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Aim. Development of a biotechnological system based on a non-pathogenic coronavirus strain for humans and a sensitive cell line to the selected strain aimed at identifying compounds with potential antiviral activity. Methods. The study was conducted on the cell lines CEF, CEFs, and ВНК-21, which are sensitive to the avian infectious bronchitis virus (IBV). Cell cultivation was performed in flasks and microplates with adhesive surfaces at 37 °C in a 5% CO2 atmosphere. To detach the cells from the growth surface, a Versene solution (0.02%) was used, and for trypsinization of CEF, a trypsin solution (0.25%) was applied. Growth media for all cell cultures were prepared based on a mixture of RPMI-1640 and DMEM in a 1:1 ratio, supplemented with 5% fetal serum. Results. The adaptation of the model virus IBV strain H120 to cultivation in ВНК-21 cell cultures was carried out using intermediate CEF and CEF cultures. In ВНК-21 cells, IBV induced a pronounced cytopathic effect and demonstrated high infectious titers, reaching 5.5 lg TCID50/mL. The use of intermediate CEF and CEF cell cultures facilitated the gradual adaptation of the virus to the new cultivation conditions due to the antigenic affinity between chicken embryo fibroblast cells and avian embryos. Conclusions: As a result of the conducted research, the vaccine virus IBV H-120 was successfully adapted to cultivation conditions in ВНК-21 cell cultures, using primary trypsinized chicken embryo fibroblast cells as an intermediate system. The obtained system "ВНК-21 cell culture + IBV H-120," cultivated at 37 °C in a 5% CO2 atmosphere, can be recommended for use in further biotechnological and virological studies, particularly for evaluating the antiviral activity of potential drugs against coronaviruses.

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.

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 (TCID50) 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 TCID50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID50 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 TCID50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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).

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Accurate measurements of the physical and infectious titers of adenoviral vectors are crucial for evaluating preclinical studies and for the safety and efficacy of clinical studies. Unfortunately, there are no standardized methods of measurement, consequently variable and unreliable values are the result. Furthermore, infectious titers of helper-dependent adenoviral vectors (HDAd) are difficult to measure because traditional cytopathic effect assays cannot be employed, thus hindering their potential clinical application. In response to this problem, a fully characterized Adenovirus Reference Material (ARM) has been developed to be used as a reference standard for clinical grade adenoviral vectors. However, no specific protocols for this purpose have been provided. To fulfill this important need, we have developed a simple assay involving co-infection of 293 cells with the adenoviral vector and the ARM to permit direct comparisons of their physical and infectious titers. We demonstrate, using a HDAd, that this co-infection assay is reliable, sensitive, and reproducible. Importantly, this assay is inherently unaffected by variables that plague other methods of determining vector titers. This assay is applicable to all human serotype 5 adenoviral vectors and will permit reliable comparisons within and between studies as well as meet an important prerequisite for clinical studies. [Copyright &y& Elsevier]

Methods currently used for HIV-1 viral load measurements are very sensitive, but cannot distinguish between infectious and noninfectious particles. Here we describe the development of a novel, sensitive, and highly reproducible method that allows rapid isolation and quantification of infectious particles from patient plasma.By immobilizing HIV-1 particles in human plasma to platelets using polybrene, we observed a 10- to 1000-fold increase in infectivity over infection protocols using free virus particles. Using this method, we evaluated infectivity in plasma from 52 patients at various disease stages. At plasma viral loads of 1000–10 000 HIV-1 RNA copies/ml 18%, at 10 000–50 000 copies/ml 73%, at 50 000–100 000 copies/ml 90%, and above 100 000 copies 96% of cultures were positive. We found that infectious titers among patients vary distinctively but are characteristic for a patient over extended time periods. Furthermore, we demonstrate that by evaluating infectious titers in conjunction with total HIV RNA loads, subtle effects of treatment intervention on viremia levels can be detected. The immobilization procedure does not interfere with viral entry and does not restore the infectivity of neutralized virus. Therefore, this assay system can be utilized to investigate the influence of substances that specifically affect virion infectivity such as neutralizing antibodies, soluble CD4, or protease inhibitors. Measuring viral infectivity may thereby function as an additional, useful marker in monitoring disease progression and evaluating efficacy of antivirals in vivo. [Copyright &y& Elsevier]