Titers Of Infectious Virus Research Articles

A comprehensive approach for evaluating the virucidal performance of domestic laundry detergents under practical conditions.

We aimed to develop a method to assess the virucidal performance of domestic laundry in a lab-scale washing machine (Rotawash) based on EN 17658. For method development, virus recovery was investigated after drying on cotton carriers for three test viruses murine norovirus (MNV), modified vaccinia virus Ankara (MVA), and bovine coronavirus (BCoV), followed by washing simulations in flasks and Rotawash. MNV and MVA demonstrated sufficient recovery from carriers after drying and washing (up to 40°C and 60min). BCoV exhibited lower recovery, indicating less relevance as a test virus. Rotawash efficacy tests conducted with MNV, a resistant, non-enveloped virus, showed limited efficacy of a bleach-free detergent, aligning with results from a domestic washing machine. Rotawash washes achieved higher reductions in infectious virus titers than suspension tests, indicating the role of washing mechanics in virus removal. This study established a practical method to test the virucidal efficacy of laundry detergents in Rotawash, simulating domestic washing.

Extreme infectious titer variability in individual Aedes aegypti mosquitoes infected with Sindbis virus is associated with both differences in virus population structure and dramatic disparities in specific infectivity.

Variability in how individuals respond to pathogens is a hallmark of infectious disease, yet the basis for individual variation in host response is often poorly understood. The titer of infectious virus among individual mosquitoes infected with arboviruses is frequently observed to vary by several orders of magnitude in a single experiment, even when the mosquitoes are highly inbred. To better understand the basis for this titer variation, we sequenced populations of Sindbis virus (SINV) obtained from individual infected Aedes aegypti mosquitoes that, despite being from a highly inbred laboratory colony, differed in their titers of infectious virus by approximately 10,000-fold. We observed genetic differences between these virus populations that indicated the virus present in the midguts of low titer mosquitoes was less fit than that of high titer mosquitoes, possibly due to founder effects that occurred during midgut infection. Furthermore, we found dramatic differences in the specific infectivity or SI (the ratio of infectious units/viral genome equivalents) between these virus populations, with the SI of low titer mosquitoes being up to 10,000-fold lower than that of high titer mosquitoes. Despite having similar amounts of viral genomes, low titer mosquitoes appeared to contain less viral particles, suggesting that viral genomes were packaged into virions less efficiently than in high titer mosquitoes. Finally, antibiotic treatment, which has been shown to suppress mosquito antiviral immunity, caused an increase in SI. Our results indicate that the extreme variation that is observed in SINV infectious titer between individual Ae. aegypti mosquitoes is due to both genetic differences between virus populations and to differences in the proportion of genomes that are packaged into infectious particles.

Production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Cell culture-based production of vector-based vaccines and virotherapeutics is of increasing interest. The vectors used not only retain their ability to infect cells but also induce robust immune responses. Using two recombinant vesicular stomatitis virus (rVSV)-based constructs, we performed a proof-of-concept study regarding an integrated closed single-use perfusion system that allows continuous virus harvesting and clarification.Using suspension BHK-21 cells and a fusogenic oncolytic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV), a modified alternating tangential flow device (mATF) or tangential flow depth filtration (TFDF) systems were used for cell retention. As the hollow fibers of the former are characterized by a large internal lumen (0.75 mm; pore size 0.65 μm), membrane blocking by the multi-nucleated syncytia formed during infection could be prevented. However, virus particles were completely retained. In contrast, the TFDF filter unit (lumen 3.15 mm, pore size 2–5 μm) allowed not only to achieve high viable cell concentrations (VCC, 16.4–20.6×106 cells/mL) but also continuous vector harvesting and clarification. Compared to an optimized batch process, 11-fold higher infectious virus titers were obtained in the clarified permeate (maximum 7.5×109 TCID50/mL).Using HEK293-SF cells and a rVSV vector expressing a green fluorescent protein, perfusion cultivations resulted in a maximum VCC of 11.3×106 cells/mL and infectious virus titers up to 7.1×1010 TCID50/mL in the permeate. Not only continuous harvesting but also clarification was possible. Although the cell-specific virus yield decreased relative to a batch process established as a control, an increased space-time yield was obtained.Key points• Viral vector production using a TFDF perfusion system resulted in a 460% increase in space-time yield• Use of a TFDF system allowed continuous virus harvesting and clarification• TFDF perfusion system has great potential towards the establishment of an intensified vector production

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.

Comparing the Infectivity of Recent SARS-CoV-2 Omicron Sub-Variants in Syrian Hamsters.

Since the emergence of the first omicron SARS-CoV-2 variant at the end of 2021, several sub-variants have evolved and become predominant in the human population, showing enhanced transmissibility and ability to (partly) escape the adaptive immune response. The XBB sub-variants (e.g., EG.5.1) have become globally dominant. Besides the XBB sub-variants, a phylogenetically distinct variant, i.e., BA.2.86, is also circulating; it carries several mutations in the spike protein as compared to its parental BA.2 variant. Here, we explored the infectivity of the BA.2.86 and EG.5.1 sub-variants compared to the preceding BA.5 sub-variant in Syrian hamsters. Such preclinical models are important for the evaluation of updated vaccine candidates and novel therapeutic modalities. Following intranasal infection with either variant, throat swabs and lung samples were collected on days 3 and 4 post infection. No significant differences in viral RNA loads in throat swabs were observed between these sub-variants. However, the infectious virus titers in the lungs of EG.5.1- and BA.2.86-infected animals were significantly lower compared to the BA.5-infected ones. The lung pathology scores of animals infected with EG.5.1 and BA.2.86 were also markedly lower than that of BA.5 sub-variant. Together, we show that EG.5.1 and BA.2.86 sub-variants exhibit an attenuated replication in hamsters' lungs as compared to the BA.5 sub-variant.

PH-dependent virucidal effects of weak acids against pathogenic viruses

BackgroundWeak acids, such as acetic acid, show virucidal effects against viruses, and disinfectants are considered effective virucidal agents possibly because of their low pH, depending on the proton concentration. This study aimed to evaluate the efficacy of different weak acids (acetic, oxalic, and citric acids) and eligible vinegars under different pH conditions by comparing their inactivation efficacies against enveloped and non-enveloped viruses.MethodsAcetic, oxalic, and citric acids were adjusted to pH values of 2, 4 and 6, respectively. They were also diluted from 1 M to 0.001 M with distilled water. Enveloped influenza A virus (FulV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and non-enveloped feline calicivirus (FCV) were treated with adjusted weak acids for up to 30 min. These viruses were also reacted with white distilled vinegar (WDV) and grain-flavored distilled vinegar (GV) for up to 30 min. Infectious viral titers after the reactions were expressed as plaque-forming units per mL.ResultsAcetic acid showed virucidal effects against FulV at pH 4, whereas citric and oxalic acids did not. Acetic and citric acids inactivated SARS-CoV-2 at pH 2, whereas oxalic acid did not. All acids showed virucidal effects against FVC at pH 2; however, not at pH 4. The virucidal effects of the serially diluted weak acids were also reflected in the pH-dependent results. WDV and GV significantly reduced FulV titers after 1 min. SARS-CoV-2 was also susceptible to the virucidal effects of WDV and GV; however, the incubation period was extended to 30 min. In contrast, WDV and GV did not significantly inactivate FCV.ConclusionsThe inactivation efficacy of weak acids is different even under the same pH conditions, suggesting that the virucidal effect of weak acids is not simply determined by pH, but that additional factors may also influence these effects. Moreover, eligible vinegars, the main component of which is acetic acid, may be potential sanitizers for some enveloped viruses, such as FulV, in the domestic environment.

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.

Development of recombinant oyster heat shock protein 70 mediated in situ capture RT-qPCR to detect human norovirus and Tulane virus

Human noroviruses (HuNoVs) are the major foodborne pathogens that cause non-bacterial gastroenteritis globally. Conventional RT-qPCR is prone to counting free RNA in the sample, resulting in an inflated infectious virus titer. Porcine gastric mucin (PGM), broadly used as the capture unit of in situ capture RT-qPCR (ISC-RT-qPCR), can precisely capture intact norovirus without adsorbing free RNA. However, PGM is a complex mixture that is difficult to obtain, and the concentration of histo-blood group antigens in it is unclear, hampering its widespread use. This study introduced a newly discovered capture unit, recombinant oyster heat shock protein 70 (roHSP70), to replace PGM. The roHSP70-mediated ISC-RT-qPCR (roHSP-ISC-RT-qPCR) were developed and compared with the PGM-mediated ISC-RT-qPCR (PGM-ISC-RT-qPCR) and conventional column-based RNA extraction RT-qPCR (CBE-RT-qPCR). The results showed that roHSP70 can capture various genotypes of HuNoVs. The smaller concentration of roHSP70 (20.0 μg/mL) captured HuNoVs to a similar extent compared to PGM (1.0 mg/mL). In addition, roHSP-ISC-RT-qPCR can accurately detect the amount of authentic intact virus particles in heat-treated Tulane virus (TV) samples with an accuracy comparable to that of TCID50. Interestingly, roHSP-ISC-RT-qPCR gave lower titers than CBE-RT-qPCR when measuring cell-cultured TV with high titers. However, when measuring lower-titer TV in artificially contaminated oysters, roHSP-ISC-RT-qPCR instead gave significantly higher titers than CBE-RT-qPCR. Overall, roHSP-ISC-RT-qPCR had a similar excellent performance as the PGM-ISC-RT-qPCR in detecting intact norovirus in samples. Moreover, roHSP70 has broader application scenarios than PGM due to its single-identified component and easier accessibility.

Generation of quality-controlled SARS-CoV-2 variant stocks.

One of the main challenges in the fight against coronavirus disease 2019 (COVID-19) stems from the ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into multiple variants. To address this hurdle, research groups around the world have independently developed protocols to isolate these variants from clinical samples. These isolates are then used in translational and basic research-for example, in vaccine development, drug screening or characterizing SARS-CoV-2 biology and pathogenesis. However, over the course of the COVID-19 pandemic, we have learned that the introduction of artefacts during both in vitro isolation and subsequent propagation to virus stocks can lessen the validity and reproducibility of data. We propose a rigorous pipeline for the generation of high-quality SARS-CoV-2 variant clonal isolates that minimizes the acquisition of mutations and introduces stringent controls to detect them. Overall, the process includes eight stages: (i) cell maintenance, (ii) isolation of SARS-CoV-2 from clinical specimens, (iii) determination of infectious virus titers by plaque assay, (iv) clonal isolation by plaque purification, (v) whole-virus-genome deep-sequencing, (vi and vii) amplification of selected virus clones to master and working stocks and (viii) sucrose purification. This comprehensive protocol will enable researchers to generate reliable SARS-CoV-2 variant inoculates for in vitro and in vivo experimentation and will facilitate comparisons and collaborative work. Quality-controlled working stocks for most applications can be generated from acquired biorepository virus within 1 month. An additional 5-8 d are required when virus is isolated from clinical swab material, and another 6-7 d is needed for sucrose-purifying the stocks.

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFUmL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

Clinical, Virological, and Immunological Features in Cosmopolitan Genotype DENV-2-Infected Patients during a Large Dengue Outbreak in Sri Lanka in 2017.

In 2017, Sri Lanka experienced its largest dengue epidemic and reported severe and unusual presentations of dengue with high morbidity. This outbreak was associated with the reemergence of dengue virus-2 (DENV-2), with the responsible strain identified as a variant of the previously circulating DENV-2 cosmopolitan genotype. In this study, we characterized the DENV-2 cosmopolitan genotype from patients during this epidemic. Also, we identified host factors that contributed to the severity of dengue infection in patients infected with this particular virus. Ninety-one acute serum samples from patients at the National Hospital in Kandy were randomly selected. Of these, 40.2% and 48.9% were positive for dengue IgM and IgG, respectively. NS1 antigen levels were significantly higher in primary infections. The severe dengue (SD) and dengue with warning signs (DWWS) groups exhibited significantly higher viral genome and infectivity titers than the dengue without warning signs (DWoWS) group. The highest viremia level was observed in SD patients. As for host cytokine response, interferon α (IFN-α) levels were significantly higher in the DWoWS group than in the DWWS and SD groups, whereas interleukin (IL)-12p40 and tumor necrosis factor α (TNF-α) levels in SD patients were significantly higher than in the other two groups. The TNF-α, IL-4, and monocyte chemoattractant protein-1 concentrations were positively correlated with NS1 antigen levels. From whole-genome analysis, NS4 had the highest frequency of amino acid variants, followed by the E gene. Our study suggests that viremia levels and immune responses contributed to SD outcomes, and these findings may help in identifying an effective therapeutic strategy against SD infection.

Evaluation of Activity of Zinc Oxide Nanoparticles on Human Rotavirus and Multi-Drug Resistant Acinetobacter Baumannii.

Rotaviruses are the cause of acute gastroenteritis and severe diarrheal diseases in children worldwide. Children under the age of five are more susceptible to rotavirus infections. Due to such as the lack of effective drugs and supportive therapy only, the development of new antiviral agents against rotaviruses. Multi-drug-resistant Acinetobacter baumannii is also one of the most challenging Gram-negative bacteria to control and treat due to its antibiotic resistance, particularly in intensive care units. This study aimed to investigate the activity of zinc oxide nanoparticles against human rotavirus and multi-drug resistant Acinetobacter baumannii. The standard 50% tissue culture infectious dose method and the real-time polymerase chain reaction assay were used to investigate the effects of zinc oxide nanoparticles on rotaviruses. The well diffusion and the minimum inhibitory concentration method were used to assess the antibacterial activity of zinc oxide nanoparticles against Acinetobacter baumannii. 300 μg/ml of zinc oxide nanoparticles demonstrated the highest anti-rotavirus effects, resulting in a 3.16 logarithmic decrease in virus infectious titer, and a four-unit increase in the cycle threshold value of the real-time polymerase chain reaction assay compared to the untreated control (P value <0.001 and P value = 0.005, respectively). The diameter of the inhibition zone of zinc oxide nanoparticles solution against Acinetobacter baumannii was 17 mm. The minimum inhibitory concentration results of the zinc oxide nanoparticles solution against Acinetobacter baumannii was 1.56 mg/ml. Our findings showed that zinc oxide nanoparticles could be considered a promising antimicrobial compound.

Rosmarinic Acid Inhibits Rift Valley Fever Virus: In Vitro , Computational and Analytical Studies

Aim: The antiviral potentials of rosmarinic acid (RA) against Rift Valley fever (RVF) virus were investigated. Methods: Antiviral activity was investigated by evaluating the reduction in the viral infectivity titer. Computational and LC–MS studies were performed for investigating the mechanism of action. This is via testing the interaction between RA and its major metabolite with the key infectivity proteins and determination of RA cellular permeability. Results: A superior reduction in RVF infectivity titer (45.5%) was observed when RA was applied post-infection compared to 17.7% reduction following its application before infection in addition to time-dependent inactivation kinetics. Recorded data showed in-silico inhibitory potential of RA and its metabolite against RVFV cap-binding protein and glycoprotein C, which are integral for viral transcription. LC–MS revealed cellular inclusion of RA, supporting its intracellular viral interaction. Conclusion: These antiviral potentials might suggest a promising foundation for future anti-RVF drug development.

Pharmacological inhibition of bromodomain and extra-terminal proteins induces an NRF-2-mediated antiviral state that is subverted by SARS-CoV-2 infection.

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19.

Oral Favipiravir Exposure and Pharmacodynamic Effects in Adult Outpatients With Acute Influenza.

The pharmacokinetics of oral favipiravir and the relationships of plasma concentrations to antiviral effects are incompletely studied in influenza. Serial plasma samples were collected from adults with uncomplicated influenza who were randomized to favipiravir (1800 mg twice a day on day 1, 800 mg twice a day on days 2 to 5; n = 827) or placebo (n = 419) in 2 phase 3 trials. Post hoc analyses assessed the frequency of reaching an average minimum concentration (Cmin) ≥20 µg/mL, its association with antiviral efficacy, and factors associated with reduced favipiravir exposure. Wide interindividual variability existed in favipiravir concentrations, and this regimen failed to reach an average Cmin>20 µg/mL in 41%-43% of participants. Those attaining this threshold showed greater reductions in nasopharyngeal infectious virus titers on treatment days 2 and 3 and lower viral titer area under the curve compared to those who did not. Those with average Cmin <20 µg/mL had over 2-fold higher mean ratios of the metabolite T-705M1 to favipiravir, consistent with greater metabolism, and were more likely to weigh >80 kg (61.5%-64%). Higher favipiravir levels with average Cmin>20 µg/mL were associated with larger antiviral effects and more rapid illness alleviation compared to placebo and to favipiravir recipients with lower average Cmin values in uncomplicated influenza. Clinical Trials Registration . NCT1068912 and NCT01728753.

Resveratrol Treatment-induced Nuclear HMGB1 Retention is Critical for Inducing Host Interferon Responses Against Zika Virus

Background: Zika virus (ZIKV) infection is a public health concern and currently there is no specific therapeutic or approved vaccine. Resveratrol (RESV), a natural antiviral compound, has been shown to possess antiviral properties against ZIKV and other viral infections, but the mechanisms of action against ZIKV remain unknown. Objective: This study aimed to investigate the role of the high mobility group box 1 protein (HMGB1) in the underlying anti-ZIKV mechanisms of RESV. Methods: HMGB1 protein expression and ZIKV replication in both the RESV-treated wildtype (WT) and HMGB1-knockdown (shHMGB1) Huh7 cells were analyzed using ELISA, immunofluorescence assay, immunoblot assay, focus-forming assay and qRT-PCR. HMGB1’s role was explored by evaluating the changes in the type-1 interferon (IFN) response genes using the qRT-PCR and immunoblot assays. Results: The treatment of the ZIKV-infected WT Huh7 cells with RESV significantly reduced ZIKV titers by &gt;90% (P &lt; 0.001) at 48 and 72 hr pi in a dose-dependent manner and inhibited ZIKV-induced HMGB1 translocation (P &lt; 0.001), resulting in nuclear HMGB1 accumulation. Compared to the WT Huh7 cells, shHMGB1 Huh7 cells without RESV treatment showed a significant increase in the infectious virus titers and RNA with a maximum rise of 74% (P &lt; 0.001) and 65% (P &lt; 0.01), respectively. RESV treatment of the ZIKV-infected WT Huh7 cells significantly increased the MxA (one of the classical interferon-stimulated genes, ISGs) and IFN-β levels (P &lt; 0.05). The treatment of the infected shHMGB1 Huh7 cells with RESV showed a less effective antiviral response (P &gt; 0.05) and did not cause changes in the expressions of MxA and IFN-β. Conclusion: RESV possesses therapeutic activity against ZIKV infection and the mechanism of action is mainly attributed to HMGB1 nuclear retention, which could upregulate the type-1 IFN and ISGs.

Characterization of a quail suspension cell line for production of a fusogenic oncolytic virus.

The development of efficient processes for the production of oncolytic viruses (OV) plays a crucial role regarding the clinical success of virotherapy. Although many different OV platforms are currently under investigation, manufacturing of such viruses still mainly relies on static adherent cell cultures, which bear many challenges, particularly for fusogenic OVs. Availability of GMP-compliant continuous cell lines is limited, further complicating the development of commercially viable products. BHK21, AGE1. CR and HEK293 cells were previously identified as possible cell substrates for the recombinant vesicular stomatitis virus (rVSV)-based fusogenic OV, rVSV-NDV. Now, another promising cell substrate was identified, the CCX.E10 cell line, developed by Nuvonis Technologies. This suspension cell line is considered non-GMO as no foreign genes or viral sequences were used for its development. The CCX.E10 cells were thus thoroughly investigated as a potential candidate for OV production. Cell growth in the chemically defined medium in suspension resulted in concentrations up to 8.9 × 106 cells/mL with a doubling time of 26.6 h in batch mode. Cultivation and production of rVSV-NDV, was demonstrated successfully for various cultivation systems (ambr15, shake flask, stirred tank reactor, and orbitally shaken bioreactor) at vessel scales ranging from 15 mL to 10 L. High infectious virus titers of up to 4.2 × 108 TCID50 /mL were reached in orbitally shaken bioreactors and stirred tank reactors in batch mode, respectively. Our results suggest that CCX.E10 cells are a very promising option for industrial production of OVs, particularly for fusogenic VSV-based constructs.

Comparing molnupiravir and nirmatrelvir/ritonavir efficacy and the effects on SARS-CoV-2 transmission in animal models

Therapeutic options against SARS-CoV-2 are underutilized. Two oral drugs, molnupiravir and paxlovid (nirmatrelvir/ritonavir), have received emergency use authorization. Initial trials suggested greater efficacy of paxlovid, but recent studies indicated comparable potency in older adults. Here, we compare both drugs in two animal models; the Roborovski dwarf hamster model for severe COVID-19-like lung infection and the ferret SARS-CoV-2 transmission model. Dwarf hamsters treated with either drug survive VOC omicron infection with equivalent lung titer reduction. Viral RNA copies in the upper respiratory tract of female ferrets receiving 1.25 mg/kg molnupiravir twice-daily are not significantly reduced, but infectious titers are lowered by >2 log orders and direct-contact transmission is stopped. Female ferrets dosed with 20 or 100 mg/kg nirmatrelvir/ritonavir twice-daily show 1–2 log order reduction of viral RNA copies and infectious titers, which correlates with low nirmatrelvir exposure in nasal turbinates. Virus replication resurges towards nirmatrelvir/ritonavir treatment end and virus transmits efficiently (20 mg/kg group) or partially (100 mg/kg group). Prophylactic treatment with 20 mg/kg nirmatrelvir/ritonavir does not prevent spread from infected ferrets, but prophylactic 5 mg/kg molnupiravir or 100 mg/kg nirmatrelvir/ritonavir block productive transmission. These data confirm reports of similar efficacy in older adults and inform on possible epidemiologic benefit of antiviral treatment.

In vitro and in silico studies of the antiviral activity of polyhydrated fullerenes against influenza A (H1N1) virus

As of today, influenza viruses remain a relevant target for the development of antiviral compounds due to their rapid evolution and acquisition of the resistance to existing drugs. Fullerene derivatives have already shown the ability to successfully interact with viruses, and polyhydrated fullerenes (or fullerenols) are particularly attractive due to their compatibility with biological fluids and low toxicity. Therefore, the goal of this work was to study the effect of two batches of a mixture of polyhydrated fullerenes with a mass ratio of 78.1% C60/C70 and 21.9% C76/C78/C84 on the influenza A (H1N1) virus. It was determined that the mixture of fullerenols, along with the low toxicity, showed high antiviral activity with a decrease in the viral infectious titer up to 4 orders of magnitude. In addition, studied fullerenols did not affect the hemagglutination process and did not show any significant prophylactic activity. With the help of molecular docking and molecular dynamics simulation, the likely target of fullerenols' action was determined—the binding site of the RNA primer of the viral RNA-dependent RNA polymerase. Therefore, we assume that the high antiviral effect of polyhydrated fullerenes on influenza A virus is related to their interaction with the viral RNA polymerase.

Improvement of Precision in Recombinant Adeno-Associated Virus Infectious Titer Assay with Droplet Digital PCR as an Endpoint Measurement.

Recombinant adeno-associated virus (rAAV) has been utilized successfully for in vivo gene delivery for treatment of a variety of human diseases. To sustain the growth of recombinant AAV gene therapy products, there is a critical need for the development of accurate and robust analytical methods. Fifty percent tissue culture infectious dose (TCID50) assay is an in vitro cell-based method widely used to determine AAV infectivity, and this assay is historically viewed as a challenge due to its high variability. Currently, quantitative PCR (qPCR) serves as the endpoint method to detect the amount of replicated viral genome after infection. In this study, we optimize the TCID50 assay by adapting endpoint detection with droplet digital PCR (ddPCR). We performed TCID50 assays using ATCC AAV-2 reference standard stock material across 18 independent runs. The cell lysate from TCID50 assay was then analyzed using both qPCR and ddPCR endpoint to allow for direct comparison between the two methods. The long-term 1-year side-by-side comparison between qPCR and ddPCR as endpoint measurement demonstrated improved interassay precision when the ddPCR method was utilized. In particular, after the addition of a novel secondary set threshold for infectivity scoring of individual wells, the average infectious titer of 18 runs is 6.45E+08 with % coefficient of variation (CV) of 42.5 and 5.63E+08 with % CV of 34.9 by qPCR and ddPCR, respectively. In this study, we offer improvements of infectious titer assay with (1) higher interassay precision by adapting ddPCR as an endpoint method without the need of standard curve preparation; (2) identification of a second "set threshold" value in infectivity scoring that improves assay precision; and (3) application of statistical analysis to identify the acceptance range of infectious titer values. Taken together, we provide an optimized TCID50 method with improved interassay precision that is important for rAAV infectious titer testing during process development and manufacturing.