Infectious Particles Research Articles

Inactivation of SARS-CoV-2 on salt-coated surfaces: an in vitro study

In the COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), face masks have become a very important safety measure against the main route of transmission of the virus: droplets and aerosols. Concerns that masks contaminated with SARS-CoV-2 infectious particles could be a risk for self-contamination have emerged early in the pandemic as well as solutions to mitigate this risk. The coating of masks with sodium chloride, an antiviral and non-hazardous to health chemical, could be an option for reusable masks. To assess the antiviral properties of salt coatings deposited onto common fabrics by spraying and dipping, the present study established an in vitro bioassay using three-dimensional airway epithelial cell cultures and SARS-CoV-2 virus. Virus particles were given directly on salt-coated material, collected, and added to the cell cultures. Infectious virus particles were measured by plaque forming unit assay and in parallel viral genome copies were quantified over time. Relative to noncoated material, the sodium chloride coating significantly reduced virus replication, confirming the effectiveness of the method to prevent fomite contamination with SARS-CoV-2. In addition, the lung epithelia bioassay proved to be suitable for future evaluation of novel antiviral coatings.

Herpes simplex virus infection

Two of the most prevalent human viruses worldwide, herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, respectively), cause a variety of diseases, including cold sores, genital herpes, herpes stromal keratitis, meningitis and encephalitis. The intrinsic, innate and adaptive immune responses are key to control HSV, and the virus has developed mechanisms to evade them. The immune response can also contribute to pathogenesis, as observed in stromal keratitis and encephalitis. The fact that certain individuals are more prone than others to suffer severe disease upon HSV infection can be partially explained by the existence of genetic polymorphisms in humans. Like all herpesviruses, HSV has two replication cycles: lytic and latent. During lytic replication HSV produces infectious viral particles to infect other cells and organisms, while during latency there is limited gene expression and lack of infectious virus particles. HSV establishes latency in neurons and can cause disease both during primary infection and upon reactivation. The mechanisms leading to latency and reactivation and which are the viral and host factors controlling these processes are not completely understood.

Inhalation exposure assessment techniques on ventilation dilution of infectious respiratory particles in a retrofitted hospital lung function room

Controlling the spread of respiratory infections in healthcare settings is important to prevent nosocomial infections. To assess the effectiveness of commonly used techniques in the hospital for evaluating the performance of building retrofit to reduce the risk of nosocomial infection, we employed computational fluid dynamics (CFD) simulation, real-time carbon dioxide (CO2) monitoring, microorganism culturing, and microorganism sequencing. These techniques can quantitatively assess the direct and indirect inhalation exposure risk of healthcare workers (HCWs) to infectious respiratory particles (IRPs) exhaled by patients in a hospital lung function room under two ventilation configurations. Originally, the ventilation system provided 2 air changes per hour (ACH) for clean air and 2 ACH for recirculated air. After retrofitting, the ventilation system was modified to increase clean air to 6 ACH and remove recirculated air. However, the assessment techniques yielded conflicting results regarding the percentage differences in exposure risk before and after the retrofit. The findings indicated that the retrofit system reduced indirect inhalation exposure risk, as estimated by real-time CO2 monitoring and microorganism culture, by over 50.00%. However, the CFD simulation indicated an increase in inhalation exposure risk when 6 ACH of clean air was supplied. These results suggest that removing recirculated air and increasing the air change rate are effective in reducing long-range indirect inhalation exposure in the lung function room. However, they are insufficient to minimize the direct inhalation exposure of patients exhaling IRPs in close proximity. Given the conflicting results obtained from the commonly accepted techniques used in our study, it is necessary to provide a better understanding of infection control associated with the ventilation dilution of IRPs for hospital architects, hospital healthcare workers, and built-environment researchers.

The CD133 and CD34 cell types in human umbilical cord blood have the capacity to produce infectious dengue virus particles

Although dengue virus (DENV) can establish infections in hematopoietic stem progenitor cells (HSPCs), there is little information on dengue virus persistent infection in CD34+ and CD133+ cell surface glycoproteins of hematopoietic stem cells (HSCs). CD34 and CD133 also function as cell–cell adhesion factors, which are present in umbilical cord blood (UCB). In this study, we sought to establish a persistent infection model of DENV infection in UCB using a prolonged period of infection lasting 30 days. Post-infection, the results exhibited a productive and non-productive phase of DENV production. Using a plaque assay, Western blot, and confocal microscopy, we demonstrated that CD133 and CD34 cells are target cells for DENV infection. Moreover, we showed that DENV particles can be recovered from the non-productive phase of DENV-infected CD34 and CD133 cells after co-incubation with Vero cells. We concluded that CD133 and CD34 retain their capacity to produce the infectious virus due to proliferation and their ability to repopulate, as deduced from a BrdU proliferation assay and flow cytometry analysis using t-distributed stochastic neighbor embedding. In summary, the platform to co-culture infected primitive HSCs from their non-productive phase onto Vero cells will give new insights into understanding the DENV dynamics in cell-to-cell transmission and reactivation of the virus.

The recombinant truncated envelope protein of West Nile virus adjuvanted with Alum/CpG induces potent humoral and T cell immunity in mice

West Nile virus (WNV) is a mosquito-transmitted flavivirus distributed globally for decades and can cause disease in humans and animals. So far, no WNV vaccine has been licensed for human use. Therefore, the development of novel candidate vaccines and the improvement of vaccination strategies is imperative. As the WNV envelope (E) glycoprotein plays an important role in mediating viral binding to cellular receptors and virus-cell membrane fusion, it is a critical target for the host humoral response. Here, we prepared a recombinant truncated envelope protein of WNV (rWNV-80E) and developed a WNV subunit vaccine formulation with a combination of aluminum hydroxide (alum) and a synthetic oligonucleotide CpG as adjuvants. C57BL/6 mice were immunized twice intramuscularly at 28-day intervals with 5 µg purified rWNV-80E adjuvanted with Alum/CpG. WNV E-specific IgG was detected by enzyme-linked immunosorbent assay and neutralizing antibodies (nAbs) was detected using single-round infectious particles of WNV. Furthermore, T cell immunity was detected by enzyme-linked immunospot assay and intracellular cytokine staining assay. Notably, rWNV-80E was highly immunogenic and elicited potent humoral and cell immunity, as evidenced by significant levels of IFN-γ and TNF-α secretion in the T cells of mice. In summary, the Alum/CpG-adjuvanted rWNV-80E subunit vaccine elicited potent and balanced B- and T-cell immunity in mice, and therefore it is a promising candidate vaccine that warrants further investigation for use in human or veterinary applications.

Paired Capture and FISH Detection of Individual Virions Enable Cell-Free Determination of Infectious Titers.

Early detection of viruses can prevent the uncontrolled spread of viral infections. Determination of viral infectivity is also critical for determining the dosage of gene therapies, including vector-based vaccines, CAR T-cell therapies, and CRISPR therapeutics. In both cases, for viral pathogens and viral vector delivery vehicles, fast and accurate measurement of infectious titers is desirable. The most common methods for virus detection are antigen-based (rapid but not sensitive) and polymerase chain reaction (PCR)-based (sensitive but not rapid). Current viral titration methods heavily rely on cultured cells, which introduces variability within labs and between labs. Thus, it is highly desirable to directly determine the infectious titer without using cells. Here, we report the development of a direct, fast, and sensitive assay for virus detection (dubbed rapid capture fluorescence in situ hybridization (FISH) or rapture FISH) and cell-free determination of infectious titers. Importantly, we demonstrate that the virions captured are "infectious," thus serving as a more consistent proxy of infectious titers. This assay is unique because it first captures viruses bearing an intact coat protein using an aptamer and then detects genomes directly in individual virions using fluorescence in situ hybridization (FISH); thus, it is selective for infectious particles (i.e., positive for coat proteins and positive for genomes).

Kinetics of serum O-glycosylated M-hepatitis B surface antigen with hepatocellular carcinoma history and nucleos(t)ide analogue therapy in hepatitis B patients.

A newly developed O-glycosylated M-hepatitis B surface antigen (HBsAgGi) measurement system can detect hepatitis B surface antigen (HBsAg) associated with infectious particles. We investigated the association of HBsAgGi levels with clinical parameters and a history of hepatocellular carcinoma (HCC) development in a cross-sectional cohort analysis (Study 1) as well as the quantitative changes in HBsAgGi during nucleos(t)ide analogue (NA) therapy in a longitudinal cohort analysis (Study 2). A total of 124 patients with genotype C chronic HBV infection were analysed in Study 1 to evaluate correlations of HBsAgGi with conventional HBV markers and HCC history. Among those, 36 patients receiving NA therapy were enrolled in Study 2 for quantitative comparisons between pre-treatment baseline and 48 weeks of NA therapy. In Study 1, serum HBsAgGi was significantly associated with HBsAg (r = .5857, p < .00001) and weakly but significantly correlated with HBV DNA (r = .2936, p = .001). Although HBsAgGi (p = .111) was comparable between HCC history (+) group and HCC history (-) group, the HBsAgGi/HBsAg ratio (p = .011) was significantly higher in HCC history (+) patients. In Study 2, HBsAgGi was significantly decreased after 48 weeks of NA therapy (p < .001). HBsAg findings were similar (p = .005) along with an HBV DNA reduction (p < .001). In the baseline hepatitis B e antigen (HBeAg) (+) subgroup, HBsAgGi decreased significantly between baseline and 48 weeks of NA (p = .005), while HBsAg was comparable (p = .051). Low HBsAg and high HBsAgGi were associated with a history of HCC development. HBsAgGi decreased significantly by 48-week NA therapy.

The Roles of the 5' and 3' Untranslated Regions in Human Astrovirus Replication.

Astroviruses are small nonenveloped single-stranded RNA viruses with a positive sense genome. They are known to cause gastrointestinal disease in a broad spectrum of species. Although astroviruses are distributed worldwide, a gap in knowledge of their biology and disease pathogenesis persists. Many positive-sense single-stranded RNA viruses show conserved and functionally important structures in their 5' and 3' untranslated regions (UTRs). However, not much is known about the role of the 5' and 3' UTRs in the viral replication of HAstV-1. We analyzed the UTRs of HAstV-1 for secondary RNA structures and mutated them, resulting in partial or total UTR deletion. We used a reverse genetic system to study the production of infectious viral particles and to quantify protein expression in the 5' and 3' UTR mutants, and we established an HAstV-1 replicon system containing two reporter cassettes in open reading frames 1a and 2, respectively. Our data show that 3' UTR deletions almost completely abolished viral protein expression and that 5' UTR deletions led to a reduction in infectious virus particles in infection experiments. This indicates that the presence of the UTRs is essential for the life cycle of HAstV-1 and opens avenues for further research.

Mild COVID-19 has no detrimental effect on semen quality

BackgroundAs of today, the effect of coronavirus disease 2019 (COVID-19) on male fertility remains unclear. Studies published so far have partly contradictory results, likely due to very small sample sizes and heterogeneous populations.To gain a deeper understanding of the impact of COVID-19 on male fertility, we performed a prospective case–control study, in which we examined the ejaculate of 37 subjects, including 25 subjects in the acute phase of mild COVID-19 and 12 subjects who did not suffer from COVID-19. Determination of semen parameters, severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) qPCR, and infectivity analysis were performed in the acute phase of the disease and in series.ResultsSemen parameter values did not differ significantly between subjects with mild COVID-19 and the control group. The serial examination of semen parameters revealed no significant changes between 4, 18, and 82 days after the onset of symptoms. SARS-CoV-2 RNA or infectious particles could not be detected in any ejaculate.ConclusionMild COVID-19 seems to have no detrimental effect on semen parameter values.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Tomato brown rugose fruit virus in aqueous environments - survival and significance of water-mediated transmission.

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.

2-Deoxy-D-glucose inhibits lymphocytic choriomeningitis virus propagation by targeting glycoprotein N-glycosylation

BackgroundIncreased glucose uptake and utilization via aerobic glycolysis are among the most prominent hallmarks of tumor cell metabolism. Accumulating evidence suggests that similar metabolic changes are also triggered in many virus-infected cells. Viral propagation, like highly proliferative tumor cells, increases the demand for energy and macromolecular synthesis, leading to high bioenergetic and biosynthetic requirements. Although significant progress has been made in understanding the metabolic changes induced by viruses, the interaction between host cell metabolism and arenavirus infection remains unclear. Our study sheds light on these processes during lymphocytic choriomeningitis virus (LCMV) infection, a model representative of the Arenaviridae family.MethodsThe impact of LCMV on glucose metabolism in MRC-5 cells was studied using reverse transcription-quantitative PCR and biochemical assays. A focus-forming assay and western blot analysis were used to determine the effects of glucose deficiency and glycolysis inhibition on the production of infectious LCMV particles.ResultsDespite changes in the expression of glucose transporters and glycolytic enzymes, LCMV infection did not result in increased glucose uptake or lactate excretion. Accordingly, depriving LCMV-infected cells of extracellular glucose or inhibiting lactate production had no impact on viral propagation. However, treatment with the commonly used glycolytic inhibitor 2-deoxy-D-glucose (2-DG) profoundly reduced the production of infectious LCMV particles. This effect of 2-DG was further shown to be the result of suppressed N-linked glycosylation of the viral glycoprotein.ConclusionsAlthough our results showed that the LCMV life cycle is not dependent on glucose supply or utilization, they did confirm the importance of N-glycosylation of LCMV GP-C. 2-DG potently reduces LCMV propagation not by disrupting glycolytic flux but by inhibiting N-linked protein glycosylation. These findings highlight the potential for developing new, targeted antiviral therapies that could be relevant to a wider range of arenaviruses.

Spatiotemporal transmission of infectious particles in environment: A case study of Covid-19

This study explores the dynamic transmission of infectious particles due to COVID-19 in the environment using a spatiotemporal epidemiological approach. We proposed a novel multi-agent model to simulate the spread of COVID-19 by considering several influencing factors. The model divides the population into susceptible and infected and analyzes the impact of different prevention and control measures, such as limiting the number of people and wearing masks on the spread of COVID-19. The findings suggest that reducing population density and wearing masks can significantly reduce the likelihood of virus transmission. Specifically, the research shows that if the population moves within a fixed range, almost everyone will eventually be infected within 1 h. When the population density is 50%, the infection rate is as high as 96%. If everyone does not wear a mask, nearly 72.33% of the people will be infected after 1 h. However, when people wear masks, the infection rate is consistently lower than when they do not wear masks. Even if only 25% of people wear masks, the infection rate with masks is 27.67% lower than without masks, which is strong evidence of the importance of wearing a mask. As people's daily activities are mostly carried out indoors, and many super-spreading events of the new crown epidemic also originated from indoor gatherings, the research on indoor epidemic prevention and control is essential. This study provides decision-making support for epidemic preventions and controls and the proposed methodology can be used in other regions and future epidemics.

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography.

Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.

Revealing viral and cellular dynamics of HIV-1 at the single-cell level during early treatment periods

While combination therapy completely suppresses HIV-1 replication in blood, functional virus persists in CD4+ Tcell subsets in non-peripheral compartments that are not easily accessible. To fill this gap, we investigated tissue-homing properties of cells that transiently appear in the circulating blood. Through cell separation and invitro stimulation, the HIV-1 "Gag and Envelope reactivation co-detection assay" (GERDA) enables sensitive detection of Gag+/Env+ protein-expressing cells down to about one cell per million using flow cytometry. By associating GERDA with proviral DNA and polyA-RNA transcripts, we corroborate the presence and functionality of HIV-1 in critical body compartments utilizing t-distributed stochastic neighbor embedding (tSNE) and density-based spatial clustering of applications with noise (DBSCAN) clustering with low viral activity in circulating cells early after diagnosis. We demonstrate transcriptional HIV-1 reactivation at any time, potentially giving rise to intact, infectious particles. With single-cell level resolution, GERDA attributes virus production to lymph-node-homing cells with central memory Tcells (TCMs) as main players, critical for HIV-1 reservoir eradication.

Molecular and Genetics-Based Systems for Tracing the Evolution and Exploring the Mechanisms of Human Norovirus Infections.

Human noroviruses (HuNoV) are major causes of acute gastroenteritis around the world. The high mutation rate and recombination potential of noroviruses are significant challenges in studying the genetic diversity and evolution pattern of novel strains. In this review, we describe recent advances in the development of technologies for not only the detection but also the analysis of complete genome sequences of noroviruses and the future prospects of detection methods for tracing the evolution and genetic diversity of human noroviruses. The mechanisms of HuNoV infection and the development of antiviral drugs have been hampered by failure to develop the infectious virus in a cell model. However, recent studies have demonstrated the potential of reverse genetics for the recovery and generation of infectious viral particles, suggesting the utility of this genetics-based system as an alternative for studying the mechanisms of viral infection, such as cell entry and replication.

Strategic self-limiting production of infectious HIV particles by CRISPR in permissive cells

Post-translational glycosylation of the HIV-1 envelope protein involving precursor glycan trimming by mannosyl oligosaccharide glucosidase (MOGS) is critically important for morphogenesis of virions and viral entry. Strategic editing of the MOGS gene in T lymphocytes and myeloid origin cells harboring latent proviral DNA results in the production of non-infectious particles upon treatment of cells with latency reversal agents. Controlled activation of CRISPR-MOGS by rebound HIV-1 mitigates production of infectious particles that exhibit poor ability of the virus to penetrate uninfected cells. Moreover, exclusive activation of CRISPR in cells infected with HIV-1 alleviates concern for broad off-target impact of MOGS gene ablation in uninfected cells. Combination CRISPR treatment of peripheral blood lymphocytes prepared from blood of people with HIV-1 (PWH) tailored for editing the MOGS gene (CRISPR-MOGS) and proviral HIV-1 DNA (CRISPR-HIV) revealed a cooperative impact of CRISPR treatment in inhibiting the production of infectious HIV-1 particles. Our design for genetic inactivation of MOGS by CRISPR exhibits no detectable off-target effects on host cells or any deleterious impact on cell survival and proliferation. Our findings offer the development of a new combined gene editing-based cure strategy for the diminution of HIV-1 spread after cessation of antiretroviral therapy (ART) and its elimination.

COVID-19-related hyperglycemia is associated with infection of hepatocytes and stimulation of gluconeogenesis

Occurrence of hyperglycemia upon infection is associated with worse clinical outcome in COVID-19 patients. However, it is still unknown whether SARS-CoV-2 directly triggers hyperglycemia. Herein, we interrogated whether and how SARS-CoV-2 causes hyperglycemia by infecting hepatocytes and increasing glucose production. We performed a retrospective cohort study including patients that were admitted at a hospital with suspicion of COVID-19. Clinical and laboratory data were collected from the chart records and daily blood glucose values were analyzed to test the hypothesis on whether COVID-19 was independently associated with hyperglycemia. Blood glucose was collected from a subgroup of nondiabetic patients to assess pancreatic hormones. Postmortem liver biopsies were collected to assess the presence of SARS-CoV-2 and its transporters in hepatocytes. In human hepatocytes, we studied the mechanistic bases of SARS-CoV-2 entrance and its gluconeogenic effect. SARS-CoV-2 infection was independently associated with hyperglycemia, regardless of diabetic history and beta cell function. We detected replicating viruses in human hepatocytes from postmortem liver biopsies and in primary hepatocytes. We found that SARS-CoV-2 variants infected human hepatocytesin vitrowith different susceptibility. SARS-CoV-2 infection in hepatocytes yields the release of new infectious viral particles, though not causing cell damage. We showed that infected hepatocytes increase glucose production and this is associated with induction of PEPCK activity. Furthermore, our results demonstrate that SARS-CoV-2 entry in hepatocytes occurs partially through ACE2- and GRP78-dependent mechanisms. SARS-CoV-2 infects and replicates in hepatocytes and exerts a PEPCK-dependent gluconeogenic effect in these cells that potentially is a key cause of hyperglycemia in infected patients.

The Reovirus σ1 Attachment Protein Influences the Stability of Its Entry Intermediate.

Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.