Highlights d Established live cell imaging system to visualize HCV assembly events d Determined conditions for recruitment of viral proteins to putative assembly sites d HCV induces wrapping of lipid droplets by ER membranes at putative assembly sites d Wrapping membranes are linked to double membrane vesicles, the HCV replication sites

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.

Dengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loopmore precisely, amino acid residue Q134 -as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies. IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target. D engue virus (DENV) is an enveloped plus-strand RNA virus belonging to the genus Flavivirus of the family Flaviviridae. DENV infection causes the most prevalent arthropod-borne viral disease worldwide, with approximately 100 million symptomatic cases per year. In 0.5 to 1% of the cases, the infection manifests with severe symptoms that can deve...

Seroconversion after COVID‐19 vaccination is impaired in kidney transplant recipients. Emerging variants of concern such as the B.1.617.2 (delta) and the B.1.1.529 (omicron) variants pose an increasing threat to these patients. In this observational cohort study, we measured anti‐S1 IgG, surrogate neutralizing, and anti‐receptor‐binding domain antibodies three weeks after a third mRNA vaccine dose in 49 kidney transplant recipients and compared results to 25 age‐matched healthy controls. In addition, vaccine‐induced neutralization of SARS‐CoV‐2 wild‐type, the B.1.617.2 (delta), and the B.1.1.529 (omicron) variants was assessed using a live‐virus assay. After a third vaccine dose, anti‐S1 IgG, surrogate neutralizing, and anti‐receptor‐binding domain antibodies were significantly lower in kidney transplant recipients compared to healthy controls. Only 29/49 (59%) sera of kidney transplant recipients contained neutralizing antibodies against the SARS‐CoV‐2 wild‐type or the B.1.617.2 (delta) variant and neutralization titers were significantly reduced compared to healthy controls ( p < 0.001). Vaccine‐induced cross‐neutralization of the B.1.1.529 (omicron) variants was detectable in 15/35 (43%) kidney transplant recipients with seropositivity for anti‐S1 IgG, surrogate neutralizing, and/or anti‐RBD antibodies. Neutralization of the B.1.1.529 (omicron) variants was significantly reduced compared to neutralization of SARS‐CoV‐2 wild‐type or the B.1.617.2 (delta) variant for both, kidney transplant recipients and healthy controls ( p < .001 for all).

Background and objectivesAntibody response after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is impaired in kidney transplant recipients. Emerging variants, such as B.1.617.2 (δ), are of particular concern because of their higher transmissibility and partial immune escape. Little is known about protection against these variants in immunocompromised patients.Design, setting, participants, & measurementsIn this prospective two-center study, antispike 1 IgG and surrogate neutralizing antibodies were measured in 173 kidney transplant recipients and 166 healthy controls with different vaccination schedules. In addition, different SARS-CoV-2 epitope antibodies from 135 vaccinated kidney transplant recipients were compared with antibodies in 25 matched healthy controls after second vaccination. In 36 kidney transplant recipients with seroconversion, neutralization against B.1.1.7 (α), B.1.351 (β), and B.1.617.2 (δ) was determined on VeroE6 cells and compared with neutralization in 25 healthy controls.ResultsKidney transplant recipients had significantly lower seroconversion rates compared with healthy controls. After the second vaccination, antispike 1, antireceptor-binding domain, and surrogate neutralizing antibodies were detectable in 30%, 27%, and 24% of kidney transplant recipients, respectively. This compares with 100%, 96%, and 100% in healthy controls, respectively (P<0.001). Neutralization against B.1.1.7 was detectable in all kidney transplant recipients with seroconversion, with a median serum dilution that reduces infection of cells by 50% of 80 (interquartile range, 80–320). In contrast, only 23 of 36 (64%) and 24 of 36 (67%) kidney transplant recipients showed neutralization against B.1.351 and B.1.617.2, respectively, with median serum dilutions that reduce infection of cells by 50% of 20 (interquartile range, 0–40) and 20 (interquartile range, 0–40), respectively. Neutralization against different variants was significantly higher in healthy controls (P<0.001), with all patients showing neutralization against all tested variants.ConclusionsSeroconverted kidney transplant recipients show impaired neutralization against emerging variants of concern after standard two-dose vaccination.Clinical Trial registry name and registration number:Observational study to assess the SARS-CoV-2 specific immune response in kidney transplant recipients (COVID-19 related immune response), DRKS00024668