Development of an RT-RPA assay for La Crosse virus detection provides insights into age-dependent neuroinvasion in mice.
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| Názov: | Development of an RT-RPA assay for La Crosse virus detection provides insights into age-dependent neuroinvasion in mice. |
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| Autori: | Lumkong L; Rutgers Global Health Institute, Rutgers University, New Brunswick, NJ, USA.; Department of Medicine, Division of Allergy, Immunology, and Infectious Diseases, Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA., Alatrash R; Rutgers Global Health Institute, Rutgers University, New Brunswick, NJ, USA.; Department of Medicine, Division of Allergy, Immunology, and Infectious Diseases, Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA., Sridhar S; Rutgers Global Health Institute, Rutgers University, New Brunswick, NJ, USA.; Department of Medicine, Division of Allergy, Immunology, and Infectious Diseases, Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA., Tonto PB; Rutgers Global Health Institute, Rutgers University, New Brunswick, NJ, USA.; Department of Medicine, Division of Allergy, Immunology, and Infectious Diseases, Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA., Herrera BB; Rutgers Global Health Institute, Rutgers University, New Brunswick, NJ, USA. bherrera@globalhealth.rutgers.edu.; Department of Medicine, Division of Allergy, Immunology, and Infectious Diseases, Rutgers Robert Wood Johnson Medical School, Child Health Institute of New Jersey, Rutgers University, New Brunswick, NJ, USA. bherrera@globalhealth.rutgers.edu. |
| Zdroj: | Virology journal [Virol J] 2025 Apr 09; Vol. 22 (1), pp. 95. Date of Electronic Publication: 2025 Apr 09. |
| Spôsob vydávania: | Journal Article; Research Support, Non-U.S. Gov't |
| Jazyk: | English |
| Informácie o časopise: | Publisher: BioMed Central Country of Publication: England NLM ID: 101231645 Publication Model: Electronic Cited Medium: Internet ISSN: 1743-422X (Electronic) Linking ISSN: 1743422X NLM ISO Abbreviation: Virol J Subsets: MEDLINE |
| Imprint Name(s): | Original Publication: [London] : BioMed Central, 2004- |
| Výrazy zo slovníka MeSH: | La Crosse virus*/isolation & purification , La Crosse virus*/genetics , Encephalitis, California*/diagnosis , Encephalitis, California*/virology , Nucleic Acid Amplification Techniques*/methods, Animals ; Mice ; Sensitivity and Specificity ; RNA, Viral/genetics ; Age Factors ; Disease Models, Animal ; DNA Primers/genetics ; Female |
| Abstrakt: | Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: BBH is a co-founder of Mir Biosciences, Inc., a biotechnology company focused on T cell-based diagnostics and vaccines for infectious diseases, cancer, and autoimmunity. Background: La Crosse virus (LACV) is a mosquito-borne arbovirus responsible for pediatric encephalitis in North America, predominantly affecting children under 16 years. Early and accurate diagnosis is critical to reducing morbidity in this vulnerable population. However, existing molecular and serological methods are limited in sensitivity, specificity, and accessibility. Methods: To address these limitations, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for LACV detection. Primers targeting the divergent M segment of the LACV genome were designed and screened for optimal performance. The assay's analytical sensitivity was evaluated through serial dilutions of LACV RNA prior to reverse transcription, while specificity was assessed using reverse transcribed RNA from related or geographically relevant arboviruses. We further adapted the RT-RPA test into a lateral flow assay (LFA) format for potential point-of-care use. Additionally, we employed a murine model to explore the age-dependent dynamics of LACV neuroinvasion and clearance, with the virus detected using RT-RPA and reverse transcription quantitative polymerase change reaction (RT-qPCR). Results: Primer screening identified an optimal primer pair that amplified LACV cDNA within 20 min at 39 °C, with a limit of detection (LOD) of 100 copies. The assay demonstrated high specificity, with no amplification of related or other geographically relevant arboviruses. Integration of the RT-RPA test into an LFA format preserved the LOD and specificity, enabling visual detection via test strips. In the murine model, weanling mice exhibited LACV neuroinvasion as early as 4 days post-infection (dpi), with sustained detection between 5 and 7 dpi. In adult mice, neuroinvasion was first detected at 5 dpi, plateauing between 6 and 10 dpi, and cleared entirely by 20 dpi in surviving animals. Conclusions: This study establishes the RT-RPA assay as an efficient, specific, and sensitive diagnostic platform for LACV, with potential for adaptation into field-deployable LFA tests. Moreover, our findings provide valuable insights into the age-dependent dynamics of LACV neuroinvasion and clearance, informing future diagnostic and therapeutic strategies. (© 2025. The Author(s).) |
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| Grant Information: | IFH18-24 New Jersey Health Foundation |
| Contributed Indexing: | Keywords: Age-susceptible mouse model; La Crosse virus; Lateral flow assay; Neuroinvasion; Reverse transcription recombinase polymerase amplification |
| Substance Nomenclature: | 0 (RNA, Viral) 0 (DNA Primers) |
| Entry Date(s): | Date Created: 20250409 Date Completed: 20250410 Latest Revision: 20250424 |
| Update Code: | 20250425 |
| PubMed Central ID: | PMC11984161 |
| DOI: | 10.1186/s12985-025-02720-y |
| PMID: | 40205618 |
| Databáza: | MEDLINE |
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Nájsť tento článok vo Web of Science | Abstrakt: | Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: BBH is a co-founder of Mir Biosciences, Inc., a biotechnology company focused on T cell-based diagnostics and vaccines for infectious diseases, cancer, and autoimmunity.<br />Background: La Crosse virus (LACV) is a mosquito-borne arbovirus responsible for pediatric encephalitis in North America, predominantly affecting children under 16 years. Early and accurate diagnosis is critical to reducing morbidity in this vulnerable population. However, existing molecular and serological methods are limited in sensitivity, specificity, and accessibility.<br />Methods: To address these limitations, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for LACV detection. Primers targeting the divergent M segment of the LACV genome were designed and screened for optimal performance. The assay's analytical sensitivity was evaluated through serial dilutions of LACV RNA prior to reverse transcription, while specificity was assessed using reverse transcribed RNA from related or geographically relevant arboviruses. We further adapted the RT-RPA test into a lateral flow assay (LFA) format for potential point-of-care use. Additionally, we employed a murine model to explore the age-dependent dynamics of LACV neuroinvasion and clearance, with the virus detected using RT-RPA and reverse transcription quantitative polymerase change reaction (RT-qPCR).<br />Results: Primer screening identified an optimal primer pair that amplified LACV cDNA within 20 min at 39 °C, with a limit of detection (LOD) of 100 copies. The assay demonstrated high specificity, with no amplification of related or other geographically relevant arboviruses. Integration of the RT-RPA test into an LFA format preserved the LOD and specificity, enabling visual detection via test strips. In the murine model, weanling mice exhibited LACV neuroinvasion as early as 4 days post-infection (dpi), with sustained detection between 5 and 7 dpi. In adult mice, neuroinvasion was first detected at 5 dpi, plateauing between 6 and 10 dpi, and cleared entirely by 20 dpi in surviving animals.<br />Conclusions: This study establishes the RT-RPA assay as an efficient, specific, and sensitive diagnostic platform for LACV, with potential for adaptation into field-deployable LFA tests. Moreover, our findings provide valuable insights into the age-dependent dynamics of LACV neuroinvasion and clearance, informing future diagnostic and therapeutic strategies.<br /> (© 2025. The Author(s).) |
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| ISSN: | 1743-422X |
| DOI: | 10.1186/s12985-025-02720-y |