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.

Virus Isolation of Epizootic Hemorrhagic Disease Virus in Cell Culture.

Virus isolation is used to assist in the diagnosis and confirmation of viral infections. Successful isolation of a virus is highly dependent upon the quality of starting material. Here we describe the preparation and isolation of epizootic hemorrhagic disease virus (EHDV) from blood and tissue samples in tissue culture flasks (TCFs) through the inoculation of susceptible cell lines including Vero, BHK, and KC cells.

Evaluation of a qualified MDCK cell line for virus isolation to develop cell-based influenza vaccine viruses with appropriate antigenicity.

We established a qualified Madin-Darby canine kidney cell line (qMDCK-Cs) and investigated its suitability for source virus isolation to develop cell-based seasonal influenza vaccine viruses using vaccine manufacturer cells (Manuf-Cs). When inoculated with 81 influenza-positive clinical specimens, the initial virus isolation efficiency of qMDCK-Cs was exceeded 70%. Among the qMDCK-C isolates, 100% of the A/H1N1pdm09, B/Victoria and B/Yamagata strains and >70% of the A/H3N2 strains showed antigenicity equivalent to that of the contemporary vaccine or relevant viruses in haemagglutination inhibition (HI) or virus neutralization (VN) tests using ferret antisera. These qMDCK-C isolates were propagated in Manuf-Cs (MDCK and Vero cells) (Manuf-C viruses) to develop vaccine viruses. In reciprocal antigenicity tests, ferret antisera raised against corresponding reference viruses and Manuf-C viruses recognized 29 of 31 Manuf-C viruses and corresponding reference viruses, respectively at HI or VN titres more than half of the homologous virus titres, which is the antigenicity criterion for cell culture seasonal influenza vaccine viruses specified by the World Health Organization. Furthermore, ferret antisera against these Manuf-C viruses recognized ≥95% of the viruses circulating during the relevant influenza season with HI or VN titres greater than one-quarter of the homologous virus titres. No cell line-specific amino acid substitutions were observed in the resulting viruses. However, polymorphisms at positions 158/160 of H3HA, 148/151 of N2NA and 197/199 of B/Victoria HA were occasionally detected in the qMDCK-C and Manuf-C viruses but barely affected the viral antigenicity. These results indicated that qMDCK-Cs are suitable for isolating influenza viruses that can serve as a source of antigenically appropriate vaccine viruses. The use of the qMDCK-C isolates will eliminates the need for clinical sample collection, virus isolation, and antigenicity analysis every season, and is expected to contribute to the promotion of vaccine virus development using manufacturer cells.

Propagating and banking genetically diverse human sapovirus strains using a human duodenal cell line: investigating antigenic differences between strains.

There are four genogroups and 18 genotypes of human sapoviruses (HuSaVs) responsible for acute gastroenteritis. To comprehend their antigenic and virological differences, it is crucial to obtain viral stocks of the different strains. Previously, we utilized the human duodenum-derived cell line HuTu80, and glycocholate, a conjugated bile acid, to replicate and propagate GI.1, GI.2, and GII.3 HuSaVs (H. Takagi et al., Proc Natl Acad Sci U S A 117:32078-32085, 2020, https://10.1073/pnas.2007310117). First, we investigated the impact of HuTu80 passage number on HuSaV propagation. Second, we demonstrated that taurocholate improved the initial replication success rate and viral RNA levels in fecal specimens relative to glycocholate. By propagating 15 HuSaV genotypes (GI.1-7, GII.1-5, -8, and GV.1-2) and accomplishing preparation of viral stocks containing 1.0 × 109 to 3.4 × 1011 viral genomic copies/mL, we found that all strains required bile acids for replication, with GII.4 showing strict requirements for taurocholate. The deduced VP1 sequences of the viruses during the scale-up of serial passaged virus cultures were either identical or differed by only two amino acids from the original sequences in feces. In addition, we purified virions from nine strains of different genotypes and used them as immunogens for antiserum production. Enzyme-linked immunosorbent assays (ELISAs) using rabbit and guinea pig antisera for each of the 15 strains of different genotypes revealed distinct antigenicity among the propagating viruses across genogroups and differences between genotypes. Acquisition of biobanked viral resources and determination of key culture conditions will be valuable to gain insights into the common mechanisms of HuSaV infection. IMPORTANCE: The control of human sapovirus, which causes acute gastroenteritis in individuals of all ages, is challenging because of its association with outbreaks similar to those caused by human norovirus. The establishment of conditions for efficient viral propagation of various viral strains is essential for understanding the infection mechanism and identifying potential control methods. In this study, two critical factors for human sapovirus propagation in a conventional human duodenal cell line were identified, and 15 strains of different genotypes that differed at the genetic and antigenic levels were isolated and used to prepare virus stocks. The preparation of virus stocks has not been successful for noroviruses, which belong to the same family as sapoviruses. Securing virus stocks of multiple human sapovirus strains represents a significant advance toward establishing a reliable experimental system that does not depend on limited virus-positive fecal material.

Increased production of orthoflavivirus single-round infectious particles produced in mammalian cells at a suboptimal culture temperature of 28°C.

In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.

Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells.

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.

Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines.

Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis. IMPORTANCE: Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

Innovations in cell culture-based influenza vaccine manufacturing - from static cultures to high cell density cultivations.

Influenza remains a serious global health concern, causing significant morbidity and mortality each year. Vaccination is crucial to mitigate its impact, but requires rapid and efficient manufacturing strategies to handle timing and supply. Traditionally relying on egg-based production, the field has witnessed a paradigm shift toward cell culture-based methods offering enhanced flexibility, scalability, and process safety. This review provides a concise overview of available cell substrates and technological advancements. We summarize crucial steps toward process intensification - from roller bottle production to dynamic cultures on carriers and from suspension cultures in batch mode to high cell density perfusion using various cell retention devices. Moreover, we compare single-use and conventional systems and address challenges including defective interfering particles. Taken together, we describe the current state-of-the-art in cell culture-based influenza virus production to sustainably meet vaccine demands, guarantee a timely supply, and keep up with the challenges of seasonal epidemics and global pandemics.

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.

Association of Symptoms and Viral Culture Positivity for SARS-CoV-2-Tennessee, April-July 2020.

BACKGROUND: Understanding how symptoms are associated with SARS-CoV-2 culture positivity is important for isolation and transmission control guidelines. METHODS: Individuals acutely infected with SARS-CoV-2 in Tennessee and their household contacts were recruited into a prospective study. All participants self-collected nasal swabs daily for 14 days and completed symptom diaries from the day of illness onset through day 14 postenrollment. Nasal specimens were tested for SARS-CoV-2 using RT-qPCR. Positive specimens with cycle threshold values < 40 were sent to the Centers for Disease Control and Prevention (CDC) for viral culture. First, we modeled the association between symptoms and the risk of culture positivity using an age-adjusted generalized additive model (GAM) accounting for repeated measurements within participants and a symptom-day spline. Next, we investigated how timing of symptom resolution was associated with the timing of culture resolution. RESULTS: In a GAM restricted to follow-up days after symptoms began, the odds of a specimen being culture positive was significantly increased on days when wheezing, loss of taste or smell, runny nose, nasal congestion, sore throat, fever, or any symptom were reported. For all symptoms except sore throat, it was more common for participants to have culture resolution before symptom resolution than for culture to resolve after or on the same day as symptom resolution. CONCLUSIONS: Overall, symptomatic individuals were more likely to be SARS-CoV-2 viral culture positive. For most symptoms, culture positivity was more likely to end before symptoms resolved. However, a proportion of individuals remained culture positive after symptom resolved, across all symptoms.

Development of an HSV-1 production process involving serum-reduced culturing and bead-to-bead transfer.

Herpes simplex virus type 1 (HSV-1) plays an important role in the field of gene therapy and viral vaccines, especially as an oncolytic virus. However, the mass production of HSV-1 viral vectors remains a challenge in the industry. In this study, a microcarrier-mediated serum-reduced medium culture was used to improve the bioprocess of HSV-1 production and increase HSV-1 yields. The composition of the culture media, which included a basal medium, serum concentration, and glutamine additive, was optimized. The process was successfully conducted in a 1 L bioreactor, and virus production was threefold greater than that of conventional processes with a 10% serum medium. The bead-to-bead transfer process was also developed to further increase scalability. In spinner flasks, the detachment rate increased from 49.4 to 80.6% when combined agitation was performed during digestion; the overall recovery proportion increased from 37.9 to 71.1% after the operational steps were optimized. Specifically, microcarrier loss was reduced during aspiration and transfer, and microcarriers and detached cells were separated with filters. Comparable cell growth was achieved with the baseline process using 2D culture as the inoculum by exchanging the subculture medium. To increase virus production after bead-to-bead transfer, critical parameters, including shear stress during digestion, TrypLE and EDTA concentrations in the subculture, and the CCI, were identified from 47 parameters via correlation analysis and principal component analysis. The optimized bead-to-bead transfer process achieved an average of 90.4% overall recovery and comparable virus production compared to that of the baseline process. This study is the first to report the optimization of HSV-1 production in Vero cells cultured on microcarriers in serum-reduced medium after bead-to-bead transfer. KEY POINTS: • An HSV-1 production process was developed that involves culturing in serum-reduced medium, and this process achieved threefold greater virus production than that of traditional processes. • An indirect bead-to-bead transfer process was developed with over 90% recovery yield in bioreactors. • HSV-1 production after bead-to-bead transfer was optimized and was comparable to that achieved with 2D culture as inoculum.

Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells.

Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.

Optimization of the replication of hepatitis E virus genotype 3 in vitro.

AIMS: Hepatitis E virus (HEV) is responsible for ∼20 million human infections worldwide every year. The genotypes HEV-3 and HEV-4 are zoonotic and are responsible for most of the autochthonous HEV cases in high-income countries. There are several cell culture systems that allow for propagation of different HEV genotypes in vitro. One of these systems uses human lung carcinoma cells (A549), and was further optimized for propagation of HEV-3 47832c strain. In this study, we investigated the effect of different media supplements as well as microRNA-122 (miR-122) on improving the replication of HEV-3 47832c in A549 cells. METHODS AND RESULTS: We observed that supplementation of maintenance media with 5% fetal bovine serum was sufficient for efficient replication of HEV-3, and verified the positive effect of media supplementation with Amphotericin B, MgCl2, and dimethyl sulfoxide on replication of HEV-3. We have also demonstrated that adding miR-122 mimics to the culture media does not have any significant effect on the replication of HEV-3 47832c. CONCLUSIONS: Herein, we detected over a 6-fold increase in HEV-3 replication in A549/D3 cells by adding all three supplements: Amphotericin B, MgCl2, and dimethyl sulfoxide to the culture media, while demonstrating that miR-122 might not play a key role in replication of HEV-3 47832c.

T84 Monolayer Cell Cultures Support Productive HBoV and HSV-1 Replication and Enable In Vitro Co-Infection Studies.

Based on several clinical observations it was hypothesized that herpesviruses may influence the replication of human bocaviruses, the second known parvoviruses that have been confirmed as human pathogens. While several cell lines support the growth of HSV-1, HBoV-1 was exclusively cultivated on air-liquid interface cultures, the latter being a rather complicated, slow, and low throughput system. One of the cell lines are T84 cells, which are derived from the lung metastasis of a colorectal tumor. In this study, we provide evidence that T84 also supports HBoV replication when cultivated as monolayers, while simultaneously being permissive for HSV-1. The cell culture model thus would enable co-infection studies of both viruses and is worth being optimized for high throughput studies with HBoV-1. Additionally, the study provides evidence for a supporting effect of HSV-1 on the replication and packaging of HBoV-1 progeny DNA into DNase-resistant viral particles.

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal.

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

In Vitro Modeling of Feline Morbillivirus Infections Using Primary Feline Kidney Cells.

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to 4.64 × 107 TU mL-1 in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38 × 107 TU mL-1 in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of >1 × 107 TU mL-1 day-1 for up to 29 consecutive days in a non-optimized 5 L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Establishment of a new equine embryo brain primary cell culture with long-term expansion.

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

Multivariate data analysis on multisensor measurement for inline process monitoring of adenovirus production in HEK293 cells.

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.

SARS-CoV-2 live virus culture and sample freeze-thaw stability.

The effect of freeze-thaw on SARS-CoV-2 viral viability is not well established. We isolated virus from 31 split clinical samples cultured fresh or after a 7- or 17/18-day freeze. We found that freeze-thaw did not significantly affect viral culture isolation. Therefore, frozen samples may be used to assess SARS-CoV-2 infectiousness.