Proof of stability of an RSV Controlled Human Infection Model challenge agent.

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze-thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze-thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

High-Throughput Determination of Infectious Virus Titers by Kinetic Measurement of Infection-Induced Changes in Cell Morphology.

Infectivity assays are the key analytical technology for the development and manufacturing of virus-based therapeutics. Here, we introduce a novel assay format that utilizes label-free bright-field images to determine the kinetics of infection-dependent changes in cell morphology. In particular, cell rounding is directly proportional to the amount of infectious virus applied, enabling rapid determination of viral titers in relation to a standard curve. Our kinetic infectious virus titer (KIT) assay is stability-indicating and, due to its sensitive readout method, provides results within 24 h post-infection. Compared to traditional infectivity assays, which depend on a single readout of an infection endpoint, cumulated analysis of kinetic data by a fit model results in precise results (CV < 20%) based on only three wells per sample. This approach allows for a high throughput with ~400 samples processed by a single operator per week. We demonstrate the applicability of the KIT assay for the genetically engineered oncolytic VSV-GP, Newcastle disease virus (NDV), and parapoxvirus ovis (ORFV), but it can potentially be extended to a wide range of viruses that induce morphological changes upon infection. The versatility of this assay, combined with its independence from specific instruments or software, makes it a promising solution to overcome the analytical bottleneck in infectivity assays within the pharmaceutical industry and as a routine method in academic research.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2-) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2- and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.

Postmortem Stability of SARS-CoV-2 in Mouse Lung Tissue.

The infectivity of severe acute respiratory syndrome coronavirus 2 in deceased persons and organisms remains unclear. We studied transgenic K18 hACE2 mice to determine the kinetics of virus infectivity after host death. Five days after death, virus infectivity in the lung declined by >96% and RNA copies declined by 48.2%.

Reduction of severe acute respiratory syndrome coronavirus-2 infectivity by admissible concentration of ozone gas and water.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Because complete elimination of SARS-CoV-2 appears difficult, decreasing the risk of transmission is important. Treatment with 0.1 and 0.05 ppm ozone gas for 10 and 20 hr, respectively, decreased SARS-CoV-2 infectivity by about 95%. The magnitude of the effect was dependent on humidity. Treatment with 1 and 2 mg/L ozone water for 10 s reduced SARS-CoV-2 infectivity by about 2 and 3 logs, respectively. Our results suggest that low-dose ozone, in the form of gas and water, is effective against SARS-CoV-2.

Isolation and genetic characterization of SARS-CoV-2 from Indian patients in a single family without H/O travel abroad.

In view of the rapidly progressing COVID-19 pandemic, our aim was to isolate and characterize SARS-CoV-2 from Indian patients. SARS-CoV-2 was isolated from nasopharyngeal swabs collected from the two members of a family without any history of (H/O) travel abroad. Both the virus isolates (8003 and 8004) showed CPE on day 3 post-inoculation, viral antigens by immunofluorescence assay and produced distinct, clear and uniform plaques. Infectious virus titers were 5 × 106 and 4 × 106 Pfu/ml by plaque assay and 107.5 and 107 by CPE-based TCID50/ml, respectively. Phylogenetic analysis grouped our isolates with the Italian strains. On comparison with Wuhan strain, 3 unique mutations were identified in nsp3 (A1812D), exonuclease (P1821S) of Orf1ab and spike protein (Q677H) regions, respectively. Both the viruses grouped with Italian strains of SARS-CoV-2 suggesting possible source being the virus imported from Italy. These fully characterized virus isolates will be useful in developing neutralization/virological assays for the evaluation of vaccines/antivirals.

The effect of climbing chalk powder on the infectivity of human coronavirus OC43.

There does not appear to be any studies in the published literature on the stability of SARS-CoV-2 in climbing chalk powder (magnesium carbonate and/or calcium carbonate), which has been hypothesized to pose a potential risk of fomite transmission of coronavirus disease 2019 (COVID-19) within climbing gyms. The aim of this study was to determine the infectivity of a model human coronavirus HCoV-OC43 in the presence of climbing chalk powder on a dry plastic surface. The stability of HCoV-OC43 on a plastic surface dusted with climbing chalk powders (magnesium carbonate, calcium carbonate or a blended chalk) was determined by titration on BHK-21 fibroblast cells. No chalk and no virus controls were included. HCoV-OC43 was stable on the plastic surface for 48 h. The stability of HCoV-OC43 was significantly (P ≤ 0·05) reduced in the presence of magnesium carbonate, calcium carbonate and the chalk blend; the infectivity was reduced by ≥2·29 log10 50% tissue culture infective dose (TCID50 ) immediately upon on contact and by ≥2·46 log10 TCID50 within 1 h of contact. These findings suggest that the infectivity of coronaviruses is reduced by climbing chalk, limiting the risk of potential fomite transmission.

Lentivirus expressing shRNAs inhibit the replication of contagious ecthyma virus by targeting DNA polymerase gene.

BACKGROUND: Contagious ecthyma or Orf is known as a zoonotic disease remains prevalently worldwide despite the application of some control strategies against it. RNAi particularly shRNA provides us with the chance to tackle this obstacle by an encouraging new approach. The current study indicates the design and experiment of third-generation lentivirus packaging systems delivering shRNAs to inhibit Orf virus (ORFV) replication and infection. Given the importance of DNA-pol gene in virus replication, in this study, three shRNAs against this gene were designed and cloned into lentiviral vectors to stabilize the expression of shRNAs. After producing lentivectors expressing ORFV-DNA- pol in HEK293T cells, the synthesized shRNAs were applied to downregulate viral replication and gene expression. The reduction in viral titer and RNA was evaluated by TCID50 test as well as real-time RT-PCR. The results were then analyzed in comparison with the control group. RESULTS: Designed shRNAs significantly reduced virus yield approximately 90 to 97% and 96.8 to 99.4%, respectively compared to the control groups (cells infected with ORFV and infected with ORFV and scrambled vector) by TCID50 test. Real-time RT-PCR revealed a dramatic reduction in the expression of viral RNA approximately 99% compared to cells infected with ORFV and from 92.6 to 99%, respectively compared to cells infected with ORFV and scrambled vector. CONCLUSIONS: Therefore, it can be stated that RNAi is capable of being used as a potent therapeutically option against viruses like ORFV.

Vectorial Release of Hepatitis E Virus in Polarized Human Hepatocytes.

Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic, but immunocompromised patients infected with HEV genotype 3 (HEV3), HEV4, or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV), while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (oriented toward the blood) and apical (oriented toward the bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A cells. F2 cells cultured on semipermeable collagen inserts and infected basolaterally with nHEV3 released 94.6% of virus particles apically, those infected with eHEV3 released 96.8% apically, and eHEV1-infected cells released 99.3% apically. Transcytosis was not involved. Density gradient centrifugation and NP-40 treatment showed that HEV particles released both apically and basolaterally were lipid associated. The apically released HEV3 and HEV1 particles were six and nine times more infectious than those released basolaterally, respectively. Confocal microscopy indicated that the open reading frame 2 (ORF2) capsid protein colocalized apically with ORF3 virus protein, the apical marker DPP4, and the recycling endosome GTPase Rab27a. The amounts of soluble glycosylated ORF2 secreted apically and basolaterally were similar. These polarized-hepatocyte data suggest that infectious HEV particles are mainly released into bile, while the small fraction released into blood could spread HEV throughout the host.IMPORTANCE Hepatitis E virus (HEV) in stools is naked, while that in culture supernatants and patients' blood is lipid associated. Its life cycle in hepatocytes, polarized cells with a basolateral side communicating with blood and an apical side connected with bile, is incompletely understood. We have developed a polarized hepatocyte model and used the cells to analyze the supernatants bathing the apical and basolateral sides and HEV subcellular distribution. HEV particles from both sides were lipid associated, and most infectious HEV particles left the cell via its apical side. Similar amounts of the open reading frame 2 (ORF2) soluble capsid protein were secreted from both sides of the hepatocytes. This model mimicking physiological conditions should help clarify the HEV cell cycle in polarized hepatocytes.

Human Herpesvirus 6A Exhibits Restrictive Propagation with Limited Activation of the Protein Kinase R-eIF2α Stress Pathway.

The eIF2α protein plays a critical role in the regulation of translation. The production of double-stranded RNA (dsRNA) during viral replication can activate protein kinase R (PKR), which phosphorylates eIF2α, leading to inhibition of the initial step of translation. Many viruses have evolved gene products targeting the PKR-eIF2a pathway, indicating its importance in antiviral defense. In the present study, we focused on alternations of PKR-eIF2a pathway during human herpesvirus 6A (HHV-6A) infection while monitoring viral gene expression and infectious viral yields. We have found increased phosphorylated PKR as well as phosphorylated eIF2α coincident with accumulation of the late gp82-105 viral protein. The level of total PKR was relatively constant, but it decreased by 144 h postinfection. The phosphorylation of eIF2a led to a moderate increase in activating transcription factor 4 (ATF4) accumulation, indicating moderate inhibition of protein translation during HHV-6A infection. The overexpression of PKR led to decreased viral propagation coincident with increased accumulation of phosphorylated PKR and phosphorylated eIF2a. Moreover, addition of a dominant negative PKR mutant resulted in a moderate increase in viral replication. HHV-6A exhibits relatively low efficiency of propagation of progeny virus secreted into the culture medium. This study suggests that the replicative strategy of HHV-6A involves a mild infection over a lengthy life cycle in culture, while preventing severe activation of the PKR-eIF2α pathway.IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are common, widely prevalent viruses, causing from mild to severe disease. Our study focused on the PKR-eIF2α stress pathway, which limits viral replication. The HHV-6 genome carries multiple genes transcribed from the two strands, predicting accumulation of dsRNAs which can activate PKR and inhibition of protein synthesis. We report that HHV-6A induced the accumulation of phosphorylated PKR and phosphorylated eIF2α and a moderate increase of activating transcription factor 4 (ATF4), which is known to transcribe stress genes. Overexpression of PKR led to increased eIF2α phosphorylation and decreased viral replication, whereas overexpression of a dominant negative PKR mutant resulted in a moderate increase in viral replication. These results suggest that the HHV-6A replication strategy involves restricted activation of the PKR-eIF2α pathway, partial translation inhibition, and lower yields of infectious virus. In essence, HHV-6A limits its own replication due to the inability to bypass the eIF2α phosphorylation.

Development of indirect immunofluorescence assay for TCID50 measurement of grass carp reovirus genotype II without cytopathic effect onto cells.

Grass carp reovirus (GCRV) caused severe hemorrhagic disease with significant losses of fingerling and yearling grass carp, Cyenopharyngodon idellus, in southeast Asian. It was first isolated in 1983 in China, and clade analysis of the different GCRV isolates indicates there are at least three different genotypes I, II, and III. In recent years, GCRV genotype II has been determined as a dominant virus type which cause severe obvious clinical signs in fish but no cytopathic effect onto presently available cell culture. TCID50 is one of standard method to quantity infectious virus particles. In the present study, an indirect immunofluorescence assay (IFA) was developed using antibody against a protein encoded by segment 10 of GCRV genotype II. Moreover, the specific assay to differentitate GCRV of different genotypes and a sensitive assay for determination of GCRV genotype II were developed respectively. The results showed the IFA only can recognize genotype II virus at the lowest initial concentration of 550 genomic copies/ml. Furthermore, comparison of results obtained from qPCR and the TCID50 assay combined IFA was conducted. The results indicated that TCID50 of GCRV isolates JX0901 and HZ08 differs with 2 log steps reduction in the numbers of viruses compared with the number of genome copies detected by qPCR. The immunofluorescence assay developed is sensitive, specific, and the TCID50 combined with IFA will be a standardizable technique for the quantitation and detection of infectious GCRV in cell culture without cytolysis.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Alternative methods to determine infectivity of Tulane virus: a surrogate for human nororvirus.

Culturable animal caliciviruses are widely-used as surrogates for human norovirus (HuNoV). The infectivity of a culturable virus was traditionally determined by plaque assay and/or 50% tissue culture infectious dose (TCID50) assay, both of which are time-consuming and labor-intensive. Molecular approaches, such as quantitative real time RT-PCR (qRT-PCR) and RT-PCR, could be used for detection of the viral genome but yet fail to determine the infectivity of a virus. In this study, we evaluated different assays for determination of infectivity of Tulane virus (TV), a surrogate for HuNoV. The infectivity of TV was measured by RNase exposure assay, RT-PCR assays, cellular-receptor-mediated capture qRT-PCR assay, receptor-mediated in situ capture qRT-PCR assay, cell-culture-mediated amplification qRT-PCR, and confirmed by TCID50 assay. RNase exposure assay was only useful for measuring TV inactivation caused by heat. Short template RT-PCR assay did not reflect inactivation status of TV. Partial reduction in viral RNA signal could be measured by long-template RT-PCR only when TV was inactivated by thermal or chlorine treatments at full-inactivation levels. Cellular-receptor-mediated capture qRT-PCR exhibited low sensitivity and specificity for the evaluation of virus infectivity. The in situ capture qRT-PCR assay could be used to evaluate virus inactivation deriving from damage to viral capsid caused by heat and chlorine. The cell-culture-mediated amplification qRT-PCR could be used as an alternative method to rapidly determine the infectivity of TV.

Oncolytic measles and vesicular stomatitis virotherapy for endometrial cancer.

OBJECTIVE: Current adjuvant therapy for advanced-stage, recurrent, and high-risk endometrial cancer (EC) has not reduced mortality from this malignancy, and novel systemic therapies are imperative. Oncolytic viral therapy has been shown to be effective in the treatment of gynecologic cancers, and we investigated the in vitro and in vivo efficacy of the Edmonston strain of measles virus (MV) and vesicular stomatitis virus (VSV) on EC. METHODS: Human EC cell lines (HEC-1-A, Ishikawa, KLE, RL95-2, AN3 CA, ARK-1, ARK-2, and SPEC-2) were infected with Edmonston strain MV expressing the thyroidal sodium iodide symporter, VSV expressing either human or murine IFN-ß, or recombinant VSV with a methionine deletion at residue 51 of the matrix protein and expressing the sodium iodide symporter. Xenografts of HEC-1-A and AN3 CA generated in athymic mice were treated with intratumoral MV or VSV or intravenous VSV. RESULTS: In vitro, all cell lines were susceptible to infection and cell killing by all 3 VSV strains except KLE. In addition, the majority of EC cell lines were defective in their ability to respond to type I IFN. Intratumoral VSV-treated tumors regressed more rapidly than MV-treated tumors, and intravenous VSV resulted in effective tumor control in 100% of mice. Survival was significantly longer for mice treated with any of the 3 VSV strains compared with saline. CONCLUSION: VSV is clearly more potent in EC oncolysis than MV. A phase 1 clinical trial of VSV in EC is warranted.

Epizootic Hemorrhagic Disease Virus Titration.

The titration of viruses onto susceptible cell lines is an important virological technique used to quantify infectious viral titers. It forms an integral component of epizootic hemorrhagic disease virus (EHDV) research, including estimating infectivity, calculating multiplicity of infection, and confirming virus propagation in cell culture. However, the ability to quantify infectious EHDV is also critical for disease control, particularly in the event of an outbreak. Routine EHD diagnostics do not accurately quantify infectious virus, which would allow accurate prediction of the onward transmission risk, but instead are typically more qualitative in nature (e.g., virus isolation) or only quantify viral genome copies (e.g., real-time PCR) which often remain detectable long after infectious virus is cleared from the host.Infectious EHDV titers are typically quantified through the detection of visible cytopathic effect (CPE) in the monolayer of susceptible mammalian cell cultures. However, not all susceptible cell lines demonstrate visible CPE upon EHDV infection, including cell lines such as KC cells, which are derived from the EHDV biological insect vector, Culicoides sonorensis. This chapter presents a comprehensive method for the titration of EHDV-positive samples onto relevant, susceptible mammalian (Vero) and insect (KC) cell lines and describes alternative methods that can be used to visualize EHDV infection, by CPE or immunofluorescent labeling of viral proteins, to enable the calculation of infectious EHDV titers.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Guidelines for In Vitro Production and Quantification of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an arthropod-borne virus (arbovirus) responsible for a severe zoonotic disease affecting a wide range of domestic and wild ruminants as well as humans. RVFV is endemic in many African countries and has also caused outbreaks in Madagascar and Arabian Peninsula. With regard to its wide geographical distribution, its potential to emerge in a new area, and its capability to trigger major health and economic crisis, it is essential to study and better understand several aspects of its life cycle and, in particular, its interactions with mammalian hosts and arthropod vectors. To do so, it is key for researchers to be able to amplify in vitro viral strains isolated from the field and determine accurately the viral titers of RVFV stocks. In this chapter, we present protocols that can be easily implemented to produce and titrate RVFV stocks in your laboratory.

Cytokines as fast indicator of infectious virus titer during process development.

Measuring infectious titer is the most time-consuming method during the production and process development of live viruses. Conventionally, it is done by measuring the tissue culture infectious dose (TCID50) or plaque forming units (pfu) in cell-based assays. Such assays require a time span of more than a week to the readout and significantly slow down process development. In this study, we utilized the pro-inflammatory cytokine response of a Vero production cell line to a recombinant measles vaccine virus (MVV) as model system for rapidly determining infectious virus titer within several hours after infection instead of one week. Cytokines are immunostimulatory proteins contributing to the first line of defence against virus infection. The probed cytokines in this study were MCP-1 and RANTES, which are secreted in a virus dose as well as time dependent manner and correlate to TCID50 over a concentration range of several logarithmic levels with R2 = 0.86 and R2 = 0.83, respectively. Furthermore, the pro-inflammatory cytokine response of the cells was specific for infectious virus particles and not evoked with filtered virus seed. We also discovered that individual cytokine candidates may be more suitable for off- or at-line analysis, depending on the secretion profile as well as their sensitivity towards changing process conditions. Furthermore, the method can be applied to follow a purification procedure and is therefore suited for process development and control.

Evaluation of recombinant baculovirus clearance during rAAV production in Sf9 cells using a newly developed fluorescent-TCID50 assay.

Introduction: Recombinant adeno-associated virus (rAAV) vectors provide a safe and efficient means for in vivo gene delivery, although its large-scale production remains challenging. Featuring high manufacturing speed, flexible product design, and inherent safety and scalability, the baculovirus/Sf9 cell system offers a practical solution to the production of rAAV vectors in large quantities and high purity. Nonetheless, removal and inactivation of recombinant baculoviruses during downstream purification of rAAV vectors remain critical prior to clinical application. Methods: The present study utilized a newly developed fluorescent-TCID50 (F-TCID50) assay to determine the infectious titer of recombinant baculovirus (rBV) stock after baculovirus removal and inactivation, and to evaluate the impact of various reagents and solutions on rBV infectivity. Results and discussion: The results showed that a combination of sodium lauryl sulfate (SLS) and Triton X-100 lysis, AAVx affinity chromatography, low pH hold (pH3.0), CsCl ultracentrifugation, and NFR filtration led to effective removal and/or inactivation of recombinant baculoviruses, and achieved a log reduction value (LRV) of more than 18.9 for the entire AAV purification process. In summary, this study establishes a standard protocol for downstream baculovirus removal and inactivation and a reliable F-TCID50 assay to detect rBV infectivity, which can be widely applied in AAV manufacturing using the baculovirus system.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy.

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.