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Published on: December 3, 2011
Propidium Monoazide Coupled with PCR Predicts Infectivity of Enteric Viruses in Swine Manure and Biofertilized Soil
Gislaine Fongaro1, Marta Hernández2,3, María Cruz García-González2
1Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, UFSC, Florianópolis, Brazil.
Abstract:
The use of propidium monoazide (PMA) coupled with real-time PCR (RT-qPCR or qPCR for RNA or DNA viruses, respectively) was assessed to discriminate infectious enteric viruses in swine raw manure, swine effluent from anaerobic biodigester (AB) and biofertilized soils. Those samples were spiked either with infectious and heat-inactivated human adenovirus-2 (HAdV-2) or mengovirus (vMC0), and PMA-qPCR/RT-qPCR allowed discriminating inactivated viruses from the infective particles, with significant reductions (>99.9%). Then, the procedure was further assayed to evaluate the presence and stability of two non-cultivable viruses (porcine adenovirus and rotavirus A) in natural samples (swine raw manure, swine effluent from AB and biofertilized soils); it demonstrated viral inactivation during the storage period at 23 °C. As a result, the combination of PMA coupled to real-time PCR can be a promising alternative for prediction of viral infectivity in comparison to more labour-intensive and costly techniques such as animal or tissue-culture infectivity methods, and for those viruses that do not have currently available cell culture techniques.
Insights
Propidium monoazide (PMA) combined with real-time PCR effectively distinguishes infectious enteric viruses from inactivated ones in environmental samples. This method offers a reliable alternative for assessing viral infectivity in swine manure, effluent, and soils.
Area of Science:
- Environmental microbiology
- Virology
- Molecular biology
Background:
- Assessing viral infectivity in environmental matrices like swine manure, effluent, and soils is crucial for public health and agriculture.
- Traditional methods for determining viral infectivity, such as animal or cell culture assays, are often labor-intensive, costly, and not feasible for non-cultivable viruses.
- Developing rapid and accurate methods to differentiate between infectious and non-infectious viral particles is essential for risk assessment.
Purpose of the Study:
- To evaluate the efficacy of propidium monoazide (PMA) coupled with real-time PCR (qPCR/RT-qPCR) in discriminating infectious enteric viruses from inactivated ones.
- To assess the presence and stability of non-cultivable viruses (porcine adenovirus, rotavirus A) in environmental samples using the PMA-qPCR/RT-qPCR method.
- To determine if PMA-qPCR/RT-qPCR can serve as a viable alternative to traditional infectivity assays for various viruses, including those without established cell culture methods.
Main Methods:
- Samples of swine raw manure, anaerobic biodigester effluent, and biofertilized soils were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) or mengovirus (vMC0).
- Propidium monoazide (PMA) treatment followed by quantitative PCR (qPCR for DNA viruses) or reverse transcription qPCR (RT-qPCR for RNA viruses) was employed to differentiate between live and dead viral particles.
- The developed PMA-qPCR/RT-qPCR procedure was applied to natural samples to evaluate the presence and stability of non-cultivable porcine adenovirus and rotavirus A under storage conditions at 23 °C.
Main Results:
- PMA-qPCR/RT-qPCR significantly reduced the detection of inactivated viruses by over 99.9%, effectively discriminating them from infectious particles.
- The method demonstrated viral inactivation during the storage period at 23 °C for non-cultivable porcine adenovirus and rotavirus A in natural environmental samples.
- The study confirmed the presence and stability of these viruses in swine manure, effluent, and biofertilized soils, with inactivation observed over time.
Conclusions:
- The combination of propidium monoazide (PMA) with real-time PCR (qPCR/RT-qPCR) is a promising and effective tool for predicting viral infectivity in environmental samples.
- This method provides a significant advancement over traditional, more laborious, and expensive infectivity assays.
- PMA-qPCR/RT-qPCR offers a valuable alternative for the detection and quantification of infectious enteric viruses, particularly for non-cultivable viruses, in various environmental matrices.
