Discrimination of infectious hepatitis A viruses by propidium monoazide real-time RT-PCR

Area of Science:

• Virology
• Molecular Biology
• Public Health

Background:

• Distinguishing infectious from inactivated viruses is crucial for accurate viral quantification, particularly for enteric viruses.
• Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is a common method, but it cannot differentiate between infectious and non-infectious viral particles.
• Hepatitis A virus (HAV) quantification is important for assessing infection risk and treatment efficacy.

Purpose of the Study:

• To evaluate propidium monoazide (PMA) and RNase pretreatments for detecting and quantifying infectious hepatitis A virus (HAV).
• To determine the effectiveness of PMA treatment in differentiating infectious from inactivated HAV.
• To establish a reliable method for quantifying infectious HAV in media suspensions.

Main Methods:

• Hepatitis A virus (HAV) samples were subjected to propidium monoazide (PMA) or RNase pretreatment.
• Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify viral titers.
• Comparison of PMA and RNase effectiveness in reducing titers of thermally inactivated HAV.

Main Results:

• PMA treatment significantly reduced the titers of thermally inactivated HAV by over 2.4 log(10) units, outperforming RNase treatment.
• Combining 50 μM of PMA with RT-qPCR allowed for the selective quantification of infectious HAV in media suspensions.
• PMA pretreatment demonstrated high efficacy in differentiating infectious from non-infectious viral particles.

Conclusions:

• Propidium monoazide (PMA) pretreatment is a highly effective method for discriminating between infectious and inactivated hepatitis A virus (HAV).
• The combination of PMA and RT-qPCR provides a selective and accurate approach for quantifying infectious HAV.
• PMA treatment prior to RT-qPCR detection is a promising alternative for assessing HAV infectivity in various sample types.