Viral adventitious agent detection using laser force cytology: Intrinsic cell property changes with infection and comparison to in vitro testing.

Adventitious agent testing in biomanufacturing requires assays of broad detection capability to screen for as many infectious agents as possible. The current gold standard for general infectious adventitious virus screening is the in vitro assay in which test articles are cultured onto a panel of different cell lines and observed for cytopathic effect (CPE). However, this assay is inherently subjective due to the nature of visual observation of cell morphology and labor and time intensive, requiring highly trained personnel to identify CPE. Laser force cytology (LFC) is an alternative, automated analytical method that uses a combination of optical and fluidic forces along with imaging to objectively and quantitatively assess CPE in cell culture. Importantly, because LFC uses no labels or antibodies, the assay is appropriate for general adventitious agent testing. Using LFC, changes in cellular features associated with virally infected cells were identified using principal component analysis. Using these features of infected cells, the sensitivity and earliness of detection with LFC was directly compared with the in vitro assay for a diverse panel of viruses incubated with chinese hamster ovary (CHO), Vero, and Medical Research Council cell strain 5 (MRC-5) cells. LFC detected viral infection with a sensitivity equal to the in vitro assay on average, but in certain virus and cell combinations including mouse minute virus (MMV) and reovirus 3 in CHO cells, detection was 4 days earlier and for MMV, the limit of detection was 10-fold lower. Overall, these results demonstrate the ability of LFC to serve as a biopharmaceutical adventitious agent testing methodology with sensitivity equivalent to the in vitro assay, but in an objective and automated manner.

Generation of recombinant vaccinia virus and analysis of virus-induced cell death.

Vaccinia virus is a large double-stranded DNA virus that is widely used to express foreign genes from different origins. We generated recombinant vaccinia virus that expresses a viral inhibitor to examine its effect on virus-induced necroptosis. We provide a detailed protocol to describe the generation of recombinant vaccinia virus, validation of protein expression, and determination of necroptosis using live cell imaging. This approach can be adapted to examine the effect of other cell death regulators on virus-induced cell death. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Statins significantly repress rotavirus replication through downregulation of cholesterol synthesis.

Rotavirus is the most common cause of severe diarrhea among infants and young children and is responsible for more than 200,000 pediatric deaths per year. There is currently no pharmacological treatment for rotavirus infection in clinical activity. Although cholesterol synthesis has been proven to play a key role in the infections of multiple viruses, little is known about the relationship between cholesterol biosynthesis and rotavirus replication. The models of rotavirus infected two cell lines and a human small intestinal organoid were used. We investigated the effects of cholesterol biosynthesis, including inhibition, enhancement, and their combinations on rotavirus replication on these models. The knockdown of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was built by small hairpin RNAs in Caco2 cells. In all these models, inhibition of cholesterol synthesis by statins or HMGCR knockdown had a significant inhibitory effect on rotavirus replication. The result was further confirmed by the other inhibitors: 6-fluoromevalonate, Zaragozic acid A and U18666A, in the cholesterol biosynthesis pathway. Conversely, enhancement of cholesterol production increased rotavirus replication, suggesting that cholesterol homeostasis is relevant for rotavirus replication. The effects of all these compounds toward rotavirus were further confirmed with a clinical rotavirus isolate. We concluded that rotavirus replication is dependent on cholesterol biosynthesis. To be specific, inhibition of cholesterol synthesis can downregulate rotavirus replication; on the contrary, rotavirus replication is upregulated. Statin treatment is potentially an effective novel clinical anti-rotavirus strategy.

Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

Isolation of peste des petits ruminants virus using primary goat kidney cell culture from kidneys obtained at slaughter.

BACKGROUND: Traditionally isolation of peste des petits ruminant virus (PPRV) is performed in Vero cells that takes several blind passages before observing typical cytopathic effects (CPEs). As an alternate, researchers have been using lamb kidney (LK) cells but day-old lambs are difficult to obtain and requires animal sacrifice. OBJECTIVE: We established a primary goat kidney (GK) cell culture from the kidneys obtained at slaughter. METHODS: The kidney of Black Bengal goats were collected from slaughter house and processed to make single cell suspension. The cells were resuspended in appropriate culture medium and maintained under optimum culture condition. RESULTS: The 80% confluent monolayer of GK cells was obtained after 15-20 days post seeding. Upon infection with a field isolate of PPRV, the well-developed CPEs characterized by cell rounding, vacuolation in the cytoplasm and fusion of cells were observed after 48 hr post infection. Virus quantification in the culture supernatant revealed more viral RNA in GK cells than LK cells. The multicycle growth analysis of PPRV showed a steady increase in the virus loads in the culture supernatant of infected GK cells, suggesting an adaptation of the PPRV in GK cells. CONCLUSIONS: The findings suggest that primary GK cells can be successfully prepared from the mature kidney cortical tissues and can be used for the isolation of PPRV. This system could reduce the unnecessary sacrifice of lambs or kids. Since kidneys of slaughtered goats are available throughout the year, using this protocol primary cell culture from mature goat kidney can provide primary cells to the laboratory throughout the year.

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease 2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000 compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4 µM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies.

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

QuantIF: An ImageJ Macro to Automatically Determine the Percentage of Infected Cells after Immunofluorescence.

Counting labeled cells, after immunofluorescence or expression of a genetically fluorescent reporter protein, is frequently used to quantify viral infection. However, this can be very tedious without a high content screening apparatus. For this reason, we have developed QuantIF, an ImageJ macro that automatically determines the total number of cells and the number of labeled cells from two images of the same field, using DAPI- and specific-stainings, respectively. QuantIF can automatically analyze hundreds of images, taking approximately one second for each field. It is freely available as supplementary data online at MDPI.com and has been developed using ImageJ, a free image processing program that can run on any computer with a Java virtual machine, which is distributed for Windows, Mac, and Linux. It is routinely used in our labs to quantify viral infections in vitro, but can easily be used for other applications that require quantification of labeled cells.

Comparison of the sensitivity of three cell cultures to ORFV.

BACKGROUND: Contagious ecthyma (CE) appears in the countries and regions containing goat and sheep farms, and it is considered a global epidemic. CE not only severely endangers the healthy development of the sheep and goat industries but also threatens human health. For viral infectious diseases, fast and effective isolation and culture of the pathogen is critical for CE diagnosis, and for disease prevention and control. Therefore, the sensitivity of bovine Sertoli cells to ORFV was estimate in this study. RESULTS: The sensitivities of bovine Sertoli cells, primary neonatal bovine testicular cells, and Madin-Darby bovine kidney (MDBK) cell line to ORFV were compared. Our results showed that the isolated bovine Sertoli cells were sensitive to inoculated ORFV, and viral titers were approximately 1 log higher than those in primary neonatal bovine testicular cells and in MDBK cell lines. CONCLUSION: Appropriately sensitive cells for the highly efficient isolation and culture of the ORFV were obtained. Culture of ORFV using the Sertoli cells showed good consistency and stability and also avoided the risk of other pathogens presenting during viral culture using a primary cell line. In addition, using these passaged bovine Sertoli cells to proliferate ORFV may simplify the CE diagnosis process, thereby reducing detection time and cost. Hence, this test has important practical significance for the diagnosis of CE and the research on the pathogenic mechanism of ORFV.

Accelerated ISAV replication detection by cell culture methods combined with time-monitoring RT-qPCR.

Infectious salmon anaemia (ISA) is a viral disease that affects farmed Atlantic salmon (Salmo salar L.), often leading to mass mortalities. A quick detection of the ISA virus (ISAV) is crucial for decision-making and can prevent the occurrence of future outbreaks. Screening done by Canada's National Aquatic Animal Health Laboratory System (NAAHLS) uses quantitative reverse transcription PCR (RT-qPCR) followed by sequencing of PCR amplicons. As neither technique provides information regarding the infectivity of the virus, suspected virulent strains are subsequently tested using viral isolation. However, this stepwise process can require significant time to deliver results. To speed up this delivery, we have improved on these pre-existing techniques by combining the use of cell culture with RT-qPCR to detect replicative virus in as little as 5 days. Preliminary assays enabled the establishment of a minimal shift in Ct values over time, which is representative of viral replication in cultured cells. Subsequent blind panel analyses allowed the establishment of the optimal sampling days, as well as diagnostic sensitivity (DSe) and specificity (DSp) estimates. This method could be adopted not only by laboratories conducting diagnostic analyses for ISAV, but also for other slow-replicating viral agents that replicate through a budding mechanism.

Betanodavirus infection in primary neuron cultures from sole.

Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in sole.

Development of Human-Derived Cell Culture Lines for Recurrent Respiratory Papillomatosis.

Recurrent respiratory papillomatosis (RRP) is mainly caused by human papillomavirus (HPV) 6 and 11. While various adjuvant therapies have been reported, no effective therapy has been documented to universally "cure" this disease. In the era of precision medicine, it would be valuable to identify effective intervention based on drug sensitivity testing and/or molecular analysis. It is essential to be able to successfully carry out in vitro culture and expand tumor cells directly from patients to accomplish this goal. Here we report the result of successful culture of HPV-infected cell lines (success rate 70%, 9/13) that express the E6/E7 RNA transcript, using pathologic tissue biopsies from patients treated at our institution. The availability of such a system would enable ex vivo therapeutic testing and disease modeling.

Heterogeneity of HIV-1 Replication in Ectocervical and Vaginal Tissue Ex Vivo.

In clinical trials evaluating HIV-1 prevention products, ex vivo exposure of mucosal tissue to HIV-1 is performed to inform drug levels needed to suppress viral infection. Understanding assay and participant variables that influence HIV-1 replication will help with assay implementation. Demographic and behavioral data were obtained from 61 healthy women aged 21-45. Paired cervical tissue (CT) and vaginal tissue (VT) biopsies were collected and treated with HIV-1BaL or HIV-1JR-CSF, washed, and cultured. On days 3, 7, and/or 11, culture supernatant was collected, and viral replication was monitored by p24 ELISA. Tissue was extracted at study end, and HIV-1 relative RNA copies were determined by polymerase chain reaction. Cumulative p24 and RNA were log-transformed and analyzed using a linear mixed model, t-test, and an intraclass correlation coefficient (ICC). HIV replication was similar between CT and VT for each virus, but HIV-1BaL had 1.5 log10 and 0.9 log10 higher levels of p24 than HIV-1JR-CSF in CT and VT, respectively (p < .001), which correlated with HIV-1 relative RNA copies. Cumulative p24 and RNA copies in both tissues demonstrated low intraperson correlation for both viruses (ICC ≤0.513 HIV-1BaL; ICC ≤0.419 HIV-1JR-CSF). Enrollment into previous clinical studies in which genital biopsies were collected modestly decreased the HIV-1BaL cumulative p24 for CT, but not for VT. To improve the ex vivo challenge assay, viruses should be evaluated for replication in mucosal tissue before study implementation, baseline mucosal tissue is not needed if a placebo/no treatment group is included within the clinical trial, and previous biopsy sites should be avoided.

Two-Center Evaluation of Disinfectant Efficacy against Ebola Virus in Clinical and Laboratory Matrices.

Ebola virus (EBOV) in body fluids poses risk for virus transmission. However, there are limited experimental data for such matrices on the disinfectant efficacy against EBOV. We evaluated the effectiveness of disinfectants against EBOV in blood on surfaces. Only 5% peracetic acid consistently reduced EBOV titers in dried blood to the assay limit of quantification.

Comparison of Selected Nonlethal Samples from Adult Steelhead for Detection of Infectious Hematopoietic Necrosis Virus Using Cell Culture.

Nonlethal sampling techniques have previously been evaluated for detection of a variety of viral salmonid pathogens. However, many of these studies have used molecular assays in lieu of widely accepted cell culture techniques to evaluate the sampled tissues. Samples were collected from female steelhead Oncorhynchus mykiss broodstock using three potential nonlethal sampling methods (mucus/skin scrape, pectoral fin clip, and gill tissue biopsy) and evaluated for the presence of infectious hematopoietic necrosis virus (IHNV) via cell culture techniques. The results were compared with those from samples collected using a standard lethal sampling method (pooled anterior kidney and spleen tissues) applied to the same fish. Of the three nonlethal sampling techniques that were evaluated, fin clipping was the easiest and least invasive method. Furthermore, fin tissue was as sensitive as or more sensitive than kidney/spleen tissue for detecting IHNV in this population of fish. However, with the exception of gill tissue, the nonlethal samples did not appear to be appropriate surrogates for lethally collected tissues with regard to identifying an active infection in a particular fish. Nevertheless, nonlethal sampling coupled with cell culture appears to be suitable for helping to define the IHNV status of a steelhead population. Received July 27, 2016; accepted December 11, 2016.

A Highly Efficient and Simple Construction Strategy for Producing Recombinant Baculovirus Bombyx mori Nucleopolyhedrovirus.

The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.

IFN-γ Primes Keratinocytes for HSV-1-Induced Inflammasome Activation.

Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1ß and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1ß and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1ß and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1ß expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells.