Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

Establishment, verification and application of rapid detection of baculovirus infectious titer by flow cytometry

Titer detection of baculovirus usually is time-consuming.It is important to establish a rapid detection method for baculovirus titer. In this report, Staining of cells with a fluorescently labeled anti-gp64 antibody allows for identification of infected insect cells. By inoculating cultures with a series of log dilutions of virus, and staining of the cultures 13-22 hours post inoculation, the ratio of infected to un-infected insect cells can be determined by flow cytometry. Statistical analysis of the percentage of infected cells in the virus dilution series enables accurate infectious titer determination. The culture time, cell growth state, the concentration of GP64-APC antibody and the concentration of inactivated FBS in diluent were optimized.The generality, repeatability and intermediate precision of the method were verified. The FCM method has the advantages of simplicity, accuracy, low cost and good repeatability.

Phage susceptibility testing and infectious titer determination through wide-field lensless monitoring of phage plaque growth

The growing number of drug-resistant bacterial infections worldwide is driving renewed interest in phage therapy. Based on the use of a personalized cocktail composed of highly specific bacterial viruses, this therapy relies on a range of tests on agar media to determine the most active phage on a given bacterial target (phage susceptibility testing), or to isolate new lytic phages from an environmental sample (enrichment of phage banks). However, these culture-based techniques are still solely interpreted through direct visual detection of plaques. The main objective of this work is to investigate computer-assisted methods in order to ease and accelerate diagnosis in phage therapy but also to study phage plaque growth kinetics. For this purpose, we designed a custom wide-field lensless imaging device, which allows continuous monitoring over a very large area sensor (3.3 cm2). Here we report bacterial susceptibility to Staphylococcus aureus phage in 3 hr and estimation of infectious titer in 8 hr 20 min. These are much shorter time-to-results than the 12 to 24 hours traditionally needed, since naked eye observation and counting of phage plaques is still the most widely used technique for susceptibility testing prior to phage therapy. Moreover, the continuous monitoring of the samples enables the study of plaque growth kinetics, which enables a deeper understanding of the interaction between phage and bacteria. Finally, thanks to the 4.3 μm resolution, we detect phage-resistant bacterial microcolonies of Klebsiella pneumoniae inside the boundaries of phage plaques and thus show that our prototype is also a suitable device to track phage resistance. Lensless imaging is therefore an all-in-one method that could easily be implemented in cost-effective and compact devices in phage laboratories to help with phage therapy diagnosis.

#36: Can Astrovirus VA1, a Novel Human Astrovirus, Cause Diseases of the Central Nervous and Cardiovascular Systems?

Background Since their original isolation from human stool samples in 1975, astroviruses have been assumed to be pathogens exclusive to the gastrointestinal tract. However, astroviruses have been recently identified from brain tissue of humans and other mammals suffering from neurological diseases, suggesting that members of this family of viruses have a previously unrecognized neurotropism. One human astrovirus genotype, astrovirus VA1/HMO-C (VA1) is the most frequently identified astrovirus from cases of human encephalitis. We previously described the ability of several cell lines, including Caco-2, A549, and HEK293, to support VA1 growth in cell culture. Our goals were to develop cell culture and animal models to study the pathogenic potential of VA1. Methods VA1 was inoculated into primary astrocytes, primary neurons, and SK-N-SH cells in tissue culture. Viral RNA was measured in multistep growth curves by qRT-PCR, capsid was detected by immunofluorescence, and infectious titer was measured by a TCID50 assay. For development of an in vivo model of infection, mice were inoculated with VA1 with simultaneous intracranial, intraperitoneal, intravenous, and oral gavage inoculations with a VA1 stock. Mice were sacrificed 7 days after inoculation, RNA extracted from tissues, and viral load quantified by qRT-PCR. Tissue was also processed for histology and staining with hematoxylin and eosin. Primary human cardiac endothelial and myocytes were also inoculated with VA1 and viral RNA was measured in multistep growth curves by qRT-PCR. Results We detected the completion of the full VA1 lifecycle in primary astrocytes and SK-N-SH cells with >100-fold increase in viral RNA in a multistep growth curve, detection of intracellular viral capsid, and a >100-fold increase in the infectious titer. These results support the neurotropic potential of VA1. In the development of a mouse model of VA1 infection, we detected the virus in the brain tissue of mice 7 days after inoculation. Unexpectedly, we detected the highest titer of VA1 in heart tissue, which was associated with histological evidence of myocarditis. Furthermore, we demonstrated VA1 can replicate in primary human cardiac endothelial cells but not in primary human cardiac myocytes. Conclusions Our data demonstrate that astroviruses have tissue tropisms outside of the gastrointestinal tract. We provide the first demonstration that cultured neural-derived cells can support infection and replication of astroviruses, providing a model for studying the neuropathogenic potential of VA1. Furthermore, we have identified a novel cardiotropism for VA1, raising the possibility that VA1 may contribute to human cardiac diseases currently without an ascribed etiology.

ANTIVIRAL ACTIVITY OF AMINOCAPROIC ACID AGAINST INFECTIOUS BRONCHITIS CORONAVIRUS IN VITRO

I. V. Dziublyk, O. P. Trokhimenko, S. O. Soloviov, G. L. Gumeniuk, O. Ya. Dziublyk, N. I. Gumeniuk, O. K. Yakovenko Abstract The aim of the study is a preclinical evaluation of the antiviral activity of aminocaproic acid (ACA) against the prototype strain IBV (Infectious bronchitis virus) of Coronavirus family in vitro. Material and methods. During the research, modern methods were used to determine the cytotoxic effect of the evaluation on a monolayer of BHK-21 cell culture in vitro; cultivation, accumulation and determination of the infectious titer of IBV by cytopathic action on a monolayer of cell cultures; assessment of the antiviral effect of the drug — the establishment of the inhibitory concentration and the chemotherapeutic index (CTI) of ACA in various modes of drug administration: 2 hours before infection, simultaneously with infection and 2 hours after infection. Results. With the introduction of ACA 2 hours before infection, a decrease in the infectious titer of the IBV virus was not established. The antiviral activity of ACA was detected when the drug was added in 2 modes: simultaneously and 2 hours after infection. The introduction of ACА into the medium for cell cultivation at non-toxic concentrations of 7.91–15.82 mg / ml led to a decrease in the infectious titer of the virus by 1.4–2.0 lg TCD50 / 0.1 ml. The CTI of the ACA was 6 in the indicated concentrations and modes, which is an indicator of its promising potential for further studies of antiviral activity in vivo, including clinical studies. Conclusions. The direct antiviral effect of ACA against the prototype H-120 virus strain from the Coronaviridae family in vitro was revealed. The suppression of viral reproduction with an established low toxicity of the drug, a decrease in the infectious titer of IBV by 1.4–2.0 lg TCD50 / 0.1 ml and with a CTD equal to 6.0, indicate the prospects for further study of the antiviral properties of ACA in clinical trials. Key words: aminocaproic acid, coronavirus, antiviral activity.

Changes in infectious titer of Black sea algal virus of the microalgae tetraselmis viridis under the influence of a constant magnetic field

In three experiments aimed at studying the effect of a constant magnetic field with a magnetic induction of 600 G and a duration of experiments of 24 and 36 h, it was found that with a low initial titer of the algal virus of the microalgae Tetraselmis viridis, its stay in a magnetic field for 24 h led to an increase in the infectious titer by one order of magnitude. However, when the virus with a higher infectious titer was in a magnetic field for 24 h, no changes in the titer were observed, and after 36 h, its decrease by one order of magnitude was recorded. The results obtained indicate the need for further research in this direction, with using a wider range of algal viruses.

The Effect of GD1a Ganglioside-Expressing Bacterial Strains on Murine Norovirus Infectivity

In this study, we investigated the impact of GD1a-expressing bacterial strains on the infectivity of murine norovirus (MNV). Eligible bacterial strains were screened from a sewage sample using flow cytometry, and their genetic sequences of 16S rRNA were determined. The enzyme-linked immunosorbent assay (ELISA) was employed to analyze the binding between bacteria and MNV particles, and the plaque assay was used to assess the effects of GD1a-positive and negative strains on MNV infectivity. The result from ELISA shows that MNV particles are able to bind to both GD1a-positive and negative bacterial strains, but the binding to the GD1a-positive strain is more significant. The infectivity assay result further shows that the MNV infectious titer declined with an increasing concentration of GD1a-positive bacteria. The addition of anti-GD1a antibody in the infectivity assay led to the recovery of the MNV infectious titer, further confirming that the binding between MNV particles and bacterial GD1a ganglioside compromises MNV infectivity. Our findings highlight the role indigenous bacteria may play in the lifecycle of waterborne enteric viruses as well as the potential of exploiting them for virus transmission intervention and water safety improvement.

Modeling influenza virus infection in mature Wistar rats

It has now been established that blood vessels are target for influenza, but the mechanism by which the influenza virus affects the cardiovascular system is unknown. The aim – adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats, as these animals are the main experimental model for studying the pathology of the cardiovascular system. Material and methods. Passage of influenza A virus (IAV) in embryonated chicken eggs, intranasal inoculation of rats with virus-containing material s, production of pulmonary homogenate, determination of IAV titer in embryonated chicken eggs, detection of histological changes in lung and pulmonary vessels. Results. The article presents the results of the adaptation of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) to mature Wistar rats. The infectious titer of the virus in the homogenates of infected rats lungs at the last stage of adaptation was 7.0 lg EID50/ml. Histological studies revealed pronounced changes in the respiratory tract (spasm of bronchioles, submucosal edema, desquamation of ciliated epithelium of bronchioles) and pulmonary vessels (spasm, desquamation and swelling of endotheliocytes, dissociation and swelling of the elastic membrane and media). In order to identify IAV in blood vessels and lung tissues, an immunohistochemical study was performed using monoclonal antibodies to NP antigen of IAV. Conclusion. The data obtained allow us to conclude that the strain of influenza virus A/St. Petersburg/48/16 H1N1(pdm09) was adapted to mature Wistar rats maintaining virulent properties. The infectious titer of the virus at the last stage of adaptation was 7.0 lg EID50/ml. IAV identification is confirmed by immunohistochemical examination.

The IMPDH inhibitor merimepodib provided in combination with the adenosine analogue remdesivir reduces SARS-CoV-2 replication to undetectable levels in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for the ongoing COVID-19 pandemic, which has resulted in over 2.5 million confirmed cases and 170,000 deaths worldwide as of late April 2020. The pandemic currently presents major public health and economic burdens worldwide. No vaccines or therapeutics have been approved for use to treat COVID-19 cases in the United States despite the growing disease burden, thus creating an urgent need for effective treatments. The adenosine analogue remdesivir (REM) has recently been investigated as a potential treatment option, and has shown some activity in limiting SARS-CoV-2 replication. We previously reported that the IMPDH inhibitor merimepodib (MMPD) provides a dose-dependent suppression of SARS-CoV-2 replication in vitro. Here, we report that a 4-hour pre-treatment of Vero cells with 2.5µM MMPD reduces the infectious titer of SARS-CoV-2 more effectively than REM at the same concentration. Additionally, pre-treatment of Vero cells with both REM and MMPD in combination reduces the infectious titer of SARS-CoV-2 to values below the detectable limit of our TCID50 assay. This result was achieved with concentrations as small as 1.25 µM MMPD and 2.5 µM REM. At concentrations of each agent as low as 0.31 µM, significant reduction of viral production occurred. This study provides evidence that REM and MMPD administered in combination might be an effective treatment for COVID-19 cases.