Rapidly quantification of intact infectious H1N1 virus using ICA-qPCR and PMA-qPCR

Abstract

The increase in emerging and reemerging infectious diseases has underscored the need for the prompt monitoring of intact infectious viruses and the quick assessment of their infectivity. However, molecular techniques cannot distinguish between intact infectious and noninfectious viruses. Here, two distinct methodologies have been developed for the expeditious and dependable quantification of intact infectious H1N1 virus, and several experiments have been conducted to substantiate their efficacy. One is an integrated cell absorption quantitative polymerase chain reaction (qPCR) method (ICA-qPCR), and the other is a combined propidium monoazide qPCR method (PMA-qPCR). The quantification limit is 100 cell culture infective dose 50 % (CCID₅₀)/mL in ICA-qPCR following a 1.5-hour cell absorption or 126 CCID₅₀/mL after a 15-minute incubation. For PMA-qPCR, the limit was 2,512 CCID₅₀/mL. The number of genome copies quantified by the ICA-qPCR and PMA-qPCR methods was strongly correlated with the infectious titer determined by the CCID₅₀ assay, thereby enabling the estimation of virus infectivity. The ICA-qPCR and PMA-qPCR methods are both suitable for the identification and quantification of intact infectious H1N1 virus in inactivated samples, wastewater, and biological materials. In conclusion, the ICA-qPCR and PMA-qPCR methods have distinct advantages and disadvantages, and can be used to quantify intact infectious viruses rapidly. These methodologies can facilitate the identification of the presence of intact infectious viruses in wastewater or on pathogen-related physical surfaces in high-level biosafety laboratories and medical facilities. Furthermore, these methodologies can also be utilized to detect other highly pathogenic pathogens.