Virus-cell Interactions Research Articles

SPR-Based Kinetic Analysis of the Early Stages of Infection in Cells Infected with Human Coronavirus and Treated with Hydroxychloroquine.

Cell-based assays are a valuable tool for examination of virus–host cell interactions and drug discovery processes, allowing for a more physiological setting compared to biochemical assays. Despite the fact that cell-based SPR assays are label-free and thus provide all the associated benefits, they have never been used to study viral growth kinetics and to predict drug antiviral response in cells. In this study, we prove the concept that the cell-based SPR assay can be applied in the kinetic analysis of the early stages of viral infection of cells and the antiviral drug activity in the infected cells. For this purpose, cells immobilized on the SPR slides were infected with human coronavirus HCov-229E and treated with hydroxychloroquine. The SPR response was measured at different time intervals within the early stages of infection. Methyl Thiazolyl Tetrazolium (MTT) assay was used to provide the reference data. We found that the results of the SPR and MTT assays were consistent, and SPR is a reliable tool in investigating virus–host cell interaction and the mechanism of action of viral inhibitors. SPR assay was more sensitive and accurate in the first hours of infection within the first replication cycle, whereas the MTT assay was not so effective. After the second replication cycle, noise was generated by the destruction of the cell layer and by the remnants of dead cells, and masks useful SPR signals.

Severe Acute Respiratory Coronavirus-2: A Critical Review of Virus Biology, Genome and Pathophysiology

Severe Acute Respiratory Coronavirus-2 [SARS-CoV-2] emerged as a great threat to the world at the end of December 2019 in China. The SARS-CoV-2 evolved from a virus responsible for the SARS epidemic in 2002. The SARS-CoV-2 has a high rate of human-human transmission and originated from the bat. It has a close resemblance with bat-like-SARS-CoV compared to SARS-CoV; however, the Spike protein responsible for virus-host cell interaction possesses the least similarity with that of SARS-CoV. Cytokine Storm is associated with the severity of Covid-19 and leads to acute respiratory distress syndrome [ARDS] and/or multiple organ dysfunction syndromes [MODS]. In the current review article, the features of a novel coronavirus, including viral biology, genomic organisation, life cycle, pathophysiology and genetic diversity, have been discussed. The development of policies and plans which can prepare the world for future pandemics has also been proposed. In addition, the drug development pipelines, diagnostic facilities and management of such pandemics need an up-gradation to contain the current as well as future outbreaks.

Proteome Analysis in PAM Cells Reveals That African Swine Fever Virus Can Regulate the Level of Intracellular Polyamines to Facilitate Its Own Replication through ARG1.

In 2018, African swine fever broke out in China, and the death rate after infection was close to 100%. There is no effective and safe vaccine in the world. In order to better characterize and understand the virus–host-cell interaction, quantitative proteomics was performed on porcine alveolar macrophages (PAM) infected with ASFV through tandem mass spectrometry (TMT) technology, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). The proteome difference between the simulated group and the ASFV-infected group was found at 24 h. A total of 4218 proteins were identified, including 306 up-regulated differentially expressed proteins and 238 down-regulated differentially expressed proteins. Western blot analysis confirmed changes in the expression level of the selected protein. Pathway analysis is used to reveal the regulation of protein and interaction pathways after ASFV infection. Functional network and pathway analysis can provide an insight into the complexity and dynamics of virus–host cell interactions. Further study combined with proteomics data found that ARG1 has a very important effect on ASFV replication. It should be noted that the host metabolic pathway of ARG1-polyamine is important for virus replication, revealing that the virus may facilitate its own replication by regulating the level of small molecules in the host cell.

Studying SARS-CoV-2 with Fluorescence Microscopy.

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus–cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy—which in our opinion can provide important information about the molecular biology of SARS-CoV-2—are discussed.

Sphingolipids as Modulators of SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic with severe consequences for afflicted individuals and the society as a whole. The biology and infectivity of the virus has been intensively studied in order to gain a better understanding of the molecular basis of virus-host cell interactions during infection. It is known that SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) via its spike protein. Priming of the virus by specific proteases leads to viral entry via endocytosis and to the subsequent steps in the life cycle of SARS-CoV-2. Sphingosine and ceramide belong to the sphingolipid family and are abundantly present in cell membranes. These lipids were recently shown to interfere with the uptake of virus particles of SARS-CoV-2 into epithelial cell lines and primary human nasal cells in culture. The mechanisms of action were partly different, as sphingosine blocked, whilst ceramide facilitated viral entry. Acid sphingomyelinase (ASM) is vital for the generation of ceramide and functional inhibition of ASM by drugs like amitriptyline reduced SARS-CoV-2 entry into the epithelial cells. Recent data indicates that serum level of sphingosine-1-phosphate (S1P) is a prognostic factor for COVID-2 severity. Further, stimulation of sphingosine-1-phosphate receptor 1 (S1PR1) might also constrain the hyper-inflammatory conditions linked to SARS-CoV-2. Here, we review recent exciting findings regarding sphingolipids in the uptake of SARS-CoV-2 and in the course of COVID-19 disease. More studies are required on the mechanisms of action and the potential use of antidepressant drugs and sphingolipid modifiers in SARS-CoV-2 infections and in the treatment of the more serious and fatal consequences of the disease.

A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.

BRLF1-dependent viral and cellular transcriptomes and transcriptional regulation during EBV primary infection in B lymphoma cells

The immediate-early protein BRLF1 plays important roles in lytic infection of Epstein-Barr virus (EBV), in which it activates lytic viral transcription and replication. However, knowledge of the influence of BRLF1 on cellular gene expression and transcriptional reprogramming during the early lytic cycle remains limited. In the present study, deep RNA-sequencing analysis identified all differentially expressed genes (DEGs) and alternative splicing in B lymphoma cells subjected to wild-type and BRLF1-deficient EBV primary infection. The BRLF1-dependent cellular DEGs were annotated, and major differentially enriched pathways were related to DNA replication and transcription, immune and inflammatory responses, cytokine-receptor interactions and chemokine signaling and metabolic processes. Furthermore, analysis of BRLF1-binding proteins by mass spectrometry shows that BRLF1 binds to and cooperates with several transcription factors and components of the spliceosome and then influences both RNA polymerase II-dependent transcription and pre-mRNA splicing. The RTA-binding RRE motifs or specific motifs of unique cooperative transcription factors in viral and cellular DEG promoter regions indicate that BRLF1 employs different strategies for regulating viral and cellular transcription. Thus, our study characterized BRLF1-dependent cellular and viral transcriptional profile during primary infection and then revealed the comprehensive virus-cell interaction and alterations of transcription during EBV primary infection and lytic replication.

Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro

An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal (irreversible) activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC50) for i) reducing virus-induced cytopathic effect of 0.002–0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019–0.032 mg/mL in Vero E6 cells and 0.030–0.037 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, irreversibly reducing SARS-CoV-2 infectivity by >99.9% (>3 log10) within 1 min of exposure, and up to >99.999% (>5 log10) shown at astodrimer sodium concentrations of 10–30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to irreversible inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a topically administered agent for SARS-CoV-2 therapeutic applications.

3D Visualization of Immune Cell Populations in HIV-Infected Tissues via Clearing, Immunostaining, Confocal, and Light Sheet Fluorescence Microscopy.

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immune Deficiency Syndrome (AIDS), is a major global health concern with nearly 40 million individuals infected worldwide and no widely accessible cure. Despite intensive efforts, a detailed understanding of virus and host cell interactions in tissues during infection and in response to therapy remains incomplete. To address these limitations, water-based tissue clearing techniques CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) and CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/in situ-hybridization-compatible Tissue hYdrogel) are applied to visualize complex virus host-cell interactions in HIV-infected tissues from animal models and humans using confocal and light sheet fluorescence microscopy. Optical sectioning of intact tissues and image analysis allows rapid reconstruction of spatial information contained within whole tissues and quantification of immune cell populations during infection. These methods are applicable to most tissue sources and diverse biological questions, including infectious disease and cancer.

FIB-SEM as a Volume Electron Microscopy Approach to Study Cellular Architectures in SARS-CoV-2 and Other Viral Infections: A Practical Primer for a Virologist.

The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.

The role of microRNAs in modulating SARS-CoV-2 infection in human cells: a systematic review.

MicroRNAs are gene expression regulators, associated with several human pathologies, including the ones caused by virus infections. Although their role in infection diseases is not completely known, they can exert double functions in the infected cell, by mediating the virus infection and/or regulating the immunity-related gene targets through complex networks of virus-host cell interactions. In this systematic review, the Pubmed, EMBASE, Scopus, Lilacs, Scielo, and EBSCO databases were searched for research articles published until October 22nd, 2020 that focused on describing the role, function, and/or association of miRNAs in SARS-CoV-2 human infection and COVID-19. Following the PRISMA 2009 protocol, 29 original research articles were selected. Most of the studies reported miRNA data based on the genome sequencing of SARS-CoV-2 isolates and computational prediction analysis. The latter predicted, by at least one independent study, 1266 host miRNAs to target the viral genome. Thirteen miRNAs were identified by four independent studies to target SARS-CoV-2 specific genes, suggested to act by interfering with their cleavage and/or translation process. The studies selected also reported on viral and host miRNAs that targeted host genes, on the expression levels of miRNAs in biological specimens of COVID-19 patients, and on the impact of viral genome mutations on miRNA function. Also, miRNAs that regulate the expression levels of the ACE2 and TMPRSS2 proteins, which are critical for the virus entrance in the host cells, were reported. In conclusion, despite the limited number of studies identified, based on the search terms and eligibility criteria applied, this systematic review provides evidence on the impact of miRNAs on SARS-CoV-2 infection and COVID-19. Although most of the reported viral/host miRNAs interactions were based on in silico prediction analysis, they demonstrate the relevance of the viral/host miRNA interaction for viral activity and host responses. In addition, the identified studies highlight the potential use of miRNAs as therapeutic targets against COVID-19, and other viral human diseases (This review was registered at the International Prospective Register of Systematic Reviews (PROSPERO) database (#CRD42020199290).

In silico investigation of critical binding pattern in SARS-CoV-2 spike protein with angiotensin-converting enzyme 2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a newly-discovered coronavirus and responsible for the spread of coronavirus disease 2019 (COVID-19). SARS-CoV-2 infected millions of people in the world and immediately became a pandemic in March 2020. SARS-CoV-2 belongs to the beta-coronavirus genus of the large family of Coronaviridae. It is now known that its surface spike glycoprotein binds to the angiotensin-converting enzyme-2 (ACE2), which is expressed on the lung epithelial cells, mediates the fusion of the cellular and viral membranes, and facilitates the entry of viral genome to the host cell. Therefore, blocking the virus-cell interaction could be a potential target for the prevention of viral infection. The binding of SARS-CoV-2 to ACE2 is a protein–protein interaction, and so, analyzing the structure of the spike glycoprotein of SARS-CoV-2 and its underlying mechanism to bind the host cell receptor would be useful for the management and treatment of COVID-19. In this study, we performed comparative in silico studies to deeply understand the structural and functional details of the interaction between the spike glycoprotein of SARS-CoV-2 and its cognate cellular receptor ACE2. According to our results, the affinity of the ACE2 receptor for SARS-CoV-2 was higher than SARS-CoV. According to the free energy decomposition of the spike glycoprotein-ACE2 complex, we found critical points in three areas which are responsible for the increased binding affinity of SARS-CoV-2 compared with SARS-CoV. These mutations occurred at the receptor-binding domain of the spike glycoprotein that play an essential role in the increasing the affinity of coronavirus to ACE2. For instance, mutations Pro462Ala and Leu472Phe resulted in the altered binding energy from − 2 kcal mol−1 in SARS-COV to − 6 kcal mol−1 in SARS-COV-2. The results demonstrated that some mutations in the receptor-binding motif could be considered as a hot-point for designing potential drugs to inhibit the interaction between the spike glycoprotein and ACE2.

The Epithelial-to-Mesenchymal Transition-Like Process Induced by TGF-β1 Enhances Rubella Virus Binding and Infection in A549 Cells via the Smad Pathway.

Virus–host cell interactions in rubella virus (RuV) are of great interest in current research in the field, as their mechanism is not yet well understood. By hypothesizing that the epithelial-to-mesenchymal transition (EMT) may play a role in RuV infection, this study aimed to investigate the influence of TGF-β1-induced EMT of human lung epithelial A549 cells on the infectivity of RuV. A549 cells were cultured and treated with TGF-β1 for 1 to 2 days prior to virus infection (with a clinical strain). Viral infectivity was determined by flow cytometry analysis of cells harvested at 24 and 48 h post-infection (hpi) and by titration of supernatants collected at 48 hpi. The results showed that the percentages of the TGF-β1-treated A549 cells that were positive for RuV were at least twofold higher than those of the control, and the viral progeny titers in the supernatants collected at 48 hpi were significantly higher in the treatment group than in the control group. In addition, the virus binding assay showed a strong increase (more than threefold) in the percentages of RuV-positive cells, as determined by flow cytometry analysis and further confirmed by real-time PCR. Such an enhancement effect on RuV infectivity was abolished using LY364947 or SB431542, inhibitors of the TGF-β/Smad signaling pathway. The findings suggest that the TGF-β1-induced EMT-like process enhances RuV binding and infection in A549 cells via the Smad pathway. Further studies are necessary to identify possible proteins that facilitate viral binding and entry into treated cells.

Why Does SARS-CoV-2 Infection Induce Autoantibody Production?

SARS-CoV-2 infection induces the production of autoantibodies, which is significantly associated with complications during hospitalization and a more severe prognosis in COVID-19 patients. Such a response of the patient’s immune system may reflect (1) the dysregulation of the immune response or (2) it may be an attempt to regulate itself in situations where the non-infectious self poses a greater threat than the infectious non-self. Of significance may be the primary virus-host cell interaction where the surface-bound ACE2 ectoenzyme plays a critical role. Here, we present a brief analysis of recent findings concerning the immune recognition of SARS-CoV-2, which, we believe, favors the second possibility as the underlying reason for the production of autoantibodies during COVID-19.

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.

The aryl hydrocarbon receptor facilitates the human cytomegalovirus-mediated G1/S block to cell cycle progression

The tryptophan metabolite, kynurenine, is known to be produced at elevated levels within human cytomegalovirus (HCMV)-infected fibroblasts. Kynurenine is an endogenous aryl hydrocarbon receptor (AhR) ligand. Here we show that the AhR is activated following HCMV infection, and pharmacological inhibition of AhR or knockdown of AhR RNA reduced the accumulation of viral RNAs and infectious progeny. RNA-seq analysis of infected cells following AhR knockdown showed that the receptor alters the levels of numerous RNAs, including RNAs related to cell cycle progression. AhR knockdown alleviated the G1/S cell cycle block that is normally instituted in HCMV-infected fibroblasts, consistent with its known ability to regulate cell cycle progression and cell proliferation. In sum, AhR is activated by kynurenine and perhaps other ligands produced during HCMV infection, it profoundly alters the infected-cell transcriptome, and one outcome of its activity is a block to cell cycle progression, providing mechanistic insight to a long-known element of the virus-host cell interaction.

Simultaneous tracking of capsid VP26, envelope protein gC localization in living cells infected with double fluorescent duck enteritis virus

Duck enteritis virus (DEV) can cause an acute, contagious and lethal disease of many species of waterfowl. An infectious bacterial artificial chromosome clone of DEV vaccine strain pE1 (pDEV-EF1) has been constructed in our previous study. Based on pE1, a recombinant mutated clone pDL (pVP26CFP-gCRFP), which carries a red fluorescent protein (mRFP) gene fused to the viral envelope protein gC in combination with a cyan fluorescent protein (CFP) gene fused to the viral capsid VP26, was constructed by two-step Red/ET recombination and the recombinant virus rDL (rVP26CFP-gCRFP) was rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate transfection. Western blot analysis revealed that VP26-CFP and gC-mRFP were both expressed in fusion forms in rDL-infected CEFs, and subcellular localization study showed that gC-mRFP was mainly localized in whole cell at 36, 48 h post infection (p.i.); and then mostly migrated to the cytoplasm after 60 h.p.i., ; whereas VP26-CFP was localized in the nucleus in all stages of virus infection. Additionally, viral particles at different stages of morphogenesis (A capsids, B capsids, C capsids) were observed in virus-infected cells by transmission electron microscopy, indicating that exogenous gene insertion has no effect on virus assembly. This study has laid a foundation for visually studying localization, transportation of DEV capsid proteins and envelope glycoproteins as well as virus assembly, virion movement and virus-cell interaction.

Investigating virus–host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models

Cell cultures have been successfully used to study hepatitis C virus (HCV) for many years. However, most work has been done using traditional, 2-dimensional (2D) cell cultures (cells grown as a monolayer in growth flasks or dishes). Studies have shown that when cells are grown suspended in an extra-cellular-matrix-like material, they develop into spherical, ‘organoid’ arrangements of cells (3D growth) that display distinct differences in morphological and functional characteristics compared to 2D cell cultures. In liver organoids, one key difference is the development of clearly differentiated apical and basolateral surfaces separated and maintained by cellular tight junctions. This phenomenon, termed polarity, is vital to normal barrier function of hepatocytes in vivo. It has also been shown that viruses, and virus-like particles, interact very differently with cells derived from 2D as compared to 3D cell cultures, bringing into question the usefulness of 2D cell cultures to study virus–host cell interactions. Here, we investigate differences in cellular architecture as a function of cell culture system, using confocal scanning laser microscopy, and determine differences in binding interactions between HCV virus-like particles (VLPs) and their cognate receptors in the different cell culture systems using atomic force microscopy (AFM). We generated organoid cultures that were polarized, as determined by localization of key apical and basolateral markers. We found that, while uptake of HCV VLPs by both 2D and 3D Huh7 cells was observed by flow cytometry, binding interactions between HCV VLPs and cells were measurable by AFM only on polarized cells. The work presented here adds to the growing body of research suggesting that polarized cell systems are more suitable for the study of HCV infection and dynamics than non-polarized systems.

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth.Key points• Less and more efficient glucose utilization for suspension cell growth• Concerted alteration of metabolic enzyme activity and protein expression• Protein candidates to interfere glycolytic activity in MDCK cells

Advances in Hepatitis E Virus Biology and Pathogenesis.

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.