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Infectious porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, but noninfectious PRRSV cannot. PCR and ELISA are commonly used for PRRSV detection but they cannot discriminate PRRSV infectivity. Virus isolation is a gold standard to determine virus infectivity. However, it is time‐consuming. Therefore, we developed a propidium monoazide (PMA) qPCR assay for rapid and universal detection of infectious PRRSV in this study. After comparing the inactivation efficacies of distinct disinfectants, ultraviolet (UV) light, and heat, heat at 72°C for 15 min was determined as an effective strategy for PRRSV inactivation, which was confirmed by virus isolation and immunofluorescence assay (IFA) detection. In addition, PMA pretreatment parameters were optimized, including PMA concentration (5 μM), PMA binding time (25 min), PMA binding temperature (37°C), and photolysis time (25 min). The optimal concentration of primers and probes adapted from our previous study was redetermined. The optimized PMA‐qPCR assay exhibited satisfied specificity, sensitivity, and reproducibility. Furthermore, the new PMA‐qPCR was applied on the detection of 270 clinical samples (including 57 environmental feces, 177 lungs, 33 lymph nodes [LN], and 3 sera) and compared with previously developed qPCR. Eighty samples were qPCR positive, while only 63 samples were PMA‐qPCR positive. No virus could be isolated in the 17 qPCR‐positive but PMA‐qPCR‐negative clinical samples; meanwhile, PRRSV could be isolated in representative PMA‐qPCR‐positive samples, supporting that only live PRRSV isolates in distinct samples could be detected by this PMA‐qPCR assay. In conclusion, this study provides the first PMA‐qPCR assay for rapid and universal detection of infectious PRRSV, offering an alternative and effective method for PRRSV diagnosis, prevention, and control. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential.

Here we developed an integrated cell absorption process and quantitative (reverse transcription) polymerase chain reaction (ICAP-q(RT)PCR) assay to detect infectious viruses, which based on the detection of the viral nucleic acid (RNA or DNA) in the early stage of viral attachment and entry towards cells. The results showed that the poliovirus or adenovirus whose concentration was as low as 0.2 TCID 50 /mL could be detected by ICAP-q(RT)PCR after 4 h incubation. The ICAP-q(RT)PCR exhibited much higher sensitivity than the plaque assay. In parallel, it took shorter time to detect the viruses towards field samples compared with the integrated cell culture (ICC)-qPCR, but could still get the consistent detecting results with ICC-qPCR. This method is verified by detecting four different kinds of viruses including poliovirus, adenovirus, rotavirus, and astrovirus, which existed in the actual water samples. Among all the 24 Jinhe river samples, 50% (12/24) of river water samples were positive for poliovirus when detected by ICAP-q(RT)PCR, which was in accordance with the results detected by ICC-qPCR. However, 21% (5/24) and 68% (18/24) of the samples were detected to be positive for poliovirus by the plaque counting and the direct qPCR method, respectively. Compared with ICAP-q(PT)PCR and ICC-qPCR, the detecting results of qPCR or plaque assay displayed a marked expansion or decline, respectively, which lead to the evident deviations in the accuracy. The results demonstrated that our developed ICAP-q(RT)PCR method could dramatically reduce the test duration and quite improve the sensitivity towards infectious viruses. Therefore, the ICAP-q(RT)PCR method could be an effective and quantitative tool for detecting infectious viruses in water environments. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) transmission occurs even among fully vaccinated individuals; thus, prompt identification of infected patients is central to control viral circulation. Antigen rapid diagnostic tests (Ag-RDTs) are highly specific, but sensitivity is variable. Discordant RT-qPCR vs. Ag-RDT results are reported, raising the question of whether negative Ag-RDT in positive RT-qPCR samples could imply the absence of infectious viruses. To study the relationship between negative Ag-RDT results with virological, molecular, and serological parameters, we selected a cross-sectional and a follow-up dataset and analyzed virus culture, subgenomic RNA quantification, and sequencing to determine infectious viruses and mutations. We demonstrated that RT-qPCR positive while SARS-CoV-2 Ag-RDT negative discordant results correlate with the absence of infectious virus in nasopharyngeal samples. A decrease in sgRNA detection together with an expected increase in detectable anti-S and anti-N IgGs was also verified in these samples. The data clearly demonstrate that a negative Ag-RDT sample is less likely to harbor infectious SARS-CoV-2 and, consequently, has a lower transmissible potential., Competing Interests: LG, MR, MB, RV, GC, and JH were employed by Abbott Laboratories Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Corrêa, Faffe, Galliez, Gonçalves, Maia, da Silva, Moreira, Mariani, Campos, Leitão, de Souza, Cunha, Nascimento, Ribeiro, da Cruz, Policarpo, Gonzales, Rodgers, Berg, Vijesurier, Cloherty, Hackett Jr., Ferreira Jr., Castiñeiras, Tanuri and da Costa.)