Antiviral adsorption activity of porous silicon nanoparticles against different pathogenic human viruses.

New viral infections, due to their rapid spread, lack of effective antiviral drugs and vaccines, kill millions of people every year. The global pandemic SARS-CoV-2 in 2019-2021 has shown that new strains of viruses can widespread very quickly, causing disease and death, with significant socio-economic consequences. Therefore, the search for new methods of combating different pathogenic viruses is an urgent task, and strategies based on nanoparticles are of significant interest. This work demonstrates the antiviral adsorption (virucidal) efficacy of nanoparticles of porous silicon (PSi NPs) against various enveloped and non-enveloped pathogenic human viruses, such as Influenza A virus, Poliovirus, Human immunodeficiency virus, West Nile virus, and Hepatitis virus. PSi NPs sized 60 nm with the average pore diameter of 2 nm and specific surface area of 200 m2/g were obtained by ball-milling of electrochemically-etched microporous silicon films. After interaction with PSi NPs, a strong suppression of the infectious activity of the virus-contaminated fluid was observed, which was manifested in a decrease in the infectious titer of all studied types of viruses by approximately 104 times, and corresponded to an inactivation of 99.99% viruses in vitro. This sorption capacity of PSi NPs is possible due to their microporous structure and huge specific surface area, which ensures efficient capture of virions, as confirmed by ELISA analysis, dynamic light scattering measurements and transmission electron microscopy images. The results obtained indicate the great potential of using PSi NPs as universal viral sorbents and disinfectants for the detection and treatment of viral diseases.

Mass spectrometry analysis of influenza virus reassortant clones does not reveal an influence of other viral proteins on S-acylation of hemagglutinin.

Hemagglutinin (HA) of influenza virus is S-acylated with stearate at a transmembrane cysteine and with palmitate at two cytoplasmic cysteines. The amount of stearate varies from 35 (in avian strains) to 12% (in human strains), although the acylation region exhibits only minor or even no amino acid differences between HAs. To address whether matrix proteins and neuraminidase affect stearoylation of HA, we used mass spectrometry to analyze laboratory reassortants containing avian virus HA and the internal proteins from a human virus. Only minor fluctuations in the amount of stearate were observed, implying that other viral proteins do not affect acylation of HA.

Comparative safety, immunogenicity, and efficacy of several anti-H5N1 influenza experimental vaccines in a mouse and chicken models (Testing of killed and live H5 vaccine).

OBJECTIVE: Parallel testing of inactivated (split and whole virion) and live vaccine was conducted to compare the immunogenicity and protective efficacy against homologous and heterosubtypic challenge by H5N1 highly pathogenic avian influenza virus. METHOD: Four experimental live vaccines based on two H5N1 influenza virus strains were tested; two of them had hemagglutinin (HA) of A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site, and two others had hemagglutinins from attenuated H5N1 virus A/Chicken/Kurgan/3/05, with amino acid substitutions of Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2 while maintaining the polybasic HA cleavage site. The neuraminidase and non-glycoprotein genes of the experimental live vaccines were from H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/Gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). Inactivated H5N1 and H1N1 and live H1N1 vaccine were used for comparison. All vaccines were applied in a single dose. Safety, immunogenicity, and protectivity against the challenge with HPAI H5N1 virus A/Chicken/Kurgan/3/05 were estimated. RESULTS: All experimental live H5 vaccines tested were apathogenic as determined by weight loss and conferred more than 90% protection against lethal challenge with A/Chicken/Kurgan/3/05 infection. Inactivated H1N1 vaccine in mice offered no protection against challenge with H5N1 virus, while live cold-adapted H1N1 vaccine reduced the mortality near to zero level. CONCLUSIONS: The high yield, safety, and protectivity of VN-Len and Ku-Len made them promising strains for the production of inactivated and live vaccines against H5N1 viruses.

Influenza virus hemagglutinin spike neck architectures and interaction with model enzymes evaluated by MALDI-TOF mass spectrometry and bioinformatics tools.

Interactions between model enzymes and the influenza virus hemagglutinin (HA) homotrimeric spike were addressed. We digested influenza virions (naturally occurring strains and laboratory reassortants) with bromelain or subtilisin Carlsberg and analyzed by MALDI-TOF mass spectrometry the resulting HA2 C-terminal segments. All cleavage sites, together with (minor) sites detected in undigested HAs, were situated in the linker region that connects the transmembrane domain to the ectodomain. In addition to cleavage at highly favorable amino acids, various alternative enzyme preferences were found that strongly depended on the HA subtype/type. We also evaluated the surface electrostatic potentials, binding cleft topographies and spatial dimensions of stem bromelain (homologically modeled) and subtilisin Carlsberg (X-ray resolved). The results show that the enzymes (∼45Å(3)) would hardly fit into the small (∼18-20Å) linker region of the HA-spike. However, the HA membrane proximal ectodomain region was predicted to be intrinsically disordered. We propose that its motions allow steric adjustment of the enzymes' active sites to the neck of the HA spike. The subtype/type-specific architectures in this region also influenced significantly the cleavage preferences of the enzymes.

Linker and/or transmembrane regions of influenza A/Group-1, A/Group-2, and type B virus hemagglutinins are packed differently within trimers.

Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1-H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.