Cell-Adapted Mutations and Antigenic Diversity of Influenza B Viruses in Missouri, 2019–2020 Season

Influenza B viruses (IBVs) are causing an increasing burden of morbidity and mortality, yet the prevalence of culture-adapted mutations in human seasonal IBVs are unclear. We collected 368 clinical samples from patients with influenza-like illness in Missouri during the 2019–2020 influenza season and recovered 146 influenza isolates including 38 IBV isolates. Of MDCK-CCL34, MDCK-Siat1, and humanized MDCK (hCK), hCK showed the highest virus recovery efficiency. All Missourian IBVs belonged to the Victoria V1A.3 lineage, all of which contained a three-amino acid deletion on the HA protein and were antigenically distant from the Victoria lineage IBV vaccine strain used during that season. By comparing genomic sequences of these IBVs in 31 paired samples, eight cell-adapted nonsynonymous mutations were identified, with the majority in the RNA polymerase. Analyses of IBV clinical sample–isolate pairs from public databases further showed that cell- and egg-adapted mutations occurred more widely in viral proteins, including the receptor and antibody binding sites on HA. Our study suggests that hCK is an effective platform for IBV isolation and that culture-adapted mutations may occur during IBV isolation. As culture-adapted mutations may affect subsequent virus studies and vaccine development, the knowledge from this study may help optimize strategies for influenza surveillance, vaccine strain selection, and vaccine development.

Performance evaluation of virus concentration methods for implementing SARS-CoV-2 Wastewater-based epidemiology emphasizing quick data turnaround.

Wastewater based epidemiology (WBE) has drawn significant attention as an early warning tool to detect and predict the trajectory of COVID-19 cases in a community, in conjunction with public health data. This means of monitoring for outbreaks has been used at municipal wastewater treatment centers to analyze COVID-19 trends in entire communities, as well as by universities and other community living environments to monitor COVID-19 spread in buildings. Sample concentration is crucial, especially when viral abundance in raw wastewater is below the threshold of detection by RT-qPCR analysis. We evaluated the performance of a rapid ultrafiltration-based virus concentration method using InnovaPrep Cp Select pipette tips and compared this to the established electronegative membrane filtration (EMF) method. We evaluated the sensitivity of SARS-CoV-2 quantification, surrogate virus recovery rate, and sample processing time. Results suggest that the Cp Select concentrator is more efficient at concentrating SARS-CoV-2 from wastewater compared to the EMF method. 30% of samples that tested negative when concentrated with the EMF method produced a positive signal with the Cp Select protocol. Increased recovery of the surrogate virus control using the Cp Select confirms this observation. We optimized the Cp Select protocol by adding AVL lysis buffer and sonication, to increase the recovery of virus. Sonication increased BCoV recovery by 19%, which seems to compensate for viral loss during centrifugation. Filtration time decreases by approximately 30% when using the Cp Select protocol, making this an optimal choice for building surveillance applications where a quick turnaround time is necessary.

Preclinical evaluation of an engineered oncolytic herpes simplex virus for pediatric osteosarcoma.

10040 Background: Osteosarcoma is the most common primary bone tumor in children. For those with relapsed or metastatic disease, the five-year survival rate is approximately 20%, and survivors often suffer from long-term disability from current therapies. The high morbidity and mortality for these patients highlight a great need for improved therapies. One such novel therapeutic approach is oncolytic herpes simplex virus (oHSV) immunovirotherapy. We previously demonstrated that M002, an engineered oHSV that contains deletions of the neurovirulence gene preventing infection of normal cells, effectively infects and kills neuroblastoma and rhabdomyosarcoma. Currently, similar oHSVs are being evaluated in early phase clinical trials for children and adults with relapsed or refractory brain tumors. To date, there has been limited investigation of oncolytic virotherapy in osteosarcoma. Thus, we sought to examine the ability of oHSV, M002, to infect and kill osteosarcoma cells in vitro. Methods: We evaluated two long-term passaged human osteosarcoma cell lines, U2-OS and MG-63. Flow cytometry was used to assess baseline expression of oHSV viral entry-mediated receptors (CD111, CD112, syndecan, HVEM). Single and multi-step viral recovery experiments measured virus infectivity and replication. Cells were infected with increasing multiplicity of infection (MOI) of M002, and cell viability was measured 72 hours post-infection via alamarBlue assay. Results: Both MG-63 and U2-OS cells expressed HSV entry molecules (Table) including high levels of the primary HSV entry molecule CD111. Single step virus recovery experiments in MG-63 cells infected at a MOI of 10 plaque-forming units (PFU)/cell demonstrated a 3 log-fold increase in virus titer from 12 to 24 hours post-infection. For multi-step experiments, MG-63 cells were infected with a MOI of 0.1 PFU/cell; viral replication significantly increased from 1.1x103 PFU at 6 hours post-infection to 3.8x1010 PFU at 72 hours post-infection. M002 successfully decreased osteosarcoma viability with a lethal dose in 50% of cells (LD50)of 2.82 and0.67 PFU/cell for MG-63 and U2-OS cells, respectively. Notably, at a virus MOI of 5 PFU/cell, viability was decreased by 64% ± 0.1% (p<0.001 vs control) in MG-63 cells and 96% ± 0.1% (p<0.001 vs control) in U2-OS cells. Conclusions: MG-63 and U2-OS osteosarcoma cells express high levels of HSV entry receptors. Virus recovery experiments demonstrated the ability of M002 to infect cells and replicate over time. The viability of osteosarcoma cells significantly decreased following infection with M002. These data suggest M002 may be a promising novel therapeutic option for patients with osteosarcoma and warrant further investigation for translation to the clinical setting.[Table: see text]

“ AFRICAN OFFICIAL DEVELOPMENT ASSISTANCE (ODA) DEMAND AND POLICY DIRECTIONIN THE POST-CORONA ERA.”

This study is based on a social network analysis of newspaper articles relating to the Corona pandemic contained in the African daily newspaper African News. The aim of the study is to analyse what life-changes have been brought about in Africa as a result of the Corona pandemic, so that this information can be used in official development assistance policies for Africa. For this purpose, a text network analysis method was used. As a result of the analysis, the life-changes experienced by African residents as a consequence of the Corona pandemic are summarized into five broad categories. The five topics are: the Government’s countermeasures against the virus; recovery measures in African countries; measures relating to personal health; Government measures relating to schools; and an increase in the number of deaths from Coronavirus. After the Coronavirus pandemic, the ​​aid that should be provided most urgently to Africa is in the fields of health and education. It is hoped that aid donors will refer to the points found in this analysis and reflect them in their aid policy.

Transcriptomic and genomic analysis highlights the basis of ZYMV tolerance in zucchini

Background: Cucurbita pepo is high susceptible to Zucchini yellow mosaic virus (ZYMV) and the resistance found in several wild species does not provide complete or broad-spectrum resistance. In this study, a source of tolerance introgressed in C. pepo (381e) from C. moschata, in True French (TF) background, was investigated 12 days post inoculation (DPI) at transcriptomic and genomic levels. Results: A comparative RNA-seq experiment on TF (susceptible to ZYMV) and 381e (tolerant to ZYMV), allowed to evaluate 33,000 expressed transcripts and to identify 146 differentially expressed genes (DEGs) in 381e, mainly involved in photosynthesis, transcription, cytoskeleton organization and callose synthesis. By contrast, the susceptible line True French triggered oxidative processes related to response to biotic stimulus, and key regulators of plant virus intercellular movement. Moreover, the genome mapping of transcripts allowed the identification of two chromosome regions rich in SNPs (Single Nucleotide Polymorphisms), putatively introgressed from C. moschata, containing genes exclusively expressed in 381e. Conclusion: The transcriptome reprogramming of 381e ZYMV challenged revealed a globally restoring of cellular activities and a reduced virus movement and replication. Furthermore, genes putatively involved in ZYMV tolerance were detected in C. moschata introgressed genomic regions. Our work provides new insight into the plant virus recovery process and a better understanding of the molecular basis of 381e tolerance.

Development of a downstream process for the production of an inactivated whole hepatitis C virus vaccine

There is a large unmet need for a prophylactic hepatitis C virus (HCV) vaccine to control the ongoing epidemic with this deadly pathogen. Many antiviral vaccines employ whole viruses as antigens. For HCV, this approach became feasible following the development of infectious cell culture systems for virus production. However, the lack of efficient downstream processes (DSP) for HCV purification poses a roadblock for the development of a whole virus vaccine. Using cell culture-derived genotype 1a HCV we developed a scalable and efficient DSP train, employing commonly used clarification and ultrafiltration techniques, followed by two membrane-based chromatography steps. For virus capture, steric exclusion chromatography using cellulose membranes was established, resulting in a virtually complete virus recovery with > 99% protein and 84% DNA depletion. Virus polishing was achieved by sulphated cellulose membrane adsorbers with ~ 50% virus recovery and > 99% protein and 90% DNA depletion. Additional nuclease digestion resulted in 99% overall DNA depletion with final DNA concentrations of 2 ng/mL. Process results were comparable for cell culture-derived HCV of another major genotype (5a). This study provides proof-of-concept for establishment of an efficient and economically attractive DSP with potential application for production of an inactivated whole virus vaccine against HCV for human use.

EU pacts help Vietnam’s recovery from virus crisis

Headline VIETNAM/EU: Pacts help Vietnam's post-virus recovery

Greece’s virus recovery forecasts are over-optimistic

Significance The authorities are gradually lifting pandemic-induced restrictions. The need to protect public health has given way to preserving the productive capacity so painfully built up in three years of economic recovery. The authorities hope for a V-shape recovery in 2021, driven primarily by a large fiscal stimulus. Impacts An unprecedented fall in the tourism sector will depress real estate market values. Greater flexibility in the EU fiscal framework would rekindle Athens’ efforts to reduce its primary budget surplus target. The government’s handling of the pandemic could promote the image of Greece as a relatively safe country to visit.

An IMF package will aid Egypt’s virus recovery efforts

Headline EGYPT: An IMF package will aid virus recovery efforts