SARS-CoV-2 Production in a Scalable High Cell Density Bioreactor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has demonstrated the value of pursuing different vaccine strategies. Vaccines based on whole viruses, a widely used vaccine technology, depend on efficient virus production. This study aimed to establish SARS-CoV-2 production in the scalable packed-bed CelCradleTM 500-AP bioreactor. CelCradleTM 500-AP bottles with 0.5 L working volume and 5.5 g BioNOC™ II carriers were seeded with 1.5 × 108 Vero (WHO) cells, approved for vaccine production, in animal component-free medium and infected at a multiplicity of infection of 0.006 at a total cell number of 2.2–2.5 × 109 cells/bottle seven days post cell seeding. Among several tested conditions, two harvests per day and a virus production temperature of 33 °C resulted in the highest virus yield with a peak SARS-CoV-2 infectivity titer of 7.3 log10 50% tissue culture infectious dose (TCID50)/mL at 72 h post-infection. Six harvests had titers of ≥6.5 log10 TCID50/mL, and a total of 10.5 log10 TCID50 were produced in ~5 L. While trypsin was reported to enhance virus spread in cell culture, addition of 0.5% recombinant trypsin after infection did not improve virus yields. Overall, we demonstrated successful animal component-free production of SARS-CoV-2 in well-characterized Vero (WHO) cells in a scalable packed-bed bioreactor.

Bioactive compounds from Matricaria chamomilla: structure identification, in vitro antiproliferative, antimigratory, antiangiogenic, and antiadenoviral activities

During our exploring the anticancer activity of some medicinal plants and their major metabolites, the aerial parts of the Egyptian Matricaria chamomilla (flowers and stems) were studied. GC–MS analysis of the organic soluble extracts of the flowers and stems fractions revealed the presence of 43 and 45 compounds, respectively. Individual chromatographic purification of the flowers and stems’ extracts afforded three major compounds. Structures of these compounds were identified by 1D- and 2D-NMR and HRESI-MS spectroscopic data as bisabolol oxide A (1) and (E)-tonghaosu (2) (as mixture of ratio 2:1) from the flowers extract, meanwhile apigenin-7-β-d-glucoside (3) from the stems fraction. Biologically, the chamomile extracts announced significant antiproliferative activities exceeded in potency by ∼1.5 fold in case of the stem, recording GI50 13.16 and 17.04 μg/mL against Caco-2 and MCF-7, respectively. Both fractions were approximately equipotent against the migration of the same cell type down to 10 μg/mL together, compounds 1, 2 but not 3, showed considerable growth inhibition of the same cells at GI50 13.36 and 11.83 μg/mL, respectively. Interestingly, they were able to suppress Caco-2 colon cancer cells migration at 5.8 μg/mL and potently inactivate the VEGFR2 angiogenic enzyme (1.5-fold relative to sorafenib. The obtained compounds and corresponding chamomile extracts were evaluated against Adeno-7 virus, revealing that both chamomiles’ extracts (flowers and stems) and their corresponding obtained compounds (1–3) were potent in their depletion to the Adeno 7 infectivity titer, however, the flower extract and compounds 1–2 were more effective than those of the stem extract and its end-product (3).

Trehalose improves PPR vaccine virus stability in diluent

Background: Specialized freeze-drying process is being used in the field for different thermostable vaccine preparation worldwide. The thermostability remains only in undiluted conditions. If dilution is made at the morning and used for the whole day, the vaccine efficacy is compromised at high ambient temperature. In this study, trehalose based specialized vaccine diluent was used to improve the stability of Peste des Petits Ruminants (PPR) vaccine in diluted condition. Methods: The available PPR vaccine was reconstituted with conventional diluent and with trehalose based test diluent. The diluted vaccine was kept at ambient temperature without maintaining any cool chain. Stability of diluted vaccine virus was further assessed in vivo and in vitro at different temperatures. Goats were vaccinated and Vero cells were infected with reconstituted vaccines and were assessed at 0, 3, 6, 9 and 24 hours post dilution. Antibody titer was measured and virus infectivity titer was determined in both cultured cell lysate and supernatant. The presence of the virus particles in Vero cell was confirmed by standard RT-PCR targeting Fusion (F) gene of PPR virus. Results: In vivo results revealed that the number of goats possessed antibodies to PPR virus was higher in trehalose based vaccine formulation than the conventional PBS based diluent. Reconstituted vaccine virus (using PBS and trehalose diluent) infected Vero cells produced 70-80% cytopathic effect (CPE) in 5th days of post infection. Both diluents produced and maintained infectivity titer from log10 TCID50 5.5 to log10 TCID50 3.6, until the use of vaccines incubated for 9 hours after dilution. On the other hand, at 24 hours of post dilution only trehalose formulated vaccine produced log10 TCID502.5 whereas no infectivity titer was observed at the same time using conventional one. Conclusion: The present study suggests that trehalose preserves the quality of reconstituted vaccine in terms of infectivity titers. Trehalose can be a diluent of choice for reconstitution of PPR vaccine in field.

Whole Transcriptome Analysis of Aedes albopictus Mosquito Head and Thorax Post-Chikungunya Virus Infection

Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes prolonged arthralgia in patients. After crossing the mosquito midgut barrier, the virus disseminates to tissues including the head and salivary glands. To better understand the interaction between Aedes albopictus and CHIKV, we performed RNASeq analysis on pools of mosquito heads and parts of the thorax 8 days post infection, which identified 159 differentially expressed transcripts in infected mosquitos compared to uninfected controls. After validation using RT-qPCR (reverse transcriptase-quantitative polymerase chain reaction), inhibitor of Bruton’s tyrosine kinase (BTKi), which has previously been shown to be anti-inflammatory in mammals after viral infection, was further evaluated for its functional significance. Knockdown of BTKi using double-stranded RNA in a mosquito cell line showed no significant difference in viral RNA or infectivity titer. However, BTKi gene knocked-down cells showed increased apoptosis 24 hours post-infection compared with control cells, suggesting involvement of BTKi in the mosquito response to viral infection. Since BTK in mammals promotes an inflammatory response and has been shown to be involved in osteoclastogenesis, a hallmark of CHIKV pathogenesis, our results suggest a possible conserved mechanism at play between mosquitoes and mammals. Taken together, these results will add to our understanding of Aedes Albopictus interactions with CHIKV.

Thermostability of Subpopulations of H2N3 Influenza Virus Isolates from Mallard Ducks

ABSTRACT Maintenance of avian influenza virus in waterfowl populations requires that virions remain infectious while in the environment. Temperature has been shown to negatively correlate with persistence time, which is the duration for which virions are infectious. However, thermostability can vary between isolates regardless of subtype, and it is not known whether this variation occurs when host and geographic location of isolation are controlled. In this study, we analyzed the thermostabilities of 7 H2N3 viruses isolated from mallard ducks in Alberta, Canada. Virus samples were incubated at 37°C and 55°C, and infectivity titers were calculated at different time points. Based on the rate of infectivity inactivation at 37°C, isolates could be grouped into either a thermosensitive or thermostable fraction for both egg- and MDCK-grown virus populations. Titers decreased more rapidly for isolates incubated at 55°C, and this loss of infectivity occurred in a nonlinear, 2-step process, which is in contrast with the consensus on thermostability. This suggests that stock samples contain a mixture of subpopulations with different thermostabilities. The rate of decrease for the sensitive fraction was approximately 14 times higher than that for the stable fraction. The presence of subpopulations is further supported by selection experiments and plaque purification, both of which result in homogenous populations that exhibit linear decreases of infectivity titer. Therefore, variation of thermostability of influenza virus isolates begins at the level of the population. The presence of subpopulations with high thermostability suggests that avian viruses can persist in water longer than previously estimated, thus increasing the probability of transmission to susceptible hosts.

Identification of Residues outside of the Receptor Binding Domain That Influence the Infectivity and Tropism of Porcine Endogenous Retrovirus

ABSTRACT Identification of determinants of human tropism of porcine endogenous retrovirus (PERV) is critical to understanding the risk of transmission of PERV to recipients of porcine xenotransplantation products. Previously, we showed that a chimeric envelope cDNA encoding the 360 N-terminal residues of the human-tropic PERV envelope class A (PERV-A) SU and the 130 C-terminal residues of the pig-tropic PERV-C SU and all of TM (PERV-A/C) showed a 100-fold decrease in infectivity titer on human cells (M. Gemeniano, O. Mpanju, D. R. Salomon, M. V. Eiden, and C. A. Wilson, Virology 346:108-117, 2006). To identify residues important for human cell infection, we performed site-directed mutagenesis on each of the nine residues, singly or in combination, that distinguish the C-terminal region of PERV-C from PERV-A. Of the nine amino acids, two single-amino-acid substitutions, Q374R and I412V, restored the infectivity of human cells to the chimeric PERV-A/C to a titer equivalent to that of PERV-A. In contrast, PERV-A/C mutant envelope Q439P resulted in undetectable infection of human cells and an approximately 1,000-fold decrease in control pig cells. Mutation of K441R rescued mutants that carried Q439P, suggesting an incompatibility between the proline residue at this position and the presence of KK in the proteolytic cleavage signal. We confirmed this incompatibility with vectors carrying PERV-A envelope mutant R462K that were also rendered noninfectious. Finally, tropism of vectors carrying PERV-C envelope mutants with only four amino acid changes in the C terminus of PERV-C envelope, NHRQ436YNRP plus K441R, was shifted to one similar to that of PERV-A. Our results show an important and previously unrecognized role for infectivity and tropism for residues at the C terminus of SU.