Abstract P-37: Phosphorous Mapping in Inactivated SARS-Cov-2 Particles by Electron Energy Loss Spectroscopy

Background: The severe COVID-19 pandemic started in December 2019 is caused by the SARS-CoV-2 virus. The SARS-CoV-2 virion consists of a positive-sense single-stranded RNA (ssRNA), bound with the nucleocapsid N protein and surrounded by a lipid membrane with the embedded glycoprotein S and the transmembrane proteins M and E. The structure of inactivated SARS-CoV-2 virions is crucial for the development of vaccine-induced immunity. Here we characterized the nucleic acid distribution within β-propiolactone inactivated whole-virion SARS-CoV-2 vaccine CoviVac. Methods: We used EELS to verify the presence of phosphorus (P) inside the β-propiolactone inactivated virions. Electron microscopy was performed with a JEM-2100 200kV LaB6 transmission electron microscope (JEOL, Japan) equipped with a Gatan GIF Quantum ER energy filter (Gatan, USA) operating in spectrometer mode, along with a High-Angle Annular Dark-Field (HAADF) scanning transmission electron microscopy (STEM) detector. The cooling holder model 21090 (JEOL, Japan) was operated at -182 °С to reduce the contamination effects and to enhance the specimen's stability under the electron beam. We employ a negative stain with 2% (NH4)2MoO4 rather than uranyl acetate since the Uranium O4,5 peak (edge at 96 eV) is close to the P L2,3 peak (edge at 132 eV) and interferes with the accurate background interpolation. Results: The intensity under the P peak after the background subtraction was used for STEM-EELS mapping. We observed the characteristic P signal from the inner part of the virion but not from the bare grid. The observed P signal could arise from either viral RNA or lipids of the virus membrane, and since the P signal is highly heterogeneous, it is more likely to originate from RNA. Conclusion: So far, phosphorous mapping in individual virions using EELS was done only with samples prepared using highly specialized techniques, which minimized the sample thickness, including the substrate thickness. Here, we performed elemental mapping on ordinary samples of whole viruses. All investigated virions contained P signal, but its spatial distribution and intensity differed significantly. This clearly reflects the non-even distribution of the genomic RNA, which, apparently, accompanies their inner heterogeneity, previously observed by in-situ cryo-electron tomography

Abstract OR-8: Cryo-EM Structure of Mature Yellow Fever Virus

Background: Yellow fever virus (YFV) is the prototype virus of the genus Flavivirus. It is endemic to sub-Saharan Africa and tropical South America. YF disease ranges from asymptomatic to severe jaundice and hemorrhagic fever. The flavivirus virion core is enveloped by a lipid membrane with integrated membrane (M) proteins and envelope (E) proteins that form the outer surface of the virion. The Е protein provides stability to the viral particle and is responsible for early infection stages. Flaviviruses are heterogeneous in nature, which is related to their maturation process. Samples always contain mature, immature, half-mature, and damaged particles. Thus, cryo-EM is a method of choice for their structure determination. During the early stages of cryo-EM development, structures of flaviviruses were studied at 10–20 Å resolution. However, due to the progress of recent years, it became possible to determine flavivirus structures at a resolution of 5.6-2.6 Å. The cryo-EM method was used to obtain structural data of virions of dengue fever virus, Zika virus, TBE virus, etc. For YFV, only the cryo-EM structure of the immature virions at a low resolution of 25 Å was determined (Y. Zhang et al. 2003 doi:10.1093/emboj/cdg270). However, the structure of mature (most infectious) YFV particles is still unknown. Methods: Virus sample was produced in Vero cell culture. YFV-17D was inactivated and purified using ultracentrifugation. The concentration of viral particles in the target inactivated YFV-17D (iYFV-17D) was evaluated by spectrophotometry and by estimating the concentration of E protein determined by PAGE electrophoresis. Preliminary quality control of iYFV-17D sample was performed using negative staining TEM. Cryo-EM data were collected using cryo-TEM Krios (Thermo-Fisher, USA) at 300kV using DED Falcon II. Dataset was preprocessed using Warp. Further processing was performed in Relion 3.1 and CisTEM. Model building was carried out using Isolde, Phenix and Coot software. Results: A protocol of production and purification of highly concentrated (~2x1012) monodisperse inactivated iYFV-17D sample was developed. Cryo-EM structure of mature iYFV was solved at 4.1 Å resolution. The structure of YFV is similar to other known flavivirus structures, with 180 copies of protein E arranged in a herringbone pattern that makes up the icosahedral shell. Conclusion: The high-resolution structure of mature iYFV-17D allowed to elucidate special features of this flavivirus and may be useful for vaccine improvement and drug development

Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines

Introduction. Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. Objective. To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. Materials and methods. Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. Results. 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. Conclusion. Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models

Comprehensive Elucidation of the Role of L and 2A Security Proteins on Cell Death during EMCV Infection

The EMCV L and 2A proteins are virulence factors that counteract host cell defense mechanisms. Both L and 2A exhibit antiapoptotic properties, but the available data were obtained in different cell lines and under incomparable conditions. This study is aimed at checking the role of these proteins in the choice of cell death type in three different cell lines using three mutants of EMCV lacking functional L, 2A, and both proteins together. We have found that both L and 2A are non-essential for viral replication in HeLa, BHK, and RD cell lines, as evidenced by the viability of the virus in the absence of both functional proteins. L-deficient infection led to the apoptotic death of HeLa and RD cells, and the necrotic death of BHK cells. 2A-deficient infection induced apoptosis in BHK and RD cells. Infection of HeLa cells with the 2A-deficient mutant was finalized with exclusive caspase-dependent death with membrane permeabilization, morphologically similar to pyroptosis. We also demonstrated that inactivation of both proteins, along with caspase inhibition, delayed cell death progression. The results obtained demonstrate that proteins L and 2A play a critical role in choosing the path of cell death during infection, but the result of their influence depends on the properties of the host cells

A Chemographic Audit of Anti-Coronavirus Structure-Activity Information from Public Databases (ChEMBL)

Discovery of drugs against newly emerged pathogenic agents like the SARS-CoV-2 coronavirus (CoV) must be based on previous research against related species. Scientists need to get acquainted with and develop a global oversight over so-far tested molecules. Chemography (herein used Generative Topographic Mapping, in particular) places structures on a human-readable 2D map (obtained by dimensionality reduction of the chemical space of molecular descriptors) and is thus well suited for such an audit. The goal is to map medicinal chemistry efforts so far targeted against CoVs. This includes comparing libraries tested against various virus species/genera, predicting their polypharmacological profiles and highlighting often encountered chemotypes. Maps are challenged to provide predictive activity landscapes against viral proteins. Definition of “anti-CoV” map zones led to selection of therein residing 380 potential anti-CoV agents, out of a vast pool of 800M organic compounds

Molecular Characteristics, Receptor Specificity, and Pathogenicity of Avian Influenza Viruses Isolated from Wild Ducks in Russia

Avian influenza viruses (AIV) of wild ducks are known to be able to sporadically infect domestic birds and spread along poultry. Regular surveillance of AIV in the wild is needed to prepare for potential outbreaks. During long-year monitoring, 46 strains of AIV were isolated from gulls and mallards in Moscow ponds and completely sequenced. Amino acid positions that affect the pathogenicity of influenza viruses in different hosts were tested. The binding affinity of the virus for receptors analogs typical for different hosts and the pathogenicity of viruses for mice and chickens were investigated. Moscow isolates did not contain well-known markers of pathogenicity and/or adaptation to mammals, so as a polybasic cleavage site in HA, substitutions of 226Q and 228G amino acids in the receptor-binding region of HA, and substitutions of 627E and 701D amino acids in the PB2. The PDZ-domain ligand in the NS protein of all studied viruses contains the ESEV or ESEI sequence. Although several viruses had the N66S substitution in the PB1-F2 protein, all Moscow isolates were apathogenic for both mice and chickens. This demonstrates that the phenotypic manifestation of pathogenicity factors is not absolute but depends on the genome context

Comparison of the Immunogenicity and Safety of Two Pediatric TBE Vaccines Based on the Far Eastern and European Virus Subtypes

Up to 10,000 cases of tick-borne encephalitis are registered annually, 20% of which occur in children under 17 years of age. A comparison of the immunogenicity and safety between a new pediatric Tick-E-Vac vaccine based on the TBEV strain Sofjin and FSME-IMMUN Junior vaccine was performed in the Sverdlovsk region. The vaccine strains differ from strains of the Siberian subtype of TBEV that dominates in the region. The study was performed on 163 children aged 1 to 15, who received one of the vaccines according to either a conventional or rapid vaccination schedule. Immunogenicity was assessed based on the seroprotection rates and titers of virus-neutralizing antibodies. There were no significant differences in either the immunogenicity or reactogenicity of the pediatric vaccines based on strains of the Far Eastern or European subtypes of TBEV. Under both vaccination schedules, 30 days after the second injection, seroprotection rates were 100% for Tick-E-Vac and greater than 95% for FSME-IMMUN Junior, while the geometric mean titer of TBEV-neutralizing antibodies was at least 2,4 log10 (1 : 250) for either vaccine. Fourteen days after the second injection according to the rapid schedule, seroprotection rates were significantly lower, ranging from 50% to 63% regardless of the vaccine used. The observed adverse reactions were mild or moderate for both vaccines under both vaccination schedules, with total adverse event rates of less than 25%. Reactogenicity was not associated with the gender or age of the recipients. There were no statistically significant differences in the incidence of adverse reactions between the group of subjects who were baseline seronegative or seropositive. However, 14 days after the second vaccine injection according to the rapid schedule, a statistically significant difference in nAbs titers was identified between groups of children with and without reported reactions

Substitution Arg140Gly in Hemagglutinin Reduced the Virulence of Highly Pathogenic Avian Influenza Virus H7N1

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses circulate in ducks, poultry and equines and have repeatedly caused outbreaks of disease in humans. The laboratory strain A/chicken/Rostock/R0p/1934 (H7N1) (R0p), which was previously derived from the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), was studied in this work to ascertain its biological property, genome stability and virulent changing mechanism. Several virus variants were obtained by serial passages in the chicken lungs. After 10 passages of this virus through the chicken lungs we obtained a much more pathogenic variant than the starting R0p. The study of intermediate passages showed a sharp increase in pathogenicity between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) was obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity in chicken and mice, R6p differs from R5p in the shape of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of receptor-binding specificity and the pH of conformational change of HA

Diversity and reassortment rate of influenza a viruses in wild ducks and gulls

Influenza A viruses (IAVs) evolve via point mutations and reassortment of viral gene segments. The patterns of reassortment in different host species differ considerably. We investigated the genetic diversity of IAVs in wild ducks and compared it with the viral diversity in gulls. The complete genomes of 38 IAVs of H1N1, H1N2, H3N1, H3N2, H3N6, H3N8, H4N6, H5N3, H6N2, H11N6, and H11N9 subtypes isolated from wild mallard ducks and gulls resting in a city pond in Moscow, Russia were sequenced. The analysis of phylogenetic trees showed that stable viral genotypes do not persist from year to year in ducks owing to frequent gene reassortment. For comparison, similar analyses were carried out using sequences of IAVs isolated in the same period from ducks and gulls in The Netherlands. Our results revealed a significant difference in diversity and rates of reassortment of IAVs in ducks and gulls.</p

Immunogenicity and Safety of Inactivated Sabin-Strain Polio Vaccine “PoliovacSin”: Clinical Trials Phase I and II

Global polio eradication requires both safe and effective vaccines, and safe production processes. Sabin oral poliomyelitis vaccine (OPV) strains can evolve to virulent viruses and result in poliomyelitis outbreaks, and conventional inactivated poliomyelitis vaccine (Salk-IPV) production includes accumulation of large stocks of neurovirulent wild polioviruses. Therefore, IPV based on attenuated OPV strains seems a viable option. To increase the global supply of affordable inactivated vaccine in the still not-polio free world we developed an IPV made from the Sabin strains–PoliovacSin. Clinical trials included participants 18–60 years of age. A phase I single-center, randomized, double-blind placebo-controlled clinical trial included 60 participants, who received one dose of PoliovacSin or Placebo. A phase II multicenter, randomized, double-blind, comparative clinical trial included 200 participants, who received one dose of PoliovacSin or Imovax Polio. All vaccinations were well tolerated, and PoliovacSin had a comparable safety profile to the Placebo or the reference Imovax Polio preparations. A significant increase in neutralizing antibody levels to polioviruses types 1–3 (Sabin and wild) was observed in PoliovacSin and Imovax Polio vaccinated groups. Therefore, clinical trials confirmed good tolerability, low reactogenicity, and high safety profile of the PoliovacSin and its pronounced immunogenic properties. The preparation was approved for clinical trials involving infants