Pasteur du Cambodge, Phnom Penh, Cambodia. 20-31 May 2024. 3. “Sample inactivation/extraction, protein expression and ELISA”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 01-12 July 2024. 4. “Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024. 5. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024.
Chindavong T.
1. “Lab Design and Basic Laboratory Quality Assurance and Quality Management System”, Regional Medical Sciences Center 8, Udon Thani, Thailand. 27-31 May 2024.
2. “Arboviral Genomic Surveillance In A Climate Evolving World”, Singapore. 03-09 July 2024.
3. “Specimen Referral System and Laboratory Quality Assurance and Quality Management System”, Vientiane Capital, Lao PDR. 19-23 August 2024.
4. “Certified Biosafety Officer”, Vientiane Capital, Lao PDR. 10-13 September 2024.
5. “Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024.
6. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024.
Bounmany P.
1. “Training in Cell Culture Techniques”. Institut Pasteur du Cambodge, Phnom Penh, Cambodia. 11-24 February 2024. 2. “Sample inactivation/extraction, protein expression and ELISA”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 01-12 July 2024. 3. “National Inventory of Dangerous Pathogens”, Vientiane Capital, Lao PDR. 26-27 September 2024.
Khamsuvat P.
1. Certified Biosafety Officer”, Vientiane Capital, Lao PDR. 10-13 September 2024.
Viengphouthong S.
1. “National Inventory of Dangerous Pathogens”, Vientiane Capital, Lao PDR. 26-27 September 2024.
Intavong K.
1. “Discussion session to learn lessons, control and respond to the dengue epidemic in 2023 and plan for year 2024”, Vang Vieng, Lao PDR. 20-21 December 2023.
2. “Biosafety and discussion on biologically selected agents and toxins (BSATs)”, Vientiane Capital, Lao PDR. 15-16 May 2024.
3. Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024.
4. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024.
Vachouaxiong L.
1. Basic Bioinformatics”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 18-27 September 2024.
2. “In silico Identification of Proteins”, Institut Pasteur du Laos, Vientiane Capital, Lao PDR. 30 September-04 October 2024