Virus Particles Research Articles

Real-Time Visualization of HIV-1 RNA Detection Using Loop-Mediated Isothermal Amplification-Enabled Particle Diffusometry.

Isothermal nucleic acid amplification tests, NAATs, such as reverse transcription-loop-mediated isothermal amplification (RT-LAMP), offer promising capabilities to perform real-time semiquantitative detection of viral pathogens. These tests provide rapid results, utilize simple instrumentation for single-temperature reactions, support efficient user workflows, and are suitable for field use. Herein, we present a novel and robust method for real-time monitoring of HIV-1 RNA RT-LAMP utilizing a novel implementation of particle diffusometry (PD), a diffusivity quantification technique using fluorescent particles, to quantify viral concentration in nuclease-free water. We monitor changes in particle diffusion dynamics of 400 nm fluorescently labeled particles throughout the RT-LAMP of HIV-1 RNA in nuclease-free water, enabling measurement within 20 min and detection of concentrations as low as 25 virus particles per μL. Moreover, in a single-blind study, we demonstrate semiquantitative detection by accurately determining the initial concentration of an unknown HIV-1 RNA within a 10% absolute error margin. These results highlight the potential of real-time PD readout for quantifying HIV-1 RNA via RT-LAMP, offering promise for viral load monitoring of HIV and other chronic infections.

Direct and Ultrasensitive Bioluminescent Detection of Intact Respiratory Viruses.

Respiratory viruses such as SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) represent pressing health risks. Rapid diagnostic tests for these viruses detect single antigens or nucleic acids, which do not necessarily correlate with the amount of the intact virus. Instead, specific detection of intact respiratory virus particles may be more effective at assessing the contagiousness of a patient. Here, we report GLOVID, a modular biosensor platform to detect intact virions against a background of "free" viral proteins in solution. Our approach harnesses the multivalent display of distinct proteins on the surface of a viral particle to template the reconstitution of a split luciferase, allowing specific, single-step detection of intact influenza A and RSV virions corresponding to 0.1-0.3 fM of genomic units. The protein ligation system used to assemble GLOVID sensors is compatible with a broad range of binding domains, including nanobodies, scFv fragments, and cyclic peptides, which allows straightforward adjustment of the sensor platform to target different viruses.

Spike and nucleocapsid antibody dynamics following SARS-CoV-2 infection and vaccination: Implications for sourcing COVID-19 convalescent plasma from routinely collected blood donations.

COVID-19 convalescent plasma (CCP) remains a treatment option for immunocompromised patients; however, the current FDA qualification threshold of ≥200 BAU/mL of spike antibody appears to be relatively low. We evaluated the levels of binding (bAb) and neutralizing antibodies (nAb) on serial samples from repeat blood donors who were vaccinated and/or infected to inform criteria for qualifying CCP from routinely collected plasma components. Donors were categorized into four groups: (1) infected, then vaccinated, (2) vaccinated then infected during the delta, or (3) omicron waves, (4) vaccinated without infection. IgG Spike and total Nuclecapsid bAb were measured, along with S variants and nAb titers using reporter viral particle neutralization. Mean S IgG bAb peaks after infection alone were lower than after primary and booster vaccinations, and higher after delta and omicron infection in previously vaccinated donors. Half-lives for S IgG ranged from 34 to 66 days after first infection/vaccination events and up to 108 days after second events. The levels of S IgG bAb and nAb were similar across different variants, except for omicron, which were lower. Better correlations of nAb with bAb were observed at higher levels (hybrid immunity) than at the current FDA CCP qualifying threshold. Routine plasma donations from donors with hybrid immunity had high S bAb and potent neutralizing activity for 3-6 months after infection. In donations with high (>4000 BAU/mL) S IgG, >95% had high nAb titers (>500) against ancestral and variant S, regardless of COVID-19 symptoms. These findings provide the basis for test-based criteria for qualifying CCP from routine blood donations.

Monitoring for PERV Following Xenotransplantation.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A, PERV-B and PERV-C can be released as infectious virus particles and PERV-A and PERV-B can infect human cells in culture. PERV-C does not infect human cells, but high-titer recombinant PERV-A/C can infect them. Retroviruses are able to induce immunosuppression and/or tumors in the infected host. Numerous methods have been developed to study PERV in donor pigs. No PERV infections were observed in infection experiments as well as in preclinical and clinical xenotransplantation trials. Despite this, several strategies have been developed to prevent PERV infection of the recipient. PCR-based and immunological methods are required to screen xenotransplant recipients. Since the proviruses are integrated into the pig genome, PERV infection has to be distinguished from microchimerism, e.g., the presence of pig cells in the recipient, which is common in xenotransplantation. Sensitive PCR methods using pig short interspersed nuclear elements (SINE) sequences allow to detect pig cells easily. Virus infection can also be detected by an increase of viral genomic or mRNA in human cells. The method of choice, however, is to screen for specific antibodies against PERV using different recombinant PERV proteins, purified viruses or peptides.

Effect of human reovirus strain R-92 on tumor cell lines

Background. Among all the new methods and approaches, virotherapy with oncolytic viruses, both in combination with immunotherapy and without it, shows high efficiency in various phases of clinical trials and good tolerance by patients.Aim. To study the sensitivity of some immortalized cancer cell lines to the R-92 strain of human reovirus with cell characteristics at the ultrastructural level.Materials and methods. The study was carried out on cell lines of HeLa, A549, U87MG. Cells were planted in an amount of 15 thousand per well of a 96-well plate and after adhesion, the virus was inoculated by adding a medium containing virus particles in 4 tenfold dilutions (approximately 10 9 –106 particles per ml). Next, the cells were cultured for 24 h, after which the number of living cells in the wells was determined indirectly using the methyl tetrazolium test, which was carried out according to standard methods. To study the ultrastructure of infected cells, cells were seeded into a T25 flask and inoculated with the virus at the maximum concentration. After 24 h of cultivation, the cells were fixed in a 2.5 % glutaraldehyde solution in phosphate buffer for 1 h, after which they were washed three times in phosphate buffer and samples were processed for TEM according to standard methods.Results. Diluting the virus 1000 times led to a decrease in the cytostatic effect in all three cultures to a level practically no different from the control. HeLa turned out to be the most sensitive culture to reovirus. In the experiment, the number of living cells decreased to 60.4 ± 10.2 % compared to the control during incubation with the maximum number of viral particles and to 63.7 ± 16.2 % with a tenfold dilution of the virus. This indicator was significantly lower than in the other two studied cultures under these cultivation conditions (p <0.001).In addition, at the maximum virus concentration, the A549 culture was less sensitive than the U87MG culture (p <0.01). At lower concentrations of viral particles, the average viability of the studied cell lines did not differ significantly from each other. Analysis of electron diffraction patterns showed that the virus successfully replicates in the cytoplasm of the studied cultures, but is not released from the cell, which is apparently due to the short incubation period. TEM also showed cell damage characteristic of apoptosis or necroptosis, uniformly expressed in all studied cultures.Conclusion. Cell lines A549, HeLa and U87MG, according to the results of the methyl tetrazolium test, demonstrate different sensitivity to the human reovirus strain P-92. The TEM picture of cells from infected cultures showed signs of the development of apoptosis or necroptosis.

Immunogenicity of various variants of Ebola and Marburg virus glycoprotein genes in recombinant adenoviral vectors

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

HACE2 upregulation and participation of macrophages and clear cells in the immune response of epididymis to SARS-CoV-2 in K18-hACE2 mice.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus caused the coronavirus disease 2019 pandemic, and the prevalence of deaths among men is higher than among women. The epididymis, divided into caput, corpus, and cauda, shows a region-specific immunity. The K18-hACE2 mouse expresses human angiotensin-converting enzyme 2 (hACE2), the receptor that allows SARS-CoV-2 infection. However, studies using this transgenic mouse to evaluate the impact of this viral infection in epididymis have not yet been performed. We evaluated the expression of hACE2 in the epididymis of SARS-CoV-2-infected K18-hACE2 mice, and assessed the epididymal immune response, focusing on F4/80+ mononuclear phagocytes and tumor necrosis factor-alpha expression. The following analyses were performed in the epididymal sections of infected mice: epithelial height and duct diameter, birefringent collagen, Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labelling, immunoreactions for detection of hACE2, spike, FGF, V-ATPase, F4/80, tumor necrosis factor-alpha, and iNOS. Viral particles were identified under electron microscopy. hACE2, Rigi, Tgfb1 and Tnfa expression were also evaluated by real-time quantitative polymerase chain reaction. All epididymal regions expressed hACE2, which increased in all epididymal regions in the infected mice. However, the caput appeared to be the most infected region. Despite this, the caput region showed minimal changes while the cauda showed significant epithelial changes associated with increased iNOS immunoexpression. The F4/80+ mononuclear phagocyte area increased significantly in both stroma and epithelium. In addition to the epithelial and stromal mononuclear phagocytes, tumor necrosis factor-alpha was also detected in clear cells, whose cytoplasm showed a significant increase of this cytokine in the infected animals. The K18-hACE2 mouse is a useful model for evaluating the impact of SARS-CoV-2 infection in the epididymis. The infection induced hACE2 upregulation, favoring the virulence in the epididymis. The epididymal regions responded differentially to infection, and the activation of F4/80+ mononuclear phagocytes associated with the increased tumor necrosis factor-alpha immunolabeling in clear cells indicates a role of clear cells/mononuclear phagocytes immunoregulatory mechanisms in the epididymal immune response to SARS-CoV-2 infection.

An overview of SARS-CoV-2 viral proteins with relevance to improved diagnostic and therapeutic platforms

In the past 25 years, the world has witnessed outbreaks of illnesses in humans from three different coronaviruses. Both the SARS-CoV outbreak of 2003 and the MERS-CoV outbreak of 2013 resulted in overall low fatalities in part due to inefficient human-to-human spread of each virus. In contrast, SARS-CoV-2, which emerged in 2019, was highly efficient at human-to-human spread and caused a global pandemic resulting in millions of casualties. Zoonotic transmission of viruses, including the three coronaviruses, poses an ongoing threat that cannot be ignored. In this review, we have focused on the diagnostics and therapeutics fronts using SARS-CoV-2 as a model. Specifically, we have selected proteins associated with the virus particles as targets and discussed various platform technologies. These insights hold the potential to inform the development of more effective therapeutics and vaccines not only for SARS-CoV-2 but also for future viral pandemics, thus contributing to global health on a broader scale.

Dose-escalation, tolerability, and efficacy of intratumoral and subcutaneous injection of hemagglutinating virus of Japan envelope (HVJ-E) against chemotherapy-resistant malignant pleural mesothelioma: a clinical trial

The hemagglutinating virus of Japan envelope (HVJ-E) is an inactivated Sendai virus particle with antitumor effect and inducing antitumor immunity. However, its dosage and efficacy have not been verified. We conducted a phase I clinical study on chemotherapy-resistant malignant pleural mesothelioma (MPM) aiming to determine the recommended dosage for a phase II study through dose-limiting toxicity and evaluate HVJ-E’s preliminary efficacy. HVJ-E was administered intratumorally and subcutaneously to the patients with chemotherapy-resistant MPM. While no serious adverse events occurred, known adverse events of HVJ-E were observed. In the preliminary antitumor efficacy using modified response evaluation criteria in solid tumors (RECIST) criteria, three low-dose patients exhibited progressive disease, while all high-dose patients achieved stable disease, yielding disease control rates (DCRs) of 0% and 100%, respectively. Furthermore, the dose-dependent effect of HVJ-E revealed on DCR modified by RECIST and the baseline changes in target lesion size (by CT and SUL-peak; p < 0.05). Comparing targeted lesions receiving intratumoral HVJ-E with non-injected ones, while no clear difference existed at the end of the study, follow-up cases suggested stronger antitumor effects with intratumoral administration. Our findings suggest that HVJ-E could be safely administered to patients with chemotherapy-resistant MPM at both study doses. HVJ-E exhibited some antitumor activity against chemotherapy-resistant MPM, and higher doses tended to have stronger antitumor effects than lower doses. Consequently, a phase II clinical trial with higher HVJ-E doses has been conducted for MPM treatment. Trial registration number: UMIN Clinical Trials Registry (#UMIN000019345).

Safety and Humoral Immunogenicity of Different Dose Levels of Ad26.COV2.S as a 2-Dose Regimen in COVID-19 Vaccine-Naïve Healthy Adults: A Phase 3 Randomized Clinical Trial.

This study aimed to support the end-of-shelf life specification (2.5 × 1010 virus particles [vp]) for the standard Ad26.COV2.S dose (5 × 1010 vp). This randomized, double-blind Phase 3 study evaluated immunogenicity, reactogenicity, and safety of several Ad26.COV2.S dose levels (range 1.25 to 9 × 1010 vp) in 1593 adults between June 2021 and July 2023. Spike-binding antibody responses 28 days post-dose 1 were non-inferior for the 9 × 1010 vp, but not the 2.5 × 1010 vp group when compared with the standard dose. Non-inferiority was demonstrated in terms of spike-binding antibody responses 14 days post-dose 2 for each dose level, including the lowest dose level of 1.25 × 1010 vp, compared to 28 days after one dose and 14 days after two doses of the standard dose. Spike-binding antibody levels correlated well with virus neutralizing titers. There was no impact of pre-existing Ad26.COV2.S neutralizing titers on immunogenicity at any dose level. All dose levels were well tolerated. This study highlights the challenges associated with conducting clinical studies in a rapidly evolving environment and underscores the importance of platform data that can guide initial vaccine specifications such as shelf life during accelerated vaccine development. The present study supports the end-of-shelf life specifications for the approved Ad26.COV2.S dose, and could provide useful information in future vaccine developments using adenovirus vector vaccines.

Novel adomavirus associated with proliferative skin lesions affecting the dermal denticles of a sand tiger shark (Carcharias taurus).

A captive sand tiger shark (Carcharias taurus) presented with progressive, hard, raised, miliary skin lesions localized to the lateral trunk and peduncle. Histopathologic evaluation of biopsy samples revealed dysplastic proliferation of odontogenic epithelium with the production of collagenous material. Inclusion bodies and viral particles were not observed with light or transmission electron microscopy, respectively. However, using next generation sequencing with Illumina MiSeq and PCR followed by Sanger sequencing, the complete genome of a novel adomavirus, tentatively named sand tiger shark adomavirus (STAdoV), was obtained from the affected tissue. The genome was circular and 18.5 kilobases with bidirectionally transcribed genes, namely EO1, EO2 & 4, EO3, LO4, LO5, LO6, LO7, LO8, and SET. In situ hybridization using RNAscope® technology and a STAdoV specific probe localized viral DNA to the nuclei of proliferating epithelial cells. Adomaviruses are an emerging viral group with structural and replicative genes sharing a complex evolutionary history with adenoviruses and small circular DNA tumor viruses including papillomaviruses and polyomaviruses. Adomaviruses are described in a number of fish species in association with both necrotizing and proliferative diseases. BLAST analysis of the viral genome revealed greatest nucleotide identity (71.29%) to guitarfish adomavirus (GAdoV), another elasmobranch virus associated with proliferative (epidermal) skin lesions. Lesions in the index animal persisted for approximately 1 year during which time four conspecifics developed similar proliferations. Ultimately, lesions in all sharks regressed spontaneously without recurrence for 2 years.

B-217 Effective Diagnostic Platform and Method to Differentiate the Infectious From Non-Infectious Viruses With Nucleic Acid Detection Workflow

Abstract Background While qPCR has demonstrated high sensitivity, specificity, and ability to deliver reliable quantitative data in clinical samples, such results do not differentiate between infectious and non-infectious viral particles. In view of the principle of the qPCR method itself, the amplification detects the nucleic acid (DNA/RNA) of the virus, not the virus itself. In many cases, it is still possible to detect DNA/RNA fragments from dead viruses in recovered patients, while they are not infectious anymore. This will lead to prolonged quarantine period or treatment for the patients, which could cause great psychological pressure to the patients and increased healthcare burden. Therefore, an effective detection method that can discriminate viable and inviable pathogens is of great significance. Methods SARS-CoV2 viral RNA, SARS-CoV-2 viruses, or clinical samples with SARS-CoV2 infection were diluted in Delta MTM (Molecular Transport Media) to the respective testing concentrations. The samples were incubated with intercalating dye (PMAxx 100nM) with or without Triton X-100 for 10 min. The mixture was then exposed to the photo-activation device for 5-15 min (@ temperature below 37° C). The samples were then subjected to the standard nucleic acid extraction &amp; purification steps with Qiagen viral RNA extraction kit. The extracted RNA was subjected to amplification with SARS-CoV2 Resolute 2.0 &amp; Fortunate 2.1 assays on BioRAD CFX96. Results The viability PCR (vPCR) efficiency was almost 100% for all SARS-CoV2 RNA input tested suggesting that without viral membrane protection, PMAxx dye effectively bound to the naked RNA after photoactivation process, and the bound RNA was not able to be amplified. The vPCR efficiency was further enhanced with addition of surfactant Triton X-100 suggesting the improved permeability of dye through the viral envelope facilitated the PMAxx binding to the nucleic acid. COVID19 positive clinical samples with viral load ≈ Ct 30 treated with or without PMAxx and photo-activation procedures showed distinctive ΔCt value ranging from 9.27±0.18 to 15.01±0.14. The Ct after dye treatment were all non-numeric (non-detectable) if cut-off Ct sets @ 42.0, indicating the complete inhibition after vPCR pretreatment. Since the clinical samples were undergone heat-inactivation pre-treatment, the result shown here indicating that all samples were non-infectious, which was consistent with the samples tested. Conclusions We developed an effective viability PCR detection method and proprietary vPCR device, which was specifically designed for effective dye photo-activation. In addition, the method takes less than 15 min and could be integrated into the existing standard nucleic acid extraction and amplification workflow. Comparing to the standard PCR test result, this method could provide the information not only on the positivity of the tested pathogen, but also its viability, which could be very essential information for public surveillance and diagnostic decisions for healthcare providers.

Tsg101 UEV Interaction with Nedd4 HECT Relieves E3 Ligase Auto-Inhibition, Promoting HIV-1 Assembly and CA-SP1 Maturation Cleavage.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101-Ub-Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

Imaging the immune sequelae of infection with SARS-CoV-2 in nonhuman primates by using two nanobody PET-tracers.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacts multiple anatomical sites. Whether this is due to the virus itself or is a secondary effect caused by the influx and activation of immune cells is not known. Positron emission tomography (PET) with immunoglobulins can provide insights into which sites and cells are activated in a living animal. Our aim is to use two nanobodies as tools to monitor (1) the distribution of antigen presenting cells (APC) by virtue of their Mafa-DR expression profile, (2) virus-infected cells and viral particles using a nanobody against the SARS-CoV-2 spike protein. Two [89Zr]-labeled nanobodies that target the SARS-CoV-2 spike protein and major histocompatability complex (MHC) class II antigens (Mafa-DR), respectively, are used to monitor their distribution during an experimental SARS-CoV-2 infection in a nonhuman primate model. Scans are obtained before infection and on Day 3 and 10 post infection (pi) in two macaques each. The [89Zr]anti-SARS-CoV-2 spike nanobody localized to SARS-CoV-2-associated lung lesions and the nasal mucosa, while the [89Zr]anti-human leukocyte antigen (HLA)-DR nanobody was predominantly found in non-affected lung tissue after infection. We also detected, pi, upregulation of the Mafa-DR signal, indicative of recruitment of professional APCs, in the superior sagittal sinus. [89Zr]-labeled nanobodies show recruitment of macrophages/monocytes in non-lesional lung tissue in cynomolgus macaques after experimental infection with SARS-CoV-2, as well as accumulation of the spike protein in both lung lesions and the nasal mucosa during infection. These results show the possibility of in vivo monitoring the quality and quantity of immune responses during the initial stages of an infection.

An improved high-resolution method for quantitative separation of empty and filled AAV8 capsids by strong anion exchange HPLC.

Cell and gene therapy (CGT) is a field of therapeutic medicine that aims to treat, prevent, and cure diseases using engineered cells (stem cells, immune cells, and differentiated adult or fetal cells), vectors [Adeno Associated Virus (AAV), Adeno Virus (AV), Herpes Simplex Virus (HSV), Baculo Virus (BV), Lenti Virus (LV), Retro Virus (RV), etc.], and other carriers [non-viral vectors, virus-like particles (VLP), Lipid Nano-Particles (LNP), etc.]. Among viral CGT vectors, adeno-associated viruses and lentiviruses (AAV and LV) are the most widely applied vector platforms. The presence of non-functional (empty or non-infectious) vectors that carry null or partial genes in the final drug product is classified as an impurity by the FDA. These impurities impair dosage accuracy and induce non-specific immunogenicity and variability in drug efficacy. These non-functional viral vectors in the drug product need to be elucidated following International Conference on Harmonization (ICH) guidelines for clinical manufacturing of the final drug product. This article showcases an ion-exchange chromatography (IEX) high-resolution method supporting ICH guidelines using commercially available AAV8 filled and empty capsids as reference standards. Our method successfully separated empty to full capsids with a resolution of 15 and sustained a linearity greater than 0.98 even under a wide range of empty or full viral particle concentrations (E+9 to E+13 vp/mL), which is an upgrade to other IEX capsid separation methods. The medium-throughput capacity and shorter sample processing time improve testing efficiency and save costs while delivering quality as value. The discussed method is a reliable and reproducible platform to precisely evaluate the presence of non-functional viral particles in AAV8 samples. Aligned with other orthogonal results, the method is a powerful tool to improve the quality of rAAV analytics.

HIV-1 assembly - when virology meets biophysics.

Cells naturally produce vesicles that bud from different lipid membranes using dedicated molecular machineries. Enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), also generate particles that bud from host cell membranes by hijacking cellular factors and signaling pathways similar to those involved in the budding of extracellular vesicles. HIV-1 buds from the host cell plasma membrane mainly via the self-assembly of Gag, a structural protein. Gag is a polyprotein that forms assembly complexes containing viral genomic RNA (gRNA), host cell lipids and proteins. HIV-1 Gag binds and segregates host cell plasma membrane lipids while self-assembling simultaneously on the gRNA and the plasma membrane. This self-assembly causes membrane bending and formation of a new viral particle with the help of host cell proteins, likely including cortical actin-associated factors. However, it is unclear whether the energy of Gag self-assembly is sufficient to generate new HIV-1 particles. In this Review, we discuss these processes in the light of the past and recent virology literature, incorporating lessons from studies on the quantitative biophysics of viral self-assembly, and explore how Gag might reorganize the plasma membrane and divert host cell membrane curving proteins and cortical actin-related factors to achieve particle assembly and budding.

Single particle tracking reveals through-membrane diffusion of bacteriophage during process disruption of virus filtration

In the biopharmaceutical industry, virus filters are crucial for ensuring the removal of endogenous and adventitious viruses as part of the viral clearance strategy. Although traditionally described as a size-exclusion mechanism, virus retention has a process-dependent nature where challenging conditions, such as process disruptions, may compromise membrane retention and significantly increase virus filtrate concentrations. The detailed mechanisms underlying this loss of retention are challenging to determine using traditional breakthrough experiments. In this work, single particle tracking and kinetic simulations were employed to connect individual particle behavior to the observed macroscopic losses in virus retention. Our experiments, using fluorescently labeled ΦX174 bacteriophage as a model parvovirus, replicated conditions representative of process disruptions within the Pegasus SV4, a homogeneous polymeric virus filtration membrane. During flow, phage particles retained were trapped within relatively large cavity spaces that had downstream constrictions aligned with the flow direction; the trapped particles were dynamic and exhibited significant intra-cavity motion. Upon flow stoppage, particles escaped from these retention locations rapidly, with approximately 90 % of previously trapped particles being remobilized for process disruption times ranging from 2 to 10 min, suggesting that local cavity escape had reached saturation at these timescales. Diffusion experiments within the membrane revealed isotropic and Fickian motion, hindered by more than an order of magnitude compared to diffusion in unconfined liquid. Despite the reduced mobility within the membrane, the substantial diffusion coefficient of 4.19 ± 0.06 μm2/s indicated that virus particles could travel tortuous but non-retentive pathways through the membrane on length scales equal to or greater than the membrane thickness during a disruption event. A 1D kinetic Monte-Carlo simulation successfully connected single-particle behavior to macroscopically observed virus release, indicating that significant diffusive release into the filtrate can occur even without the resumption of flow. This work provides crucial insights into the retention behavior of homogeneous membranes during periods of disruption, enabling the design of more robust mitigation strategies and filter designs.

Inhibitory activity of Euterpe oleracea Mart. fruit extract in West Nile virus infection

West Nile virus (WNV) is a neurovirulent arbovirus whose epidemic capacity is enhanced by the wide occurrence of competent vectors and susceptible avian amplifying hosts. In this study, we investigated the antiviral potential of Euterpe oleracea Mart. fruit extract (EoFE) in WNV infection of monkey kidney (Vero) cell cultures. A chromatographic authentication of the extract revealed a typical two-peak fingerprint attributable to the major anthocyanins of the fruit. As assessed by plaque assays in Vero cells, the extract showed a significant concentration-dependent antiviral effect when present throughout the infection procedure, reaching a maximum inhibition of 66.8% at 2 mg/mL without significant cytotoxicity or direct action on virus particles. A time-of-addition assay revealed that this anti-WNV effect was mostly exerted after virus entry, as incubation of Vero cells with EoFE before or during virus addition resulted in a nonsignificant decrease of infection efficiency. These results demonstrated a promising potential of EoFE in inhibiting WNV infection that can be further explored as an antiviral strategy.

MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals.

The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.

Generation and characterization of a novel ovariole cell line derived from Spodoptera frugiperda in China with sensitivity to both SfMNPV and AcMNPV

Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), belonging to the species Alphabaculovirus spofrugiperdae, has been recently registered as an insecticide in China. This virus has a specific effect on the global major agricultural pest Spodoptera frugiperda. To gain insights into viral infection, replication processes, and the complex formation of viral particles, in vitro studies using cell lines are essential tools. Although the IPLB-Sf9 and IPLB-Sf21 cell lines derived from S. frugiperda are widely used for studies on the infection and replication mechanisms of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), their capacity to produce viral polyhedra after SfMNPV infection is not optimal. To address this limitation, a novel cell line named IOZCAS-Sf-1 has been developed from a S. frugiperda population sourced Yunnan, China. The mitochondrial COX1 gene analysis confirmed the species origin of the IOZCAS-Sf-1 cell line. Furthermore, a comparative study was carried out to contrast the COX1 gene sequence of this novel cell line with that of IPLB-Sf9, highlighting the distinctions between the two. Importantly, the IOZCAS-Sf-1 cells exhibited a remarkable ability to generate polyhedra when infected with AcMNPV and SfMNPV, respectively. Consequently, this cellular lineage is considered a promising and valuable resource. It serves not only to investigate the molecular mechanisms of viral replication and its impact on host cells, but also to explore the transfection efficiency of SfMNPV DNA. This exploration further expands into its potential application in recombinant DNA experiments, laying a theoretical groundwork for the advancement of more effective biopesticides and sustainable agricultural practices.