Hepatitis C Virus P7—A Viroporin Crucial for Virus Assembly and an Emerging Target for Antiviral Therapy

The hepatitis C virus (HCV), a hepatotropic plus-strand RNA virus of the family Flaviviridae, encodes a set of 10 viral proteins. These viral factors act in concert with host proteins to mediate virus entry, and to coordinate RNA replication and virus production. Recent evidence has highlighted the complexity of HCV assembly, which not only involves viral structural proteins but also relies on host factors important for lipoprotein synthesis, and a number of viral assembly co-factors. The latter include the integral membrane protein p7, which oligomerizes and forms cation-selective pores. Based on these properties, p7 was included into the family of viroporins comprising viral proteins from multiple virus families which share the ability to manipulate membrane permeability for ions and to facilitate virus production. Although the precise mechanism as to how p7 and its ion channel function contributes to virus production is still elusive, recent structural and functional studies have revealed a number of intriguing new facets that should guide future efforts to dissect the role and function of p7 in the viral replication cycle. Moreover, a number of small molecules that inhibit production of HCV particles, presumably via interference with p7 function, have been reported. These compounds should not only be instrumental in increasing our understanding of p7 function, but may, in the future, merit further clinical development to ultimately optimize HCV-specific antiviral treatments

Hepatitis C Virus p7 Protein Is Crucial for Assembly and Release of Infectious Virions

Hepatitis C virus (HCV) infection is associated with chronic liver disease and currently affects about 3% of the world population. Although much has been learned about the function of individual viral proteins, the role of the HCV p7 protein in virus replication is not known. Recent data, however, suggest that it forms ion channels that may be targeted by antiviral compounds. Moreover, this protein was shown to be essential for infectivity in chimpanzee. Employing the novel HCV infection system and using a genetic approach to investigate the function of p7 in the viral replication cycle, we find that this protein is essential for efficient assembly and release of infectious virions across divergent virus strains. We show that p7 promotes virus particle production in a genotype-specific manner most likely due to interactions with other viral factors. Virus entry, on the other hand, is largely independent of p7, as the specific infectivity of released virions with a defect in p7 was not affected. Together, these observations indicate that p7 is primarily involved in the late phase of the HCV replication cycle. Finally, we note that p7 variants from different isolates deviate substantially in their capacity to promote virus production, suggesting that p7 is an important virulence factor that may modulate fitness and in turn virus persistence and pathogenesis

European College of Equine Internal Medicine consensus statement on equine flaviviridae infections in Europe

Horses and other equids can be infected with several viruses of the family Flaviviridae, belonging to the genus Flavivirus and Hepacivirus. This consensus statement focuses on viruses with known occurrence in Europe, with the objective to summarize the current literature and formulate clinically relevant evidence-based recommendations regarding clinical disease, diagnosis, treatment, and prevention. The viruses circulating in Europe include West Nile virus, tick-borne encephalitis virus, Usutu virus, Louping ill virus and the equine hepacivirus. West Nile virus and Usutu virus are mosquito-borne, while tick-borne encephalitis virus and Louping ill virus are tick-borne. The natural route of transmission for equine hepacivirus remains speculative. West Nile virus and tick-borne encephalitis virus can induce encephalitis in infected horses. In the British Isle, rare equine cases of encephalitis associated with Louping ill virus are reported. In contrast, equine hepacivirus infections are associated with mild acute hepatitis and possibly chronic hepatitis. Diagnosis of flavivirus infections is made primarily by serology, although cross-reactivity occurs. Virus neutralization testing is considered the gold standard to differentiate between flavivirus infections in horses. Hepacivirus infection is detected by serum or liver RT-PCR. No direct antiviral treatment against flavi- or hepacivirus infections in horses is currently available and thus, treatment is supportive. Three vaccines against West Nile virus are licensed in the European Union. Geographic expansion of flaviviruses pathogenic for equids should always be considered a realistic threat, and it would be beneficial if their detection was included in surveillance programs

Identification of structurally re-engineered rocaglates as inhibitors against hepatitis E virus replication

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy. In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC50 value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC50 values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future. © 2022 The Author(s

Biotechnological Potential of Bacteria Isolated from the Sea Cucumber Holothuria leucospilota and Stichopus vastus from Lampung, Indonesia

In order to minimize re-discovery of already known anti-infective compounds, we focused our screening approach on understudied, almost untapped marine environments including marine invertebrates and their associated bacteria. Therefore, two sea cucumber species, Holothuria leucospilota and Stichopus vastus, were collected from Lampung (Indonesia), and 127 bacterial strains were identified by partial 16S rRNA-gene sequencing analysis and compared with the NCBI database. In addition, the overall bacterial diversity from tissue samples of the sea cucumbers H. leucospilota and S. vastus was analyzed using the cultivation-independent Illumina MiSEQ analysis. Selected bacterial isolates were grown to high densities and the extracted biomass was tested against a selection of bacteria and fungi as well as the hepatitis C virus (HCV). Identification of putative bioactive bacterial-derived compounds were performed by analyzing the accurate mass of the precursor/parent ions (MS1) as well as product/daughter ions (MS2) using high resolution mass spectrometry (HRMS) analysis of all active fractions. With this attempt we were able to identify 23 putatively known and two previously unidentified precursor ions. Moreover, through 16S rRNA-gene sequencing we were able to identify putatively novel bacterial species from the phyla Actinobacteria, Proteobacteria and also Firmicutes. Our findings suggest that sea cucumbers like H. leucospilota and S. vastus are promising sources for the isolation of novel bacterial species that produce compounds with potentially high biotechnological potential

First detection and frequent occurrence of Equine Hepacivirus in horses on the African continent

Since the discovery of equine hepacivirus (EqHV) in 2011, the virus has been detected in horse populations from more than twelve countries across five continents. EqHV seroprevalence has been reported to be as high as 61.8% and EqHV ribonucleic acid (RNA) prevalence to range between 0.9% and 34.1%. Molecular and serological indications of EqHV infection have never been reported in equids on the African continent. Therefore, investigation of EqHV prevalence in South African horses and subsequent viral genetic characterization contribute to a better understanding of the global epidemiology of this virus. In a cross-sectional study, serum samples from 454 Thoroughbred foals (aged 58–183 days) were analysed for anti-EqHV non-structural protein 3 (NS3)-specific antibodies (abs) with a luciferase immunoprecipitation system (LIPS) and for EqHV RNA by quantitative real-time polymerase chain reaction (qRT-PCR). Farms of origin (n = 26) were situated in South Africa’s Western Cape Province. The associations between EqHV infection state and farm of origin, foal gender and foal age were subsequently described. Furthermore, nested PCRs were performed on parts of the 5′UTR, NS3 and NS5B genes of 17 samples. Samples were sequenced and phylogenetic analyses were conducted. The population’s seroprevalence was 83.70% and RNA was detected in 7.93% of samples. Increasing foal age was associated with decreasing ab prevalence and increasing prevalence of EqHV RNA. Sequences from South African EqHV strains did not show in-depth clustering with published sequences of EqHV isolates from particular continents. In conclusion, EqHV is present in the South African Thoroughbred population and appears more prevalent than reported in other horse populations worldwide.M.B. was supported by the Deutscher Akademischer Austauschdienst (DAAD). B.T. was supported by the Hannover Medical School. E.S. was supported by the Helmholtz Centre for Infection Research.http://www.elsevier.com/locate/vetmic2019-09-01hj2018Companion Animal Clinical StudiesEquine Research Centr

Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients

Background: Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known. Methods: Fifty-seven patients were included in this case-control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane-SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay. Results: Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4(+) T cells reactive against at least one SARS-CoV-2 protein. CD8(+) T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals. Conclusions: This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection

Ion-channel function and cross-species determinants in viral assembly of nonprimate hepacivirus p7

Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified

Inactivation of murine norovirus by chemical biocides on stainless steel

<p>Abstract</p> <p>Background</p> <p>Human norovirus (NoV) causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV) as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces.</p> <p>Methods</p> <p>The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA) or glutaraldehyde (GDA) for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells.</p> <p>Results</p> <p>PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v)] and 1-propanol [30% (v/v)] were able to inactivate MNV under clean conditions (0.03% BSA) on the carriers by ≥ 4 log₁₀within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v). Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes).</p> <p>Conclusion</p> <p>Products based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.</p