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Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days-3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community., Competing Interests: The authors declare no competing financial interests or personal relationships that could have influenced the work reported in this paper., (© 2024 The Authors.)

Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID 50 ) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID 50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID 50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID 50 assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID 50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications.

Objectives: The prolonged presence of infectious SARS-CoV-2 in deceased patients with COVID-19 has been reported. However, infectious virus titers have not been determined. Such information is important for public health, death investigation, and handling corpses. The aim of this study was to assess the level of SARS-CoV-2 infectivity in the corpses of patients with COVID-19., Methods: We collected 11 nasopharyngeal swabs and 19 lung tissue specimens from 11 autopsy cases with COVID-19 in 2021. We then investigated the viral genomic copy number by real-time reverse transcription-polymerase chain reaction and infectious titers by cell culture and virus isolation., Results: Infectious virus was present in six of 11 (55%) cases, four of 11 (36%) nasopharyngeal swabs, and nine of 19 (47%) lung specimens. The virus titers ranged from 6.00E + 01 plaque-forming units/ml to 2.09E + 06 plaque-forming units/g. In all cases in which an infectious virus was found, the time from death to discovery was within 1 day and the longest postmortem interval was 13 days., Conclusion: The corpses of patients with COVID-19 may have high titers of infectious virus after a long postmortem interval (up to 13 days). Therefore, appropriate infection control measures must be taken when handling corpses., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A genetically modified, recombinant form of Newcastle disease virus (rNDV) undergoes ionic strength-dependent changes in morphology, as observed by cryo-electron microscopy (cEM). In hypotonic solutions with ionic strengths ranging from < 0.01 to 0.02 M, rNDV virions are spherical or predominantly spherical. In isotonic and hypertonic solutions, rNDV displays pleomorphism and contains a mixed population of spherical and elongated particles, indicating that a change from spherical to elongated shape is induced with increasing salt concentration. This ionic strength-dependent transition is largely reversible, as determined by cEM. Concomitantly, we measured infectious titers of these same rNDV samples at different ionic strengths using a fluorescent focus assay (FFA). The infectivity of oncolytic rNDV was found to be independent of ionic strength, ranging from 0.01 M to approximately 0.5 M. These structural and functional observations, in combination, suggest that infectivity (and, by inference, oncolytic activity) of rNDV virions is fully maintained in their pleomorphic forms. IMPORTANCE Oncolytic viruses are being developed for cancer therapy, as they selectively target, infect, and kill cancer cells. NDV is particularly attractive because while it is pathogenic to avians (e.g., chickens), it does not cause significant viremia in humans. We have developed a genetically modified recombinant NDV (rNDV) that has much reduced pathogenicity in chickens but is highly oncolytic. The morphology of rNDV transitions from spherical at very low salt concentrations to a heterogeneous population of spherical and elongated virions in isotonic (physiologic salt concentration) and hypertonic solutions. The infectivity (cell-killing activity by infecting cells) of rNDV is unaltered by changes in salt concentration despite morphological changes. These observations are significant for purification and formulation of rNDV, as exposure to different salt concentrations may be needed. Importantly, at physiological salt concentration, relevant to clinical testing, infectivity and, therefore, oncolytic activity will not be compromised despite morphological heterogeneity., (Copyright © 2020 American Society for Microbiology.)

Newcastle disease (ND) is a big concern throughout the world because of the devastating losses that can occur with commercial and backyard poultry. The major problem in many countries is the loss of the vaccine's effectiveness due to inadequate use or storage conditions, particularly in hot climates. In the present study, stability of the five, most-used NDV vaccine strains (I-2, LaSota, B1, Clone 30 [C30], and VG-GA) was tested comparatively at different storage temperatures (4 and 37 C for the freeze-dried form and 4, 24, 37, and 45 C for the freeze-dried vaccine reconstituted in diluents). The vaccine stability was evaluated by the cumulative infectious titer drop and the theoretical shelf life at particular temperatures. Results showed that I-2 and LaSota are the most stable vaccine strains compared to B1, C30, and VG-GA; they registered the lowest titer drops and the longest shelf life whether at cool, high, or room temperatures and for both freeze-dried and reconstituted vaccines.