Development of a Rapid Surveillance System for Ross River Virus in Mosquitoes Through Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)
Alexandra Knox
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorGemma Zerna
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorCorresponding Author
Travis Beddoe
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorAlexandra Knox
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorGemma Zerna
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorCorresponding Author
Travis Beddoe
Department of Animal, Plant and Soil Sciences , Centre for AgriBioscience La Trobe University , Bundoora , 3082 , Victoria , Australia
Search for more papers by this authorAbstract
The global rise in arboviral diseases can be attributed to the ongoing effects of climate change. Ross River virus (RRV) is an illustrative example of such diseases, with case reports in Australia experiencing a significant surge since 2020. RRV is transmitted to susceptible species, such as horses and humans, through multiple mosquito vectors, namely Culex annulirostris, Aedes camptorhynchus, and more recently Ae. notoscriptus. This disease is not only endemic to Australia but has caused outbreaks in surrounding countries such as Fiji and Papua New Guinea. Currently, there are no therapeutic regimes or vaccinations available for RRV, leaving public health warning systems and advice relying upon disease prediction and surveillance. Commonly utilised methods, such as predictive modelling, are experiencing challenges resulting from an increased mosquito presence and extreme weather patterns, often yielding inaccurate advice. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) provided a promising solution to mitigate these challenges and is now considered the gold standard in many Australian states. However, this method must be performed in a laboratory setting and requires expensive machinery, thus rendering it inadequate for resource-poor or rural communities. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) serves as a simple and field-deployable substitute with comparable sensitivities and specificity to RT-qPCR, whilst possessing the ability to provide rapid results within 20 min. This paper describes a novel RRV RT-LAMP assay that can detect RRV in as little as one mosquito, with a limit of detection of 1 × 10−7 ng/µl (~620 copies/µl) and a clinical sensitivity of 84%. Through the addition of tetramethylammonium chloride (TMAC), our assay achieved a 100% specificity and was able to detect RRV RNA as early as 2 min in crude field samples. The simplistic sampling method coupled with our RRV RT-LAMP assay can provide an in-field and low-cost alternative to current routine surveillance techniques.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
All data are contained within this article or in the supporting information.
Supporting Information
Filename | Description |
---|---|
tbed1772438-sup-0001-f1.docxWord 2007 document , 206.4 KB | Supporting Information Figure S1. sequence pairwise alignment of closely related alphaviruses Barmah Forest virus (BFV—S1a), Semliki Forest virus (SFV—S1b), and Sindbis virus (SINV—1c) E2 gene, against the Ross River virus (RRV) E2 gene. RRV reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primers are highlighted throughout, outer primers (F3 and B3) are labelled in yellow, forward inner primers (F2 and F1c) are highlighted in blue, the backwards inner primers (B2 and B1c) are highlighted in green, and the backwards loop primer (LoopB) is highlighted in grey. Sequence consensuses are highlighted in red. Alignment was performed using Benchling online software (Benchling, San Fransico, United States) and edited in Microsoft Word (Microsoft Corporation, Washington, United States). Table S1. Sanger sequencing result example provided by AGRF (Melbourne, Victoria) from a mosquito pool spiked with the RRV synthetic positive control. Table S2. raw data results from the RRV reverse-transcription loop-mediated isothermal (RRV RT-LAMP) assay and the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) assay on collected mosquitoes. |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
References
- 1 KaY Yuen and Bielefeldt-Ohmann H., Ross River Virus Infection: A Cross-Disciplinary Review With a Veterinary Perspective, Pathogens. (2021) 10, no. 3, https://doi.org/10.3390/pathogens10030357, 357.
- 2 El-Hage C. M., Bamford N. J., Gilkerson J. R., and Lynch S. E., Ross River Virus Infection of Horses: Appraisal of Ecological and Clinical Consequences, Journal of Equine Veterinary Science. (2020) 93, https://doi.org/10.1016/j.jevs.2020.103143, 103143.
- 3 Yu W., Mengersen K., and Dale P., et al.Epidemiologic Patterns of Ross River Virus Disease in Queensland, Australia, 2001-2011, The American Journal of Tropical Medicine and Hygiene. (2014) 91, no. 1, 109–118, https://doi.org/10.4269/ajtmh.13-0455, 2-s2.0-84903891920.
- 4
Lyth A. and
Holbrook N. J., Assessing an Indirect Health Implication of a Changing Climate: Ross River Virus in a Temperate Island State, Climate Risk Management. (2015) 10, 77–94, https://doi.org/10.1016/j.crm.2015.06.004, 2-s2.0-84947484449.
10.1016/j.crm.2015.06.004 Google Scholar
- 5 Yu W., Dale P., Turner L., and Tong S., Projecting the Impact of Climate Change on the Transmission of Ross River Virus: Methodological Challenges and Research Needs, Epidemiology and Infection. (2014) 142, no. 10, 2013–2023, https://doi.org/10.1017/S0950268814000399, 2-s2.0-84906239090.
- 6 Kain M. P., Skinner E. B., van den Hurk A. F., McCallum H., and Mordecai E. A., Physiology and Ecology Combine to Determine Host and Vector Importance for Ross River Virus, Elife. (2021) 10, https://doi.org/10.7554/eLife.67018.
- 7 Qian W., Viennet E., Glass K., Harley D., and Hurst C., Prediction of Ross River Virus Incidence Using Mosquito Data in Three Cities of Queensland, Australia, Biology. (2023) 12, no. 11, https://doi.org/10.3390/biology12111429, 1429.
- 8 Claflin S. B. and Webb C. E., Ross River Virus: Many Vectors and Unusual Hosts Make for an Unpredictable Pathogen, PLoS Pathogens. (2015) 11, no. 9, https://doi.org/10.1371/journal.ppat.1005070, 2-s2.0-84943518231.
- 9 Tappe D., Pérez-Girón J. V., Gómez-Medina S., Günther S., Muñoz-Fontela C., and Schmidt-Chanasit J., Increased Proinflammatory Cytokine Levels in Prolonged Arthralgia in Ross River Virus Infection, Emerging Infectious Diseases. (2017) 23, no. 4, 702–704, https://doi.org/10.3201/eid2304.160466, 2-s2.0-85015768570.
- 10 Azuolas J. K., Wishart E., Bibby S., and Ainsworth C., Isolation of Ross River Virus from Mosquitoes and From Horses With Signs of Musculoskeletal Disease, Australian Veterinary Journal. (2003) 81, no. 6, 344–347, https://doi.org/10.1111/j.1751-0813.2003.tb11511.x, 2-s2.0-2342627982.
- 11 Barton A. J. and Bielefeldt-Ohmann H., Clinical Presentation, Progression, and Management of Five Cases of Ross River Virus Infection in Performance Horses Located in Southeast Queensland: A Longitudinal Case Series, Journal of Equine Veterinary Science. (2017) 51, 34–40, https://doi.org/10.1016/j.jevs.2016.12.010, 2-s2.0-85010669577.
- 12 Communicable Disease Network Australia, Ross River Virus Infection - Surveillance Case Definition, 2018, Canberra, Australia:.
- 13 Batovska J., Mee P. T., Lynch S. E., Sawbridge T. I., and Rodoni B. C., Sensitivity and Specificity of Metatranscriptomics as an Arbovirus Surveillance Tool, Scientific Reports. (2019) 9, no. 1, https://doi.org/10.1038/s41598-019-55741-3.
- 14 Koolhof I. S., Firestone S. M., and Bettiol S., et al.Optimising Predictive Modelling of Ross River Virus Using Meteorological Variables, PLoS Neglected Tropical Diseases. (2021) 15, no. 3, https://doi.org/10.1371/journal.pntd.0009252, e0009252.
- 15 Liu J., Hansen A., Cameron S., Williams C., Fricker S., and Bi P., Using Ecological Variables to Predict Ross River Virus Disease Incidence in South Australia, Transactions of the Royal Society of Tropical Medicine and Hygiene. (2021) 115, no. 9, 1045–1053, https://doi.org/10.1093/trstmh/traa201.
- 16 Walker L. J., Selvey L. A., Jardine A., Johansen C. A., and Lindsay M. D. A., Mosquito and Virus Surveillance as a Predictor of Human Ross River Virus Infection in South-West Western Australia: How Useful Is It?, The American Journal of Tropical Medicine and Hygiene. (2018) 99, no. 4, 1066–1073, https://doi.org/10.4269/ajtmh.18-0459, 2-s2.0-85054464565.
- 17 Murphy A. K., Clennon J. A., and Vazquez-Prokopec G., et al.Spatial and Temporal Patterns of Ross River Virus in South East Queensland, Australia: Identification of Hot Spots at the Rural-Urban Interface, BMC Infectious Diseases. (2020) 20, no. 1, https://doi.org/10.1186/s12879-020-05411-x.
- 18 Hime N. J., Wickens M., and Doggett S. L., et al.Weather Extremes Associated With Increased Ross River Virus and Barmah Forest Virus Notifications in NSW: Learnings for Public Health Response, Australian and New Zealand Journal of Public Health. (2022) 46, no. 6, 842–849, https://doi.org/10.1111/1753-6405.13283.
- 19 Trewin B. J., Kay B. H., Darbro J. M., and Hurst T. P., Increased Container-Breeding Mosquito Risk Owing to Drought-Induced Changes in Water Harvesting and Storage in Brisbane, Australia, International Health. (2013) 5, no. 4, 251–258, https://doi.org/10.1093/inthealth/iht023, 2-s2.0-84890028423.
- 20 Tall J., Ross River Virus Disease Activity Associated With Naturally Occurring Nontidal Flood Events in Australia: A Systematic Review, Journal of Medical Entomology. (2014) 51, no. 6, 1097–1108, https://doi.org/10.1603/ME14007, 2-s2.0-84910071772.
- 21 Nogrady B., Australian Floods Lead to Spread of Mosquito-Borne Disease, The Lancet Infectious Diseases. (2022) 22, no. 6, https://doi.org/10.1016/S1473-3099(22)00301-2, 771.
- 22 Carver S. and Koolhof I. S., Epidemic Host Community Contribution to Mosquito-Borne Disease Transmission: Ross River Virus, Epidemiology and Infection. (2017) 145, no. 4, 656–666, https://doi.org/10.1017/S0950268816002739, 2-s2.0-84997769569.
- 23 Lau C., Aubry M., and Musso D., et al.New Evidence for Endemic Circulation of Ross River Virus in the Pacific Islands and the Potential for Emergence, International Journal of Infectious Diseases. (2017) 57, 73–76, https://doi.org/10.1016/j.ijid.2017.01.041, 2-s2.0-85014082133.
- 24 Togami E., Gyawali N., and Ong O., et al.First Evidence of Concurrent Enzootic and Endemic Transmission of Ross River Virus in the Absence of Marsupial Reservoirs in Fiji, International Journal of Infectious Diseases. (2020) 96, 94–96, https://doi.org/10.1016/j.ijid.2020.02.048.
- 25 Aaskov J. G., Mataika J. U., and Lawrence G. W., et al.An Epidemic of Ross River Virus Infection in Fiji, 1979, The American Journal of Tropical Medicine and Hygiene. (1981) 30, no. 5, 1053–1059, https://doi.org/10.4269/ajtmh.1981.30.1053, 2-s2.0-0019614930.
- 26 Braddick M., O’Brien H. M., and Lim C. K., et al.An Integrated Public Health Response to an Outbreak of Murray Valley Encephalitis Virus Infection During the 2022-2023 Mosquito Season in Victoria, Frontiers in Public Health. (2023) 11, https://doi.org/10.3389/fpubh.2023.1256149, 1256149.
- 27 New South Wales Health, NSW Arbovirus Surveillance & Mosquito Monitoring 2020-2021, 2021, New South Wales, Australia:.
- 28
Knope K.,
Doggett S. L., and
Jansen C. C., et al.Arboviral Diseases and Malaria in Australia, 2014-15: Annual Report of the National Arbovirus and Malaria Advisory Committee, Communicable Diseases Intelligence. (2019) 43, https://doi.org/10.33321/CDI.2019.43.14.
10.33321/cdi.2019.43.14 Google Scholar
- 29 Knox A. and Beddoe T., Isothermal Nucleic Acid Amplification Technologies for the Detection of Equine Viral Pathogens, Animals. (2021) 11, no. 7, https://doi.org/10.3390/ani11072150, 2150.
- 30 Wilson I. G., Inhibition and Facilitation of Nucleic Acid Amplification, Applied and Environmental Microbiology. (1997) 63, no. 10, 3741–3751, https://doi.org/10.1128/aem.63.10.3741-3751.1997.
- 31 Notomi T., Okayama H., and Masubuchi H., et al.Loop-Mediated Isothermal Amplification of DNA, Nucleic Acids Research. (2000) 28, no. 12, e63–63, https://doi.org/10.1093/nar/28.12.e63.
- 32 Parida M., Posadas G., Inoue S., Hasebe F., and Morita K., Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of West Nile Virus, Journal of Clinical Microbiology. (2004) 42, no. 1, 257–263, https://doi.org/10.1128/JCM.42.1.257-263.2004, 2-s2.0-0345832024.
- 33 Kaneko H., Kawana T., Fukushima E., and Suzutani T., Tolerance of Loop-Mediated Isothermal Amplification to a Culture Medium and Biological Substances, Journal of Biochemical and Biophysical Methods. (2007) 70, no. 3, 499–501, https://doi.org/10.1016/j.jbbm.2006.08.008, 2-s2.0-33847621663.
- 34
Silva S.,
Paiva M., and
Guedes D., et al.Development and Validation of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) for Rapid Detection of ZIKV in Mosquito Samples from Brazil, Scientific Reports. (2019) 9, no. 1, https://doi.org/10.1038/s41598-019-40960-5, 2-s2.0-85063013099.
10.1038/s41598-019-40960-5 Google Scholar
- 35 Kurosaki Y., Magassouba N., and Bah H. A., et al.Deployment of a Reverse Transcription Loop-Mediated Isothermal Amplification Test for Ebola Virus Surveillance in Remote Areas in Guinea, The Journal of Infectious Diseases. (2016) 214, no. suppl 3, S229–33, https://doi.org/10.1093/infdis/jiw255, 2-s2.0-84990999100.
- 36 Wheeler S. S., Ball C. S., Langevin S. A., Fang Y., Coffey L. L., and Meagher R. J., Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification, PLoS One. (2016) 11, no. 1, https://doi.org/10.1371/journal.pone.0147962, 2-s2.0-84959212203, e0147962.
- 37 Vrati S., Faragher S. G., Weir R. C., and Dalgarno L., Ross River Virus Mutant With a Deletion in the E2 Gene: Properties of the Virion, Virus-Specific Macromolecule Synthesis, and Attenuation of Virulence for Mice, Virology. (1986) 151, no. 2, 222–232, https://doi.org/10.1016/0042-6822(86)90044-9, 2-s2.0-0022544939.
- 38 Bath C., Scott M., and Sharma P. M., et al.Further Development of a Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Foot-and-Mouth Disease Virus and Validation in the Field With use of an Internal Positive Control, Transboundary and Emerging Diseases. (2020) 67, no. 6, 2494–2506, https://doi.org/10.1111/tbed.13589.
- 39 Lamb L. E., Bartolone S. N., and Tree M. O., et al.Rapid Detection of Zika Virus in Urine Samples and Infected Mosquitos by Reverse Transcription-Loop-Mediated Isothermal Amplification, Scientific Reports. (2018) 8, no. 1, 1–9, https://doi.org/10.1038/s41598-018-22102-5, 2-s2.0-85042946922.
- 40 Bhadra S., Riedel T. E., Saldaña M. A., Hegde S., Pederson N., and Hughes G. L., Ellington A Direct Nucleic Acid Analysis of Mosquitoes for High Fidelity Species Identification and Detection of Wolbachia Using a Cellphone, PLoS Neglected Tropical Diseases. (2018) 12, no. 8, https://doi.org/10.1371/journal.pntd.0006671, 2-s2.0-85059289136, e0006671.
- 41 Hall R. A., Prow N. A., and Pyke A. T., L. Dongyou, Ross River Virus, Molecular Detection of Human Viral Pathogens, 2010, Taylor & Francis, Boca Raton, FL, U.S.A, 349–359.
- 42 Pankhong P., Siriprasertkul W., and Patpoparn S., et al.Molecular Serotyping Of Dengue Viruses In Field-Caught Aedes Mosquitos By In-House RNA Extraction/RT-PCR Reagent Kits, 23, 2002.
- 43 Jansen C., Shivas M., and May F., et al.Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia, Emerging Infectious Diseases. (2019) 25, no. 12, 2243–2252, https://doi.org/10.3201/eid2512.181810.
- 44
Wenjun Liu,
Kizu J., and
Le Grand L., et al.Genome Sequences of Three Ross River Virus Isolates Obtained From the Australian Defence Force, Microbiology Resource Announcements. (2019) 8, no. 14, 19, https://doi.org/10.1128/mra.00064-19, 2-s2.0-85066337184.
10.1128/mra.00064-19 Google Scholar
- 45 Aaskov J., Jones A., Choi W., Lowry K., and Stewart E., Lineage Replacement Accompanying Duplication and Rapid Fixation of an RNA Element in the nsP3 Gene in a Species of Alphavirus, Virology. (2011) 410, no. 2, 353–359, https://doi.org/10.1016/j.virol.2010.11.025, 2-s2.0-78751701774.
- 46 Parida M., Sannarangaiah S., Dash P. K., Rao P. V. L., and Morita K., Loop Mediated Isothermal Amplification (LAMP): A New Generation of Innovative Gene Amplification Technique; Perspectives in Clinical Diagnosis of Infectious Diseases, Reviews in Medical Virology. (2008) 18, no. 6, 407–421, https://doi.org/10.1002/rmv.593, 2-s2.0-57049113703.
- 47 Soroka M., Wasowicz B., and Rymaszewska A., Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?, Cells. (2021) 10, no. 8, https://doi.org/10.3390/cells10081931, 1931.
- 48 Ku J., Chauhan K., Hwang S.-H., Jeong Y.-J., and Kim D.-E., Enhanced Specificity in Loop-Mediated Isothermal Amplification With Poly(ethylene Glycol)-Engrafted Graphene Oxide for Detection of Viral Genes, Biosensors. (2022) 12, no. 8, https://doi.org/10.3390/bios12080661, 661.
- 49 Jang M. J. and Kim S., Inhibition of Non-Specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride, Biochip Journal. (2022) 16, no. 3, 326–333, https://doi.org/10.1007/s13206-022-00070-3.
- 50 Gao X., Sun B., and Guan Y., Pullulan Reduces the Non-Specific Amplification of Loop-Mediated Isothermal Amplification (LAMP), Analytical and Bioanalytical Chemistry. (2019) 411, no. 6, 1211–1218, https://doi.org/10.1007/s00216-018-1552-2, 2-s2.0-85059667919.
- 51 Kabeláč M. and Hobza P., Hydration and Stability of Nucleic Acid Bases and Base Pairs, Physical Chemistry Chemical Physics. (2007) 9, no. 8, 903–917, https://doi.org/10.1039/B614420A, 2-s2.0-84962376447.
- 52 MelchiorW. B.Jr. and Von Hippel P. H., Alteration of the Relative Stability of dA·dT and dG·dC Base Pairs in DNA, Proceedings of the National Academy of Sciences of the United States of America. (1973) 70, no. 2, 298–302, https://doi.org/10.1073/pnas.70.2.298, 2-s2.0-0015580506.
- 53 Yakovchuk P., Protozanova E., and Frank-Kamenetskii M. D., Base-Stacking and Base-Pairing Contributions Into Thermal Stability of the DNA Double Helix, Nucleic Acids Research. (2006) 34, no. 2, 564–574, https://doi.org/10.1093/nar/gkj454, 2-s2.0-32644434270.
- 54 Shapiro J. T., Stannard B. S., and Felsenfeld G., Binding of Small Cations to Deoxyribonucleic Acid. Nucleotide Specificity, Biochemistry. (1969) 8, no. 8, 3233–3241, https://doi.org/10.1021/bi00836a015, 2-s2.0-0014558176.
- 55 Abu Almakarem A. S., Heilman K. L., Conger H. L., Shtarkman Y. M., and Rogers S. O., Extraction of DNA From Plant and Fungus Tissues in Situ, BMC Research Notes. (2012) 5, no. 1, 1–11, https://doi.org/10.1186/1756-0500-5-266, 2-s2.0-84861803958.
- 56 Papadakis G., Pantazis A. K., and Fikas N., et al.Portable Real-Time Colorimetric LAMP-Device for Rapid Quantitative Detection of Nucleic Acids in Crude Samples, Scientific Reports. (2022) 12, no. 1, https://doi.org/10.1038/s41598-022-06632-7, 3775.
- 57 Silva S., Magalhães J., and Matthews Q., et al.Development and Field Validation of a Reverse Transcription Loop-Mediated Isothermal Amplification Assay (RT-LAMP) for the Rapid Detection of Chikungunya Virus in Patient and Mosquito Samples, Clinical Microbiology and Infection. (2024) 30, no. 6, 810–815, https://doi.org/10.1016/j.cmi.2024.03.004.