Hepatitis A virus subgenotyping based on RT-qPCR assays

Area of Science:

• Food Microbiology
• Virology
• Molecular Diagnostics

Background:

• Hepatitis A virus (HAV) is a major foodborne pathogen causing viral hepatitis globally.
• HAV infection incidence varies by geography, sanitation, and hygiene.
• Traditional phylogenetic analysis is challenging for low-level HAV detection in food.

Purpose of the Study:

• To develop and validate subtype-specific real-time reverse-transcription PCR (RT-qPCR) assays for hepatitis A virus (HAV).
• To enable accurate subtyping of HAV in various sample types, including food matrices.
• To improve the detection of HAV co-infections and recombinant strains.

Main Methods:

• Development of six subtype-specific RT-qPCR assays for human HAV.
• Evaluation of assay sensitivity, specificity, and cross-reactivity using reference isolates and RNA transcripts.
• Validation of assays against sequencing analysis for clinical samples.

Main Results:

• Assay limits of detection ranged from 50 to 5000 genome copies/assay for different HAV subtypes.
• High specificity was observed with no significant cross-reactivity.
• Subtyping results from clinical samples showed high concordance with sequencing (34/35).
• Potential co-infections (5 cases) and a recombinant event (1 case) were identified.

Conclusions:

• The developed RT-qPCR assays are effective for tracing HAV in low-level contaminated food samples.
• These assays facilitate accurate co-infection identification in human samples.
• The findings enhance HAV surveillance and food safety protocols.