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Background & Aims: In the absence of a hepatitis E virus (HEV)-specific antiviral treatment, sofosbuvir has recently been shown to have antiviral activity against HEV in vivo. However, a variant, A1343V, that is strongly associated with viral relapse impedes treatment success. In this study, we investigated the occurrence of variants during sofosbuvir and ribavirin treatment in vivo and assessed the sensitivity of resistance-associated variants to concurrent treatment in cell culture. Methods: Two patients with chronic HEV infection that did not clear infection under ribavirin treatment were subsequently treated with a combination of sofosbuvir and ribavirin. We determined response to treatment by measuring liver enzymes and viral load in blood and stool. Moreover, we analyzed viral evolution using polymerase-targeted high-throughput sequencing and assessed replication fitness of resistance-associated variants using a HEV replicon system. Results: Combination treatment was successful in decreasing viral load towards the limit of quantification. However, during treatment sustained virological response was not achieved. Variants associated with sofosbuvir or ribavirin treatment emerged during treatment, including A1343V and G1634R. Moreover, A1343V, as a single or double mutation with G1634R, was associated with sofosbuvir resistance during concomitant treatment in vitro. Conclusions: These results highlight the importance of variant profiling during antiviral treatment of patients with chronic infection. Understanding how intra-host viral evolution impedes treatment success will help guide the design of next-generation antivirals. Impact and implications: The lack of hepatitis E virus (HEV)-specific antivirals to treat chronic infection remains a serious health burden. Although ribavirin, interferon and sofosbuvir have been reported as anti-HEV drugs, not all patients are eligible for treatment or clear infection, since resistant-associated variants can rapidly emerge. In this study, we analyzed the efficacy of sofosbuvir and ribavirin combination treatment in terms of HEV suppression, the emergence of resistance-associated variants and their ability to escape treatment inhibition in vitro. Our results provide novel insights into evolutionary dynamics of HEV during treatment and thus will help guide the design of next-generation antivirals.

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old’s working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions.

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b–TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

Treatment options for COVID-19, a disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, are currently severely limited. Therefore, antiviral drugs that efficiently reduce SARS-CoV-2 replication or alleviate COVID-19 symptoms are urgently needed. Inhaled glucocorticoids are currently being discussed in the context of treatment for COVID-19, partly based on a previous study that reported reduced recovery times in cases of mild COVID-19 after inhalative administration of the glucocorticoid budesonide. Given various reports that describe the potential antiviral activity of glucocorticoids against respiratory viruses, we aimed to analyze a potential antiviral activity of budesonide against SARS-CoV-2 and circulating variants of concern (VOC) B.1.1.7 (alpha) and B.1.351 (beta). We demonstrate a dose-dependent inhibition of SARS-CoV-2 that was comparable between all viral variants tested while cell viability remains unaffected. Our results are encouraging as they could indicate a multimodal mode of action of budesonide against SARS-CoV-2 and COVID-19, which could contribute to an improved clinical performance.

Since its first discovery by Arnold Theiler in 1918, serum hepatitis also known as Theiler’s disease has been reported worldwide, causing idiopathic acute hepatitis and liver failure in horses. Recent studies have suggested a novel parvovirus, named equine parvovirus hepatitis (EqPV-H), to be associated with Theiler’s disease. Despite the severity and potential fatality of EqPV-H infection, little is known about the possibility of developing chronic infections and putative cross-species infection of equine sister species. In the present longitudinal study, we employed qPCR analysis, serology, and biochemical testing as well as pathology examination of liver biopsies and sequence analysis to investigate potential chronic EqPV-H infection in an isolated study cohort of in total 124 horses from Germany over five years (2013–2018). Importantly, our data suggest that EqPV-H viremia can become chronic in infected horses that do not show biochemical and pathological signs of liver disease. Phylogenetic analysis by maximum likelihood model also confirms high sequence similarity and nucleotide conservation of the multidomain nuclear phosphoprotein NS1 sequences from equine serum samples collected between 2013–2018. Moreover, by examining human, zebra, and donkey sera for the presence of EqPV-H DNA and VP1 capsid protein antibodies, we found evidence for cross-species infection in donkey, but not to human and zebra. In conclusion, this study provides proof for the occurrence of persistent EqPV-H infection in asymptomatic horses and cross-species EqPV-H detection in donkeys.

SARS-CoV-2 has spread across the globe with an astonishing velocity and lethality that has put scientist and pharmaceutical companies worldwide on the spot to develop novel treatment options and reliable vaccination for billions of people. To combat its associated disease COVID-19 and potentially newly emerging coronaviruses, numerous pre-clinical cell culture techniques have progressively been used, which allow the study of SARS-CoV-2 pathogenesis, basic replication mechanisms, and drug efficiency in the most authentic context. Hence, this review was designed to summarize and discuss currently used in vitro and ex vivo cell culture systems and will illustrate how these systems will help us to face the challenges imposed by the current SARS-CoV-2 pandemic.

The pleiotropic drug resistance (PDR) transporter Pdr11p is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae, where it facilitates the uptake of exogenous sterols. Members of the fungal PDR family contain six conserved cysteines in their extracellular loops (ECL). For the functional analysis of these cysteine residues in Pdr11p, we generated a series of single cysteine-to-serine mutants. All mutant proteins expressed well and displayed robust ATPase activity upon purification. Mass-spectrometry analysis identified two cysteine residues (C582 and C603) in ECL3 forming a disulfide bond. Further characterization by cell-based assays showed that all mutants are compromised in facilitating sterol uptake, protein stability, and trafficking to the plasma membrane. Our data highlight the fundamental importance of all six extracellular cysteine residues for the functional integrity of Pdr11p and provide new structural insights into the PDR family of transporters.