Abstract BACKGROUND Despite advances in knowledge and medicine, hepatitis C virus (HCV) infection remains a global challenge. The viral life cycle depends heavily on lipid metabolism; consequently, HCV infection is associated with significant alterations to host lipid homeostasis. One of the regulators maintaining this homeostasis is the transcription factor nuclear factor erythroid 2-related factor 1 (Nrf1). There are multiple Nrf1 proteoforms that differ in their capacity to act as a cholesterol sensor or to activate or inhibit gene expression. Previously, we identified that the amount of full-length Nrf1 protein in HCV-replicating cells is significantly reduced. In this study, we investigate whether HCV affects the formation and functionality of the different Nrf1 proteoforms, and whether this contributes to the HCV-dependent dysregulation of cellular lipid metabolism. METHODS The HCV-Nrf1 crosstalk was characterized using robust methods including: Western blot, FRET Acceptor Photobleaching assay, reverse transcriptase-polymerase chain reaction and confocal laser scanning microscopy-based approaches. RESULTS HCV infection does not alter the onset of Nrf1 proteoforms generated through proteasomal cleavage of the protein. However, the amount of different Nrf1 proteoforms is significantly reduced in HCV-positive cells due to enhanced Nrf1 turnover. Furthermore, the Nrf1 proteoforms with transcriptional activator functions are prevented from translocation into the nucleus. Reduced Nrf1 activity contributes to elevated cholesterol levels and favors lipid droplets formation. Conversely, rescue of Nrf1 activity in HCV-replicating cells is associated with decreased intracellular cholesterol levels, reduced number of lipid droplets and impaired viral release reflected by intracellular accumulation of the core protein and intact viral particles. CONCLUSIONS Overall, our results identify Nrf1 as a significant factor in the deregulation of host lipid metabolism induced by HCV. The inhibition of Nrf1 functionality driven by HCV leads to intracellular cholesterol accumulation, resulting in enhanced lipid droplet formation that supports the HCV life cycle and contributes to HCV-associated pathogenesis.

The life cycle of the hepatitis C virus (HCV) is closely linked to lipid metabolism. Recently, the stress defence transcription factor, nuclear factor erythroid 2 related factor-1 (Nrf1), has been described as a cholesterol sensor that protects the liver from excess cholesterol. Nrf1, like its homologue Nrf2, further responds to oxidative stress by binding with small Maf proteins (sMaf) to the promotor antioxidant response element (ARE). Given these facts, investigating the crosstalk between Nrf1 and HCV was a logical next step. In HCV-replicating cells, we observed reduced levels of Nrf1. Furthermore, activation of Nrf1-dependent target genes is impaired due to sMaf sequestration in replicase complexes. This results in a shortage of sMaf proteins in the nucleus, trapping Nrf1 at the replicase complexes and further limiting its function. Weakened Nrf1 activity contributes to impaired cholesterol removal, which occurs alongside an elevated intracellular cholesterol level and inhibited LXRα promoter activation. Furthermore, inhibition of Nrf1 activity correlated with a kinome profile characteristic of steatosis and enhanced inflammation—factors contributing to HCV pathogenesis. Our results indicate that activation of Nrf1-dependent target genes is impaired in HCV-positive cells. This, in turn, favours viral morphogenesis, as evidenced by enhanced replication and increased production of viral progeny.

Chronic hepatitis B virus (HBV) infection is a major health problem, affecting around 254 million people worldwide. Current treatments have side effects and can lead to resistance. Therefore, natural compounds derived from plants are being studied as potential antivirals. Isochlorogenic acid A (ICAA), which is derived from caffeoylquinic acid, exerts antiviral and hepatoprotective effects. The antioxidant effect of ICAA, triggered by upregulation of heme oxygenase 1 (HO-1), has been suggested as potential antiviral mechanism. However, the underlying mechanisms remain enigmatic. Our aim is to elucidate the mode of action of the antiviral effect of ICAA on HBV. Stable or transient transfected cells expressing HBV as well as HBV-infected cells were instrumental. (Sub)viral particles were characterized by biophysical and biochemical methods. Subcellular distribution of viral proteins was studied using confocal laser scanning microscopy. Viral genomes and transcripts were quantified by qPCR. Treatment with ICAA decreased levels of HBV surface and e antigens (HBsAg and HBeAg), as well as viral transcripts, genomes and most important cccDNA. Furthermore, impaired virus assembly was evident from accumulation of naked capsids suggesting improper capsid formation and impaired envelopment. ICAA-dependent effects on HBV correlate with upregulation of HO-1 and modulation of intracellular ROS Our data indicate a possible link between changes in the intracellular ROS level and altered free -SH groups in viral structural proteins, possibly influencing proper disulphide bond formation and thereby assembly. In conclusion ICAA-dependent effects on HBV life cycle are based on several pillars as modulation of intracellular ROS and impaired morphogenesis and replication.

BACKGROUND:A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization.METHODS:Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase-polymerase chain reaction to analyze separated particles in more detail.RESULTS:We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein's 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3.CONCLUSIONS:Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.

The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that the human endolysosomal cation channel TRPML2 agonist ML2-SA1 impairs the late life cycle stages of ZIKV, thus underscoring TRPML2 as a promising antiviral target. Upon treatment with ML2-SA1, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced. ML2-SA1-treated cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound’s negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV as well as many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed ML2-SA1 to also negatively impact HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides underscoring the potential of TRPML2 as a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.### Competing Interest StatementThe authors have declared no competing interest.* aa : amino acids cLSM : confocal laser scanning microscopy CTCF : corrected total cell fluorescence CV : coefficient of variation DENV : Dengue virus DMSO : dimethyl sulfoxide E : protein envelope protein FBS : fetal bovine serum GEs : genome equivalents HCV : Hepatitis C virus HEV : Hepatitis E virus hpi : hours post infection IAV : Influenza A virus ML2-SA1 : mucolipin 2 synthetic agonist 1 ML-SA1 : mucolipin synthetic agonist 1 MOI : multiplicity of infection MVB : multivesicular body PBS : phosphate buffered saline PFU : plaque forming units PIFA : (bis(trifluoroacetoxy)iodo)benzene PMSF : phenylmethylsulfonyl fluoride Renilla : Luciferase R Luc RIPA : radio-immunoprecipitation assay rt : room temperature RT-qPCR : real-time quantitative polymerase chain reaction SD : standard deviation STED : stimulated emission depletion TBS : tris buffered saline TCID50 : half maximal tissue culture infective dose tMOC : thresholded Mender’s overlap coefficient TPC : two-pore channels TRPML : transient receptor potential mucolipin WNV : West Nile virus YFV : yellow fever virus ZIKV : FP ZIKV French Polynesia isolate ZIKV : U ZIKV Uganda isolate ZIKV : Zika virus

Three new series of macrocyclic active site‐directed inhibitors of the Zika virus (ZIKV) NS2B–NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3‐aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d‐cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure‐based development of improved inhibitors.

Zoonoses such as ZIKV and SARS-CoV-2 pose a severe risk to global health. There is urgent need for broad antiviral strategies based on host-targets filling gaps between pathogen emergence and availability of therapeutic or preventive strategies. Significant reduction of pathogen titers decreases spread of infections and thereby ensures health systems not being overloaded and public life to continue. Based on previously observed interference with FGFR1/2-signaling dependent impact on interferon stimulated gene (ISG)-expression, we identified Pim kinases as promising druggable cellular target. We therefore focused on analyzing the potential of pan-Pim kinase inhibition to trigger a broad antiviral response. The pan-Pim kinase inhibitor AZD1208 exerted an extraordinarily high antiviral effect against various ZIKV isolates, SARS-CoV-2 and HBV. This was reflected by strong reduction in viral RNA, proteins and released infectious particles. Especially in case of SARS-CoV-2, AZD1208 led to a complete removal of viral traces in cells. Kinome-analysis revealed vast changes in kinase landscape upon AZD1208 treatment, especially for inflammation and the PI3K/Akt-pathway. For ZIKV, a clear correlation between antiviral effect and increase in ISG-expression was observed. Based on a cell culture model with impaired ISG-induction, activation of the PI3K-Akt-mTOR axis, leading to major changes in the endolysosomal equilibrium, was identified as second pillar of the antiviral effect triggered by AZD1208-dependent Pim kinase inhibition, also against HBV. We identified Pim-kinases as cellular target for a broad antiviral activity. The antiviral effect exerted by inhibition of Pim kinases is based on at least two pillars: innate immunity and modulation of the endolysosomal system.

Viral infections trigger the expression of interferons (IFNs) and interferon stimulated genes (ISGs), which are crucial to modulate an antiviral response. The human guanylate binding protein 1 (GBP1) is an ISG and exhibits antiviral activity against several viruses. In a previous study, GBP1 was described to impair replication of the hepatitis C virus (HCV). However, the impact of GBP1 on the HCV life cycle is still enigmatic. To monitor the expression and subcellular distribution of GBP1 and HCV we performed qPCR, Western blot, CLSM and STED microscopy, virus titration and reporter gene assays. In contrast to previous reports, we observed that HCV induces the expression of GBP1. Further, to induce GBP1 expression, the cells were stimulated with IFNγ. GBP1 modulation was achieved either by overexpression of GBP1-Wt or by siRNA-mediated knockdown. Silencing of GBP1 impaired the release of viral particles and resulted in intracellular HCV core accumulation, while overexpression of GBP1 favored viral replication and release. CLSM and STED analyses revealed a vesicular distribution of GBP1 in the perinuclear region. Here, it colocalizes with HCV core around lipid droplets, where it acts as assembly platform and thereby favors HCV morphogenesis and release. Collectively, our results identify an unprecedented function of GBP1 as a pro-viral factor. As such, it is essential for viral assembly and release acting through tethering factors involved in HCV morphogenesis onto the surface of lipid droplets.

The flavivirus genus includes human pathogenic viruses such as Dengue (DENV), West Nile (WNV) and Zika virus (ZIKV) posing a global health threat due to limited treatment options. Host ion channels are crucial for various viral life cycle stages, but their potential as targets for antivirals is often not fully realized due to the lack of selective modulators. Here, we observe that treatment with ML2-SA1, an agonist for the human endolysosomal cation channel TRPML2, impairs ZIKV replication. Upon ML2-SA1 treatment, levels of intracellular genomes and number of released virus particles of two different ZIKV isolates were significantly reduced and cells displayed enlarged vesicular structures and multivesicular bodies with ZIKV envelope protein accumulation. However, no increased ZIKV degradation in lysosomal compartments was observed. Rather, the antiviral effect of ML2-SA1 seemed to manifest by the compound's negative impact on genome replication. Moreover, ML2-SA1 treatment also led to intracellular cholesterol accumulation. ZIKV and many other viruses including the Orthohepevirus Hepatitis E virus (HEV) rely on the endolysosomal system and are affected by intracellular cholesterol levels to complete their life cycle. Since we observed that ML2-SA1 also negatively impacted HEV infections in vitro, this compound may harbor a broader antiviral potential through perturbing the intracellular cholesterol distribution. Besides indicating that TRPML2 may be a promising target for combatting viral infections, we uncover a tentative connection between this protein and cholesterol distribution within the context of infectious diseases.

Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection.

Although tetherin expression is strongly induced by ZIKV infection there is a reduction in the amount of tetherin protein. This is due to enhanced lysosomal degradation.

Zusammenfassung Die Hepatitisviren B und C (HBV, HCV) sind weltweit die relevantesten viralen Auslöser einer chronischen Hepatitis (Leberentzündung). Derzeit leiden weltweit mehr als 250 Mio. Menschen an einer chronischen HBV-Infektion, jährlich versterben 0,8 Mio. an den Folgen. Von einer chronischen HCV-Infektion sind ca. 70 Mio. Menschen betroffen, es versterben ca. 1 Mio. im Jahr. Bisher steht nur für HBV eine zugelassene Impfung zur Verfügung. Chronische Infektionen mit HBV und HCV gehen mit einem erhöhten Risiko für die Entwicklung einer Leberfibrose, einer Leberzirrhose und eines hepatozellulären Karzinoms (HCC) einher. Diese Übersichtsarbeit beschreibt Mechanismen der HBV- und HCV-assoziierten Pathogenese. Im Vordergrund stehen dabei die Wechselwirkung der chronischen Infektion mit intrazellulären Signaltransduktionswegen, mit einzelnen Stoffwechselwegen, insbesondere dem Lipidmetabolismus, die Fibrose- und Zirrhoseentstehung im Laufe der chronischen Infektion sowie Mechanismen der virusinduzierten HCC-Entstehung. Trotz großer Fortschritte in der Charakterisierung der viralen Lebenszyklen und der Entwicklung robuster antiviraler Strategien bleiben Herausforderungen bestehen: u. a. die Gewinnung eines noch besseren Verständnisses der Mechanismen, die zur Entwicklung der virusassoziierten Pathogenese beitragen, sowie die Erforschung der Relevanz verschiedener Genotypen für Unterschiede in der Pathogenese.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

Worldwide, the hepatitis B and hepatitis C viruses (HBV, HCV) are the most relevant causative viral agents of a chronic hepatitis (inflammation of the liver). At present, more than 250 million people suffer from a chronic HBV infection globally, resulting in 0.8 million deaths per year. A chronic HCV infection accounts for about 70 million cases worldwide, leading to a death toll of about 1 million per year. An approved vaccine is only available against an HBV infection. Both HBV and HCV infections result in a highly increased risk of developing liver fibrosis, cirrhosis, and a hepatocellular carcinoma (HCC). This review aims to describe mechanisms of the HBV- and HCV-associated pathogenesis. The focus is on the interplay between a chronic infection with intracellular signaling transduction, metabolic pathways with an emphasis on lipid metabolism, the establishment of liver fibrosis and cirrhosis during a chronic infection, and the mechanisms of the onset of a virally induced HCC. Despite there being great advances in the characterization of viral life cycles and the development of robust antiviral strategies, significant hurdles persist: gaining a better understanding of the mechanisms that drive virus-associated pathogenesis as well as increasing insights regarding different viral genotypes having impacts on alternate pathogeneses.

To reconcile the observed selectivity and the high rate of translocation of cargo-importin complexes through nuclear pores, we propose that the core of the nuclear pore complex is blocked by a metastable network of phenylalanine and glycine nucleoporins. Although the network arrests the unfacilitated passage of objects larger than its mesh size, cargo-importin complexes act as catalysts that reduce the free energy barrier between the cross-linked and the dissociated states of the Nups, and open the network. Using Brownian dynamics simulations we calculate the distribution of passage times through the network for inert particles and cargo-importin complexes of different sizes and discuss the implications of our results for experiments on translocation of proteins through the nuclear pore complex.

Zika virus (ZIKV) is a human pathogenic arbovirus. So far, neither a specific treatment nor a vaccination against ZIKV infections has been approved. Starting from our previously described lead structure, a series of 29 new macrocyclic inhibitors of the Zika virus protease containing different linker motifs have been synthesized. By selecting hydrophobic d-amino acids as part of the linker, numerous inhibitors with Ki values < 5 nM were obtained. For 12 inhibitors, crystal structures in complex with the ZIKV protease up to 1.30 Å resolution were determined, which contribute to the understanding of the observed structure-activity relationship (SAR). In immunofluorescence assays, an antiviral effect was observed for compound 26 containing a d-homocyclohexylalanine residue in its linker segment. Due to its excellent selectivity profile and low cytotoxicity, this inhibitor scaffold could be a suitable starting point for the development of peptidic drugs against the Zika virus and related flaviviruses.

ZusammenfassungBei der viralen Hepatitis handelt es sich um eine akute oder chronische Entzündung der Leber, die durch verschiedene Viren verursacht wird. Weltweit leiden derzeit ca. 325 Mio. Menschen an der chronischen Form. Jährlich versterben insgesamt ca. 1,6 Mio. an den Folgen einer viralen Hepatitis. Die Hepatitisviren werden in 5 Erregergruppen unterteilt, die mit den Buchstaben A bis E bezeichnet werden (HAV–HEV). Diese unterscheiden sich in Phylogenie, Übertragung, Epidemiologie, Wirtsspezifität, Lebenszyklus, Struktur und in speziellen Aspekten der Pathogenese.Das strikt humanpathogene HAV, Teil der Familie Picornaviridae, induziert meist nur akute Hepatitiden und ist primär in Entwicklungsländern verbreitet. Das den Hepeviridae zugeordnete HEV beschreibt eine ähnliche Epidemiologie, ist jedoch durch sein zoonotisches Potenzial auch in Industrienationen weitverbreitet und kann zusätzlich eine chronische Erkrankung induzieren. Eine Chronifizierung tritt ebenso bei dem weltweit verbreiteten HBV (Hepadnaviridae) auf, dessen Satellitenvirus HDV (Kolmioviridae) das vorhandene kanzerogene Potenzial noch einmal erhöht. Das ebenfalls weltweit verbreitete HCV (Flaviviridae) birgt ein äußerst hohes Risiko der Chronifizierung und somit ebenfalls ein stark erhöhtes, kanzerogenes Potenzial.Die Erreger der viralen Hepatitis unterscheiden sich in ihren Eigenschaften und Lebenszyklen. Eine differenzierte Betrachtung im Hinblick auf Epidemiologie, Nachweismethoden und Prävention ist daher angezeigt. Obwohl robuste Therapien, und im Falle einzelner Erreger auch Vakzine, vorhanden sind, muss die Forschung insbesondere in Hinblick auf die armutsassoziierten Erreger erheblich vorangetrieben werden.

SummaryBackgroundThe mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS‐CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30 μg versus 12 μg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera.MethodsSera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12 μg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays.ResultsBNT162b2‐elicited sera and convalescent sera have a higher titer of spike‐RBD‐specific antibodies and neutralizing antibodies as compared to the CVnCoV‐elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV‐elicited sera have a lower fraction of slow‐dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike‐ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine‐elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope.ConclusionThese data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2‐ and CVnCoV‐elicited sera, which could contribute to the apparent differences in vaccine efficacy.

Zika virus (ZIKV) is a flavivirus that is well known for the epidemic in the Americas in 2015 and 2016 in which microcephaly in newborns and other neurological complications were connected to ZIKV infection. Many aspects of the ZIKV viral life cycle, including binding and entry into the host cell, are still enigmatic. Based on the observation that CHO cells lack expression of the epidermal growth factor receptor (EGFR) and are not permissive for various ZIKV strains, the relevance of EGFR for the viral life cycle was analyzed. Infection of A549 cells by ZIKV leads to a rapid internalization of EGFR that colocalizes with the endosomal marker EEA1. Moreover, infection by different ZIKV strains is associated with an activation of EGFR and the subsequent activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling cascade. However, treatment of the cells with methyl-β-cyclodextrin (MβCD), which on the one hand leads to an activation of EGFR but on the other hand prevents EGFR internalization, impairs ZIKV infection. Specific inhibition of EGFR or of the Ras-Raf-MEK-ERK signal transduction cascade hinders ZIKV infection by inhibition of ZIKV entry. In accordance with this, knockout of EGFR expression impedes ZIKV entry. In the case of an already established infection, inhibition of EGFR or of downstream signaling does not affect viral replication. Taken together, these data demonstrate the relevance of EGFR in the early stages of ZIKV infection and identify EGFR as a target for antiviral strategies. IMPORTANCE These data deepen the knowledge about the ZIKV infection process and demonstrate the relevance of EGFR for ZIKV entry. In light of the fact that a variety of specific and efficient inhibitors of EGFR and of EGFR-dependent signaling have been developed and licensed, repurposing of these substances could be a helpful tool to prevent the spreading of ZIKV infection in an epidemic outbreak.