Infectious Particles Research Articles

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.

A Comprehensive Index for Evaluating the Effectiveness of Ventilation-Related Infection Prevention Measures with Energy Considerations: Development and Application Perspectives

In the wake of the COVID-19 pandemic, prioritizing indoor air quality has emerged as a crucial measure for preventing infections. Effective ventilation is vital in mitigating airborne pathogen transmission and maintaining a healthy indoor environment by diluting and removing infectious particles from enclosed spaces. However, increasing the supply of pathogen-free air to enhance infection control can lead to a rise in energy consumption. Nevertheless, evaluating the overall efficacy of ventilation-based infection prevention strategies while considering their energy requirements has posed challenges. This scientific paper introduces the ICEE (Infection Control’s Energy Efficiency) index, a newly developed simple integrated index to assess the effectiveness of ventilation strategies in reducing infection risks while accounting for associated energy demands. The paper reviews the current understanding of ventilation strategies, their impact on infection prevention, and their corresponding energy consumption. By employing a straightforward analytical approach, this metric offers a comprehensive framework to optimize ventilation systems for both infection prevention and energy efficiency. To quantify infection risk, a simplified equation model is utilized, incorporating factors such as ventilation effectiveness and filter efficiency, in case of recirculation. Energy demand is determined using approximations and relevant values from existing literature. Reference cases are defined, distinguishing between natural and mechanically ventilated scenarios, as these reference situations influence the energy-related effects of any implemented measures. The paper outlines the methodology employed to develop the index and illustrates its applicability through exemplary measures. The proposed index yields valuable insights for the design, operation, and retrofitting of ventilation systems, enabling informed decision-making towards fostering a healthier and more sustainable built environment.

Antiviral effect of peptoids on hepatitis B virus infection in cell culture

Although antimicrobial peptides have been shown to inactivate viruses through disruption of their viral envelopes, clinical use of such peptides has been hampered by a number of factors, especially their enzymatically unstable structures. To overcome the shortcomings of antimicrobial peptides, peptoids (sequence-specific N-substituted glycine oligomers) mimicking antimicrobial peptides have been developed. We aimed to demonstrate the antiviral effects of antimicrobial peptoids against hepatitis B virus (HBV) in cell culture. The anti-HBV activity of antimicrobial peptoids was screened and evaluated in an infection system involving the HBV reporter virus and HepG2.2.15-derived HBV. By screening with the HBV reporter virus infection system, three (TM1, TM4, and TM19) of 12 peptoids were identified as reducing the infectivity of HBV, though they did not alter the production levels of HBs antigen in cell culture. These peptoids were not cytotoxic at the evaluated concentrations. Among these peptoids, TM19 was confirmed to reduce HBV infection most potently in a HepG2.2.15-derived HBV infection system that closely demonstrates authentic HBV infection. In cell culture, the most effective administration of TM19 was virus treatment at the infection step, but the reduction in HBV infectivity by pre-treatment or post-treatment of cells with TM19 was minimal. The disrupting effect of TM19 targeting infectious viral particles was clarified in iodixanol density gradient analysis. In conclusion, the peptoid TM19 was identified as a potent inhibitor of HBV. This peptoid prevents HBV infection by disrupting viral particles and is a candidate for a new class of anti-HBV reagents.

Synthetic Cell Lines for Inducible Packaging of Influenza A Virus.

Influenza A virus (IAV) is a negative-sense RNA virus that causes seasonal infections and periodic pandemics, inflicting huge economic and human costs on society. The current production of influenza virus for vaccines is initiated by generating a seed virus through the transfection of multiple plasmids in HEK293 cells followed by the infection of seed viruses into embryonated chicken eggs or cultured mammalian cells. We took a system design and synthetic biology approach to engineer cell lines that can be induced to produce all viral components except hemagglutinin (HA) and neuraminidase (NA), which are the antigens that specify the variants of IAV. Upon the transfection of HA and NA, the cell line can produce infectious IAV particles. RNA-Seq transcriptome analysis revealed inefficient synthesis of viral RNA and upregulated expression of genes involved in host response to viral infection as potential limiting factors and offered possible targets for enhancing the productivity of the synthetic cell line. Overall, we showed for the first time that it was possible to create packaging cell lines for the production of a cytopathic negative-sense RNA virus. The approach allows for the exploitation of altered kinetics of the synthesis of viral components and offers a new method for manufacturing viral vaccines.

Vaccinia Virus Defective Particles Lacking the F17 Protein Do Not Inhibit Protein Synthesis: F17, a Double-Edged Sword for Protein Synthesis?

Vaccinia virus (Orthopoxvirus) F17 protein is a major virion structural phosphoprotein having a molecular weight of 11 kDa. Recently, it was shown that F17 synthesised in infected cells interacts with mTOR subunits to evade cell immunity and stimulate late viral protein synthesis. Several years back, we purified an 11 kDa protein that inhibited protein synthesis in reticulocyte lysate from virions, and that possesses all physico-chemical properties of F17 protein. To investigate this discrepancy, we used defective vaccinia virus particles devoid of the F17 protein (designated iF17- particles) to assess their ability to inhibit protein synthesis. To this aim, we purified iF17- particles from cells infected with a vaccinia virus mutant which expresses F17 only in the presence of IPTG. The SDS-PAGE protein profiles of iF17- particles or derived particles, obtained by solubilisation of the viral membrane, were similar to that of infectious iF17 particles. As expected, the profiles of full iF17- particles and those lacking the viral membrane were missing the 11 kDa F17 band. The iF17- particles did attach to cells and injected their viral DNA into the cytoplasm. Co-infection of the non-permissive BSC40 cells with a modified vaccinia Ankara (MVA) virus, expressing an mCherry protein, and iF17- particles, induced a strong mCherry fluorescence. Altogether, these experiments confirmed that the iF17- particles can inject their content into cells. We measured the rate of protein synthesis as a function of the multiplicity of infection (MOI), in the presence of puromycin as a label. We showed that iF17- particles did not inhibit protein synthesis at high MOI, by contrast to the infectious iF17 mutant. Furthermore, the measured efficiency to inhibit protein synthesis by the iF17 mutant virus generated in the presence of IPTG, was threefold to eightfold lower than that of the wild-type WR virus. The iF17 mutant contained about threefold less F17 protein than wild-type WR. Altogether these results strongly suggest that virion-associated F17 protein is essential to mediate a stoichiometric inhibition of protein synthesis, in contrast to the late synthesised F17. It is possible that this discrepancy is due to different phosphorylation states of the free and virion-associated F17 protein.

Nucleolin-RNA interaction modulates rotavirus replication.

Rotavirus infection is a leading cause of gastroenteritis in children worldwide; the genome of this virus is composed of 11 segments of dsRNA packed in a triple-layered protein capsid. Here, we investigated the role of nucleolin, a protein with diverse RNA-binding domains, in rotavirus infection. Knocking down the expression of nucleolin in MA104 cells by RNA interference resulted in a remarkable 6.3-fold increase in the production of infectious rhesus rotavirus (RRV) progeny, accompanied by an elevated synthesis of viral mRNA and genome copies. Further analysis unveiled an interaction between rotavirus segment 10 (S10) and nucleolin, potentially mediated by G-quadruplex domains on the viral genome. To determine whether the nucleolin-RNA interaction regulates RRV replication, MA104 cells were transfected with AGRO100, a compound that forms G4 structures and selectively inhibits nucleolin-RNA interactions by blocking the RNA-binding domains. Under these conditions, viral production increased by 1.5-fold, indicating the inhibitory role of nucleolin on the yield of infectious viral particles. Furthermore, G4 sequences were identified in all 11 RRV dsRNA segments, and transfection of oligonucleotides representing G4 sequences in RRV S10 induced a significant increase in viral production. These findings show that rotavirus replication is negatively regulated by nucleolin through the direct interaction with the viral RNAs by sequences forming G4 structures.IMPORTANCEViruses rely on cellular proteins to carry out their replicative cycle. In the case of rotavirus, the involvement of cellular RNA-binding proteins during the replicative cycle is a poorly studied field. In this work, we demonstrate for the first time the interaction between nucleolin and viral RNA of rotavirus RRV. Nucleolin is a cellular protein that has a role in the metabolism of ribosomal rRNA and ribosome biogenesis, which seems to have regulatory effects on the quantity of viral particles and viral RNA copies of rotavirus RRV. Our study adds a new component to the current model of rotavirus replication, where cellular proteins can have a negative regulation on rotavirus replication.

Respiratory particles: from analytical estimates to disease transmission

Respiratory particles containing infectious pathogens are responsible for a large number of diseases. To define health politics and save lives, it is important to study their transmission mechanisms, namely the path of particles once expelled. This path depends on several driving factors as intrinsic properties of particles, environmental aspects and morphology of the scenario. Following physical arguments and taking into account the results of experimental works, we consider a mathematical drift model for the mixture composed by two phases: air and particles. The relative motion between the two phases is described by a kinematic constitutive relation. We prove the stability of the model for fixed times and establish an a priori estimate for the total number of infectious particles. The upper bound of this estimate exhibits sound physical dependencies on the driving factors, in agreement with the experimental literature and mounting epidemiological evidences. Namely, we establish that the amount of particles expelled and their emission rate can explain why some people are superspreaders. Several numerical simulations illustrate the theoretical results.

Coordinated transcriptional response to environmental stress by a Synechococcus virus.

Viruses are a major control on populations of microbes. Often, their virulence is examined in controlled laboratory conditions. Yet, in nature, environmental conditions lead to changes in host physiology and fitness that may impart both costs and benefits on viral success. Phosphorus (P) is a major abiotic control on the marine cyanobacterium Synechococcus. Some viruses infecting Synechococcus have acquired, from their host, a gene encoding a P substrate binding protein (PstS), thought to improve virus replication under phosphate starvation. Yet, pstS is uncommon among cyanobacterial viruses. Thus, we asked how infections with viruses lacking PstS are affected by P scarcity. We show that the production of infectious virus particles of such viruses is reduced in low P conditions. However, this reduction in progeny is not caused by impaired phage genome replication, thought to be a major sink for cellular phosphate. Instead, transcriptomic analysis showed that under low P conditions, a PstS-lacking cyanophage increased the expression of a specific gene set that included mazG, hli2, and gp43 encoding a pyrophosphatase, a high-light inducible protein and DNA polymerase, respectively. Moreover, several of the upregulated genes were controlled by the host's phoBR two-component system. We hypothesize that recycling and polymerization of nucleotides liberates free phosphate and thus allows viral morphogenesis, albeit at lower rates than when phosphate is replete or when phages encode pstS. Altogether, our data show how phage genomes, lacking obvious P-stress-related genes, have evolved to exploit their host's environmental sensing mechanisms to coordinate their own gene expression in response to resource limitation.

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

Numerical evaluation of space form design for outpatient waiting space to reduce airborne infection risk in hospitals

The outpatient waiting space (OWS), with high density and frequent variations of occupants, represents a high-risk space for the spread of respiratory infectious diseases (RIDs) in hospitals. While previous studies have proposed strategies related to occupant behavior and environmental quality, there is a limited exploration of the impact of space form design on infection risk in OWSs. This study developed a numerical method for assessing the airborne infection risk, considering the non-uniform distribution of infectious particles and variations in occupant distributions. Validated computational fluid dynamics (CFD) simulations were employed to calculate the particle distributions. The intake fraction, accumulated throughout the waiting process, was then integrated into the Wells-Riley model for risk evaluation. Using the typical ventilation form of ceiling supply and ceiling return, with an air change per hour (ACH) set to 6 h−1 as the baseline, 12 typical space forms, including four space shapes (L shape, T shape, U shape, and O shape) and three entrance layouts (single-side entrance, middle entrance, and double-side entrances), were selected for comparisons. The results showed significant variations in infectious particle distributions across the 12 space forms, with average infection probabilities (IPs) ranging from 3.47 % to 19.75 %, suggesting that appropriate space form design has the potential to reduce IP by 16.28 %. These findings offer a novel perspective on mitigating airborne infection risk in hospitals by emphasizing the significance of architectural design. Moreover, precise space design guidance is proposed for OWS to safeguard human health against the spread of RIDs.

Evaluation of Non-Vector Transmission of Usutu Virus in Domestic Canaries (Serinus canaria).

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>102 TCID50). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Phosphorylation regulates viral biomolecular condensates to promote infectious progeny production

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication.

Opening a 60-year time capsule: sequences of historical poliovirus cold variants shed a new light on a contemporary strain.

Polioviruses (PVs) are positive strand RNA viruses responsible for poliomyelitis. Many PVs have been isolated and phenotypically characterized in the 1940s-50s for the purpose of identifying attenuated strains that could be used as vaccine strains. Among these historical PVs, only few are genetically characterized. We report here the sequencing of four PV strains stored for more than 60 years in a sealed box. These PVs are cold variants that were selected by Albert Sabin based on their capacity to multiply at relatively low temperatures. Inoculation of permissive cells at 25°C showed that two of the four historical virus stocks still contained infectious particles. Both viruses reached titres that were higher at 25°C than at 37°C, thus demonstrating that they were genuine cold variants. We obtained sequences that span virtually all the genome for three out of the four strains; a short sequence that partly covers the 5' untranslated region was recovered for the last one. Unexpectedly, the genome of one historical cold variant (which derives from PV-3 Glenn) displayed a very high nucleotide identity (above 95%) with that of a PV strain (PV-3 strain WIV14) sampled in China in 2014 and then classified as a highly evolved vaccine-derived PV. Our analyses made this hypothesis very unlikely and strongly suggested that Glenn and WIV14 shared a very recent common ancestor with one another. Some strains used to produce the inactivated polio vaccine were also very close to Glenn and WIV14 in the capsid-encoding region, but they had not been sequenced beyond the capsid. We therefore sequenced one of these strains, Saukett A, which was available in our collection. Saukett A and WIV14 featured an identity higher than 99% at the nucleotide level. This work provides original data on cold variants that were produced and studied decades ago. It also highlights that sequences of historical PV strains could be crucial to reliably characterize contemporary PVs in case of release from a natural reservoir or from a facility, which is of highest importance for the PV eradication program.

Monitoring HIV-1 Nuclear Import Kinetics Using a Chemically Induced Nuclear Pore Blockade Assay.

To integrate with host chromatin and establish a productive infection, HIV-1 must translocate the viral Ribonucleoprotein (RNP) complex through the nuclear pore complex (NPC). Current assay to measure HIV-1 nuclear import relies on a transient byproduct of HIV-1 integration failure called 2-LTR circles. However, 2-LTR circles require complete or near-complete reverse transcription and association with the non-homologous end joining (NHEJ) machinery in the nucleus, which can complicate interpretation of 2-LTR circle formation as a measure of nuclear import kinetics. Here, we describe an approach to measure nuclear import of infectious HIV-1 particles. This involves chemically induced dimerization of Nup62, a central FG containing nucleoporin. Using this technique, nuclear import of infectious particles can be monitored in both primary and cell culture models. In response to host factor depletion or restriction factors, changes in HIV-1 nuclear import can be effectively measured using the nuclear import kinetics (NIK) assay.

Nanoscale-localized multiplexed biological activation of field effect transistors for biosensing applications.

The rise in antibiotic-resistant pathogens, highly infectious viruses, and chronic diseases has prompted the search for rapid and versatile medical tests that can be performed by the patient. Field-effect transistor (FET)-based electronic biosensing platforms are particularly attractive due to their sensitivity, fast turn-around time, potential for parallel detection of multiple pathogens, and compatibility with semiconductor manufacturing. However, an unmet critical need is a scalable, site-selective multiplexed biofunctionalization method with nanoscale precision for immobilizing different types of pathogen-specific bioreceptors on individual FETs, preventing parallel detection of multiple targets. Here, we propose a paradigm shift in FET biofunctionalization using thermal scanning probe lithography (tSPL) with a thermochemically sensitive polymer. This polymer can be spin-coated on fully-fabricated FET chips, making this approach applicable to any FET sensor material and technology. Crucially, we demonstrate the spatially selective multiplexed functionalization capability of this method by immobilizing different types of bioreceptors at prescribed locations on a chip with sub-20 nm resolution, paving the way for massively parallel FET detection of multiple pathogens. Antibody- and aptamer-modified graphene FET sensors are then realized, achieving ultra-sensitive detection of a minimum measured concentrations of 3 aM of SARS-CoV-2 spike proteins and 10 human SARS-CoV-2 infectious live virus particles per ml, and selectivity against human influenza A (H1N1) live virus.

Mean time to infection by small diffusing droplets containing SARS-CoV-2 during close social contacts

<abstract><p>Airborne viruses such as SARS-CoV-2 are partly spread through aerosols containing viral particles. Inhalation of infectious airborne particles can lead to infection, a route that can be even more predominant than droplet or contact transmission. To study the transmission between a susceptible and an infected person, we estimated the distribution of arrival times of small diffusing aerosol particles to the inhaled region located below the nose until the number of particles reaches a critical threshold. Our results suggested that although contamination by continuous respiration can take approximately 90 min at a distance of 0.5 m, it is reduced to a few minutes when coughing or sneezing. Interestingly, there is not much difference between outdoors and indoors when the air is still. When a window is open inside an office, the infection time is reduced. Finally, wearing a mask leads to a delay in the time to infection. To conclude, diffusion analysis provides several key timescales of viral airborne transmission.</p></abstract>

Intranasal HD-Ad-FS vaccine induces systemic and airway mucosal immunities against SARS-CoV-2 and systemic immunity against SARS-CoV-2 variants in mice and hamsters.

The outbreak of coronavirus disease 19 (COVID-19) has highlighted the demand for vaccines that are safe and effective in inducing systemic and airway mucosal immunity against the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we developed a novel helper-dependent adenoviral vector-based COVID-19 mucosal vaccine encoding a full-length SARS-CoV-2 spike protein (HD-Ad-FS). Through intranasal immunization (single-dose and prime-boost regimens), we demonstrated that the HD-Ad-FS was immunogenic and elicited potent systemic and airway mucosal protection in BALB/c mice, transgenic ACE2 (hACE2) mice, and hamsters. We detected high titers of neutralizing antibodies (NAbs) in sera and bronchoalveolar lavages (BALs) in the vaccinated animals. High levels of spike-specific secretory IgA (sIgA) and IgG were induced in the airway of the vaccinated animals. The single-dose HD-Ad-FS elicited a strong immune response and protected animals from SARS-CoV-2 infection. In addition, the prime-boost vaccination induced cross-reactive serum NAbs against variants of concern (VOCs; Beta, Delta, and Omicron). After challenge, VOC infectious viral particles were at undetectable or minimal levels in the lower airway. Our findings highlight the potential of airway delivery of HD-Ad-FS as a safe and effective vaccine platform for generating mucosal protection against SARS-CoV-2 and its VOCs.

H3N2 Influenza Virus Pathogenesis, Transmission and Complications: A Narrative Review

The recent outbreak of the influenza virus has grown to be a significant issue and a matter of great concern in terms of controlling the impending influenza pandemic. The influenza virus poses a serious threat as it directly infects and impairs the immune system reaction and the respiratory tract. The virus effectively produces infectious viral particles when haemagglutinin molecules are properly cleaved at the respiratory epithelium. The mode of transmission is via droplets from an affected case. The lungs, responsible for the vital exchange of gases, can fail due to various mechanisms, including the destruction of epithelial cells, significant degradation of the extracellular matrix, and airway obstruction. Influenza is a primary cause of severe pneumonia, but it can be accompanied by further bacterial infections, commonly involving bacteria such as Streptococcus pneumoniae and S. aureus. Influenza infection increases the susceptibility to developing Acute Respiratory Distress Syndrome (ARDS) and bacterial sepsis. Both adults and children have a 30-50% chance of experiencing viral infections along with bacterial pneumonia. Notably, Influenza A virus (H3N2) influenza has been associated with a significant increase in admissions to intensive care units. Among the factors contributing to the development of ARDS, individuals between the ages of 36 and 55 years, pregnant women, and obese individuals are at a higher risk. However, infection with influenza viruses A (H3N2) or B, female sex, and influenza vaccination have been identified as protective factors against ARDS. Influenza infection increases the susceptibility to developing ARDS and bacterial sepsis. Disease progression can be limited by spreading awareness among the people about the factors responsible for transmission, clinical manifestations, and preventive methodologies.

Imaging HIV-1 Nuclear Import, Uncoating, and Proviral Transcription.

Live-cell imaging has become a powerful tool for dissecting the behavior of viral complexes during HIV-1 infection with high temporal and spatial resolution. Very few HIV-1 particles in a viral population are infectious and successfully complete replication (~1/50). Single-particle live-cell imaging enables the study of these rare infectious viral particles, which cannot be accomplished in biochemical assays that measure the average property of the entire viral population, most of which are not infectious. The timing and location of many events in the early stage of the HIV-1 life cycle, including nuclear import, uncoating, and integration, have only recently been elucidated. Live-cell imaging also provides a valuable approach to study interactions of viral and host factors in distinct cellular compartments and at specific stages of viral replication. Successful live-cell imaging experiments require careful consideration of the fluorescent labeling method used and avoid or minimize its potential impact on normal viral replication and produce misleading results. Ideally, it is beneficial to utilize multiple virus labeling strategies and compare the results to ensure that the virion labeling did not adversely influence the viral replication step that is under investigation. Another potential benefit of using different labeling strategies is that they can provide information about the state of the viral complexes. Here, we describe our methods that utilize multiple fluorescent protein labeling approaches to visualize and quantify important events in the HIV-1 life cycle, including docking HIV-1 particles with the nuclear envelope (NE) and their nuclear import, uncoating, and proviral transcription.