A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo.

BACKGROUND AND AIMS: The HEV is a small positive-sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. APPROACH AND RESULTS: We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2s mut ). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2s wt ) RNA or a variant lacking ORF2s expression (ORF2s mut ). The replication of the ORF2s mut virus was delayed by ~2 weeks compared with ORF2s wt , and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2s mut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2s mut , whereas they were long-lasting in RMs infected with ORF2s wt . Moreover, RMs infected with ORF2s mut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8 + T cells. CONCLUSIONS: These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

Type I and Type III Interferons Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures.

The newly emerged human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused a pandemic of respiratory illness. Current evidence suggests that severe cases of SARS-CoV-2 are associated with a dysregulated immune response. However, little is known about how the innate immune system responds to SARS-CoV-2. In this study, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral, surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by proinflammatory cytokines and chemokine induction, including interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and CXCL8, and identified NF-κB and ATF-4 as key drivers of this proinflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III interferon (IFN) response to SARS-CoV-2 infection. However, pretreatment and posttreatment with type I and III IFNs significantly reduced virus replication in pHAE cultures that correlated with upregulation of antiviral effector genes. Combined, our findings demonstrate that SARS-CoV-2 does not trigger an IFN response but is sensitive to the effects of type I and III IFNs. Our studies demonstrate the utility of pHAE cultures to model SARS-CoV-2 infection and that both type I and III IFNs can serve as therapeutic options to treat COVID-19 patients.IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. Our research identifies an excellent system to model SARS-CoV-2 infection of the human airways that can be used to test various treatments. Analysis of infection in this model system found that human airway epithelial cell cultures induce a strong proinflammatory cytokine response yet block the production of type I and III IFNs to SARS-CoV-2. However, treatment of airway cultures with the immune molecules type I or type III interferon (IFN) was able to inhibit SARS-CoV-2 infection. Thus, our model system identified type I or type III IFN as potential antiviral treatments for COVID-19 patients.

Plasmacytoid dendritic cells produce type I interferon and reduce viral replication in airway epithelial cells after SARS-CoV-2 infection.

Infection with SARS-CoV-2 has caused a pandemic of unprecedented dimensions. SARS-CoV-2 infects airway and lung cells causing viral pneumonia. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with inborn errors of type I IFN response or auto-antibodies against IFN-α. Plasmacytoid dendritic cells (pDCs) are a unique immune cell population specialized in recognizing and controlling viral infections through the production of high concentrations of type I IFN. In this study, we isolated pDCs from healthy donors and showed that pDCs are able to recognize SARS-CoV-2 and rapidly produce large amounts of type I IFN. Sensing of SARS-CoV-2 by pDCs was independent of viral replication since pDCs were also able to recognize UV-inactivated SARS-CoV-2 and produce type I IFN. Transcriptional profiling of SARS-CoV-2 and UV-SARS-CoV-2 stimulated pDCs also showed a rapid type I and III IFN response as well as induction of several chemokines, and the induction of apoptosis in pDCs. Moreover, we modeled SARS-CoV-2 infection in the lung using primary human airway epithelial cells (pHAEs) and showed that co-culture of pDCs with SARS-CoV-2 infected pHAEs induces an antiviral response and upregulation of antigen presentation in pHAE cells. Importantly, the presence of pDCs in the co-culture results in control of SARS-CoV-2 replication in pHAEs. Our study identifies pDCs as one of the key cells that can recognize SARS-CoV-2 infection, produce type I and III IFN and control viral replication in infected cells. IMPORTANCE: Type I interferons (IFNs) are a major part of the innate immune defense against viral infections. The importance of type I interferon (IFN) production for the control of SARS-CoV-2 infection is highlighted by the increased severity of COVID-19 in patients with defects in the type I IFN response. Interestingly, many cells are not able to produce type I IFN after being infected with SARS-CoV-2 and cannot control viral infection. In this study we show that plasmacytoid dendritic cells are able to recognize SARS-CoV-2 and produce type I IFN, and that pDCs are able to help control viral infection in SARS-CoV-2 infected airway epithelial cells.

Generation of induced pluripotent stem cells (iPSCs) from human foreskin fibroblasts.

The human iPS cell line VUZUZLi001-A (hVH-1) was generated from human foreskin fibroblasts to be used as a control line. Reprogramming was performed by retroviral transduction of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. Resource table.