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

• Published in: Biosafety and health Vol. 6; no. 6; pp. 327 - 336
• Main Authors: Liang, Chudan, Wang, Zequn, Fan, Linjin, Wang, Yulong, Zhou, Yuandong, Yang, Xiaofeng, Lin, Jingyan, Ye, Pengfei, Shi, Wendi, Huang, Hongxin, Yan, Huijun, Liu, Linna, Qian, Jun
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2024; Elsevier
• Subjects: Inactivation; Intact infectious virus; Integrated cell absorption quantitative polymerase chain reaction (ICA-qPCR); Original Research; Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR); Rapid quantification; Intact infectious virus; Integrated cell absorption quantitative polymerase chain reaction (ICA-qPCR); Rapid quantification; Inactivation; Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR)
• ISSN: 2590-0536; 2096-6962; 2590-0536
• DOI: 10.1016/j.bsheal.2024.11.004

•Scientific question: The increase in emerging and reemerging infectious diseases has underscored the necessity for prompt monitoring of intact infectious viruses and assessment of their infectivity. There is an urgent demand to determine whether rapid quantification methods can accurately indicate intact viral infectivity in a shorter time for practical applications.•Evidence before this study: Prior research has effectively used combined cell absorption or cell culture processes and molecular assays to detect infectious viruses before the cytopathic effect (CPE) is evident. The use of propidium monoazide (PMA) has been effectively demonstrated for distinguishing between active and inactive bacteria and other species.•New findings: Two optimized methodologies were developed: an integrated cell absorption (ICA) quantitative polymerase chain reaction (qPCR) method (ICA-qPCR) and a combined PMA qPCR method (PMA-qPCR). Both methods demonstrated to be useful and adaptable in the rapid quantification of intact infectious virus, achieving quantification limits of 126 cell culture infective dose 50 % (CCID50) /mL in ICA-qPCR after 15 minutes of absorption and 2,512 CCID50/mL in PMA-qPCR. These methods enable estimation of viral infectivity, applicable in inactivation validation, environmental monitoring, and detection of infectious pathogens.•Significance of the study: ICA-qPCR and PMA-qPCR techniques have the potential to replace traditional culture-based methods for quantifying intact infectious viruses. They can aid in identifying intact infectious viruses in wastewater or on pathogen-related physical surfaces in high-level biosafety laboratories and medical facilities. Additionally, these methodologies can be used to detect other highly pathogenic pathogens. 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.