Persistent hepatitis C virus infection in vitro: coevolution of virus and host.

The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host

HBV core promoter mutations and AKT upregulate S-phase kinase-associated protein 2 to promote postoperative hepatocellular carcinoma progression

Mutations in the hepatitis B virus (HBV) core promoter (CP) have been shown to be associated with hepatocellular carcinoma (HCC). The CP region overlaps HBV X gene, which activates AKT to regulate hepatocyte survival. However, the cooperation between these two cascades in HCC progression remains poorly understood. Here, we assayed virological factors and AKT expression in liver tissues from 56 HCC patients with better prognoses (BHCC, ≥5-year survival) and 58 with poor prognoses (PHCC, <5-year survival) after partial liver resection. Results showed double mutation A1762T/G1764A (TA) combined with other mutation(s) (TACO) in HBV genome and phosphorylated AKT (pAKT) were more common in PHCC than BHCC. TACO and pAKT levels correlated with proliferation and microvascularization but inversely correlated with apoptosis in HCC samples. These were more pronounced when TACO and pAKT co-expressed. Levels of p21 and p27 were decreased in TACO or pAKT overexpressing HCC due to SKP2 upregulation. Levels of E2F1 and both mRNA and protein of SKP2 were increased in TACO expressing HCC. Levels of 4EBP1/2 decreased and SKP2 mRNA level remained constant in pAKT-overexpressing HCC. Therefore, TACO and AKT are two independent predictors of postoperative survival in HCC. Their co-target, SKP2 may be a diagnostic or therapeutic marker

Rhamnolipid Nano-Micelles Inhibit SARS-CoV-2 Infection and Have No Dermal or Eye Toxic Effects in Rabbits

Hand hygiene is considered to be the key factor in controlling and preventing infection, either in hospital care settings or in the community. Alcohol-based hand sanitizers are commonly used due to their rapid action and broad spectrum of microbicidal activity, offering protection against bacteria and viruses. However, their frequent administration during COVID-19 pandemic was associated with serious hazards, such as skin toxicity, including irritation, skin dermatitis, skin dryness or cracking, along with peeling redness or itching, with the higher possibility of getting infections. Thus, there is a need to find alternative and novel approaches for hand sanitation. In our previous publications, we reported that rhamnolipids nano-micelles had a comparable antibacterial activity to alcohol-based hand sanitizer and a lower cytotoxicity against human dermal fibroblast cells. In the current study, we investigated the antiviral activity of rhamnolipids nano-micelles against SARS-CoV-2. There was no cytotoxic effect on Vero cells noted at the tested concentrations of rhamnolipids nano-micelles. The rhamnolipids nano-micelles solution at 20, 78, and 312 µg/mL all demonstrated a significant (p &lt; 0.05) decrease of virus infectivity compared to the virus only and the blank vehicle sample. In addition, an acute irritation test was performed on rabbits to further ascertain the biosafety of rhamnolipids nano-micelles. In the eye and skin irritation tests, no degree of irritation was recorded after topical application of rhamnolipids nano-micelles. In addition, histopathological, biomarker, and hematological analyses from animals treated with rhamnolipids nano-micelles were identical to those recorded for untreated animal. From the above, we can conclude that rhamnolipids nano-micelles are a good candidate to be used as a hand sanitizer instead of alcohol-based hand sanitizers. However, they must still be tested in the future among healthcare workers (HCW) in a health care setting to ascertain their antimicrobial efficacy and safety compared to alcohol-based hand sanitizers

Rhamnolipid Nano-Micelles Inhibit SARS-CoV-2 Infection and Have No Dermal or Eye Toxic Effects in Rabbits

Hand hygiene is considered to be the key factor in controlling and preventing infection, either in hospital care settings or in the community. Alcohol-based hand sanitizers are commonly used due to their rapid action and broad spectrum of microbicidal activity, offering protection against bacteria and viruses. However, their frequent administration during COVID-19 pandemic was associated with serious hazards, such as skin toxicity, including irritation, skin dermatitis, skin dryness or cracking, along with peeling redness or itching, with the higher possibility of getting infections. Thus, there is a need to find alternative and novel approaches for hand sanitation. In our previous publications, we reported that rhamnolipids nano-micelles had a comparable antibacterial activity to alcohol-based hand sanitizer and a lower cytotoxicity against human dermal fibroblast cells. In the current study, we investigated the antiviral activity of rhamnolipids nano-micelles against SARS-CoV-2. There was no cytotoxic effect on Vero cells noted at the tested concentrations of rhamnolipids nano-micelles. The rhamnolipids nano-micelles solution at 20, 78, and 312 µg/mL all demonstrated a significant (p &lt; 0.05) decrease of virus infectivity compared to the virus only and the blank vehicle sample. In addition, an acute irritation test was performed on rabbits to further ascertain the biosafety of rhamnolipids nano-micelles. In the eye and skin irritation tests, no degree of irritation was recorded after topical application of rhamnolipids nano-micelles. In addition, histopathological, biomarker, and hematological analyses from animals treated with rhamnolipids nano-micelles were identical to those recorded for untreated animal. From the above, we can conclude that rhamnolipids nano-micelles are a good candidate to be used as a hand sanitizer instead of alcohol-based hand sanitizers. However, they must still be tested in the future among healthcare workers (HCW) in a health care setting to ascertain their antimicrobial efficacy and safety compared to alcohol-based hand sanitizers

Rituximab Treatment in Hepatitis C Infection: An In Vitro Model to Study the Impact of B Cell Depletion on Virus Infectivity

Hepatitis C virus (HCV) infected patients with vasculitis are often treated with the B-cell-depleting anti-CD20 antibody rituximab. Treatment reduces the cryoglobulins that cause vasculitis, yet it also leads to a transient increase in liver enzymes and HCV genomic RNA in the periphery. The mechanism underlying the increased viral load is unclear and both direct and indirect roles have been proposed for B cells in HCV infection. We previously reported that HCV can associate with B cells and can trans-infect hepatocytes. We established an in vitro assay to study the effect(s) of rituximab on B cell-associated HCV infectivity. Rituximab-mediated lysis of B cells in vitro increases the level of infectious HCV released from B cells. Our results, using a model where virus does not replicate in B cells, recapitulate observations seen in patients and may explain in part the rapid increase in blood HCV RNA observed after rituximab treatment

A role for B cells to transmit hepatitis C virus infection

Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge on the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We and others have reported that HCV can associate with and infect immune cells and may thereby evade host immune surveillance and elimination. To evaluate whether B cells play a role in HCV transmission, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) HCV patients to infect humanized liver chimeric mice. HCV was transmitted by B cells from chronic infected patients whereas the sera were non-infectious. In contrast, B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. We observed an association between circulating anti-glycoprotein E1E2 antibodies and B cell HCV transmission. Taken together, our studies provide evidence for HCV transmission by B cells, findings that have clinical implications for prophylactic and therapeutic antibody-based vaccine design

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81