Inactivating hepatitis C virus in donor lungs using light therapies during normothermic ex vivo lung perfusion.

Availability of organs is a limiting factor for lung transplantation, leading to substantial mortality rates on the wait list. Use of organs from donors with transmissible viral infections, such as hepatitis C virus (HCV), would increase organ donation, but these organs are generally not offered for transplantation due to a high risk of transmission. Here, we develop a method for treatment of HCV-infected human donor lungs that prevents HCV transmission. Physical viral clearance in combination with germicidal light-based therapies during normothermic ex-vivo Lung Perfusion (EVLP), a method for assessment and treatment of injured donor lungs, inactivates HCV virus in a short period of time. Such treatment is shown to be safe using a large animal EVLP-to-lung transplantation model. This strategy of treating viral infection in a donor organ during preservation could significantly increase the availability of organs for transplantation and encourages further clinical development.

Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells.

Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1-8 cells were infected with this variant, viral particles were produced at >1011 copies ml-1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.

Elastografía tipo ARFI: modificación tras tratamiento antiviral en el trasplante hepático con recidiva por VHC / ARFI elastography: changes after direct-acting antiviral treatment in transplanted livers with relapse of hepatitis C virus infection

Objetivo. Evaluar la modificación de los valores en la elastografía hepática tipo ARFI tras el tratamiento antiviral en pacientes con trasplante hepático e infección por el virus de la hepatitis C (VHC) con recidiva durante el postrasplante. Material y métodos. Se han estudiado 15 pacientes receptores de trasplante hepático con infección por el VHC y recidiva sobre el injerto durante el primer año tras el trasplante que fueron tratados con antivirales de acción directa. A todos ellos se les realizó ecografía basal, Doppler y elastografía hepática tipo ARFI en los 3 meses anteriores al inicio del tratamiento y durante los 3 meses posteriores a su finalización. En la misma semana de la elastografía se realizó analítica con perfil hepático y determinación de la carga viral (ARN-VHC) en sangre periférica mediante técnica de reacción en cadena de la polimerasa. Se ha estudiado el cambio en las velocidades de cizallamiento de la elastografía antes y después del tratamiento. El análisis estadístico se ha efectuado mediante el test de Wilcoxon para datos apareados. Resultados. Todos los pacientes alcanzaron una respuesta viral mantenida a los 3 meses de finalizar el tratamiento. Se han encontrado diferencias estadísticamente significativas en las velocidades de la elastografía, con disminución de estas tras finalizar el tratamiento (la mediana de los valores de la onda de cizallamiento previos al tratamiento fue de 1,97m/s, y tras finalizarlo fue de 1,58m/s; p=0,001). Conclusión. La velocidad de la onda de cizallamiento disminuye de manera significativa tras el tratamiento antiviral en los pacientes receptores de trasplante hepático con recidiva de la infección por VHC (AU)

Objective. To evaluate the changes in the values obtained in acoustic radiation force impulse (ARFI) elastography of the liver after antiviral treatment in patients with liver transplants and hepatitis C virus (HVC) infection recurring after transplantation. Material and methods. We studied 15 patients infected with HVC who had received liver transplants where an HVC infection recurring in the graft within one year after the transplant was treated with direct-acting antivirals. All patients underwent ultrasonography including Doppler and ARFI elastography in the three months before starting treatment with direct-acting antivirals (baseline study) and again in the three months after finishing the treatment. In the same week when the elastography study was done, liver function tests were done and the viral load (HCV RNA) was determined in peripheral blood with the polymerase chain reaction method. We used the Wilcoxon test for paired samples to compare the changes in the shear wave velocities before and after treatment. Results. All patients achieved a virologic response that was sustained at 3 months after the end of treatment. On elastography, median shear wave velocities decreased after treatment (1.97m/s before treatment vs. 1.58m/s after treatment; p=0.001). Conclusion. The shear wave velocity decreased significantly after antiviral treatment in patients who had relapsed HVC infection after liver transplantation (AU)

The Paradoxical Effects of Different Hepatitis C Viral Loads on Host DNA Damage and Repair Abilities.

Hepatitis C virus (HCV)-induced hepatic stress is associated with increased oxidative DNA damage and has been implicated in hepatic inflammation. However, HCV infection and replication are uneven and vary among individual hepatocytes. To investigate the effect of the viral load on host DNA damage, we used an Enhanced Yellow Fluorescent Protein gene (EYFP)-tagged HCV virus to distinguish between HCV intracellular high viral load (HVL) cells and low viral load (LVL) cells. The cell sorting efficiency was confirmed by the high expression of the HCV polyprotein. We found DNA damage γ-H2AX foci in the HVL population. Comet assays demonstrated that HVL was related to the extent of the DNA strand breaks. Surprisingly, the DNA qPCR arrays and western blotting showed that the damage-related genes GPX2, MRE11, phospho-ATM, and OGG1 were significantly up-regulated in LVL cells but inversely down-regulated or consistently expressed in HVL cells. The colony survival assay to examine the repair abilities of these cells in response to irradiation showed that the LVL cells were more resistant to irradiation and had an increased ability to repair radiation-induced damage. This study found that intracellular viral loads drove cellular DNA damage levels but suppressed damage-related gene expression. However, the increase in damage-related gene expression in the LVL cells may be affected by ROS from the HVL cells. These findings provide new insights into the distinct DNA damage and repair responses resulting from different viral loads in HCV-infected cells.

Inactivation of HCV and HIV by microwave: a novel approach for prevention of virus transmission among people who inject drugs.

Hepatitis C virus (HCV) and human immunodeficiency virus (HIV-1) transmissions among people who inject drugs (PWID) continue to pose a challenging global health problem. Here, we aimed to analyse a universally applicable inactivation procedure, namely microwave irradiation, as a safe and effective method to reduce the risk of viral transmission. The exposure of HCV from different genotypes to microwave irradiation resulted in a significant reduction of viral infectivity. Furthermore, microwave irradiation reduced viral infectivity of HIV-1 and of HCV/HIV-1 suspensions indicating that this inactivation may be effective at preventing co-infections. To translate microwave irradiation as prevention method to used drug preparation equipment, we could further show that HCV as well as HIV-1 infectivity could be abrogated in syringes and filters. This study demonstrates the power of microwave irradiation for the reduction of viral transmission and establishment of this safety strategy could help reduce the transmission of blood-borne viruses.

Spherically-clustered porous Au-Ag alloy nanoparticle prepared by partial inhibition of galvanic replacement and its application for efficient multimodal therapy.

The polyvinylpyrrolidone (PVP)-coated spherically clustered porous gold-silver alloy nanoparticle (PVP-SPAN) was prepared by low temperature mediated, partially inhibited galvanic replacement reaction followed by silver etching process. The prepared porous nanostructures exhibited excellent photothermal conversion efficiency under irradiation of near-infrared light (NIR) and allowed a high payload of both doxorubicin (Dox) and thiolated dye-labeled oligonucleotide, DNAzyme (FDz). Especially, PVP-SPAN provided 10 times higher loading capacity for oligonucleotide than conventional hollow nanoshells due to increased pore diameter and surface-to-volume ratio. We demonstrated highly efficient chemo-thermo-gene multitherapy based on codelivery of Dox and FDz with NIR-mediated photothermal therapeutic effect using a model system of hepatitis C virus infected human liver cells (Huh7 human hepatocarcinoma cell line containing hepatitis C virus NS3 gene replicon) compared to conventional hollow nanoshells.

[Influence of Low-Frequency Electromagnetic Field on DNA Molecules in Water Solutions].

It is shown that the amplicons of hepatitis virus DNA (hepatitis B virus, hepatitis C virus) are capable of inducing radiation after an exposure to electromagnetic fields in the frequency range from 3 to 30 Hz and the field strength, 24-40 A/m, registered by means of a chemiluminescence method. The most effect of the electromagnetic field on water solutions of the amplicons of hepatitis virus DNA occurs at the frequency of 9 Hz, the change in the hydration shell of DNA amplicons is observed. It is suggested that the change in the hydration shell of DNA amplicons exposed to the low-frequency electromagnetic field leads to restoration of hydrogen bonding, stitchings formation and DNA repair as a whole.

Mechanisms of methods for hepatitis C virus inactivation.

Virus inactivation by chemical disinfectants is an important instrument for infection control in medical settings, but the mechanisms involved are poorly understood. In this study, we systematically investigated the effects of several antiviral treatments on hepatitis C virus (HCV) particles as model for enveloped viruses. Studies were performed with authentic cell culture-derived viruses, and the influence of chemical disinfectants, heat, and UV treatment on HCV was analyzed by the determination of infectious particles in a limiting-dilution assay, by quantitative reverse transcription-PCR, by core enzyme-linked immunosorbent assay, and by proteolytic protection assay. All different inactivation methods resulted in a loss of HCV infectivity by targeting different parts of the virus particle. Alcohols such as ethanol and 2-propanol did not affect the viral RNA genome integrity but disrupted the viral envelope membrane in a capsid protection assay. Heat and UV treatment of HCV particles resulted in direct damage of the viral genome since transfection of viral particle-associated RNA into permissive cells did not initiate RNA replication. In addition, heat incubation at 80°C disrupted the HCV envelope, rendering the viral capsid susceptible to proteolytic digest. This study demonstrated the molecular processes of viral inactivation of an enveloped virus and should facilitate the development of effective disinfection strategies in infection control not only against HCV but also against other enveloped viruses.

Thermostability of seven hepatitis C virus genotypes in vitro and in vivo.

Hepatitis C virus (HCV) is transmitted primarily through percutaneous exposure to contaminated blood especially in healthcare settings and among people who inject drugs. The environmental stability of HCV has been extrapolated from studies with the bovine viral diarrhoea virus or was so far only addressed with HCV genotype 2a viruses. The aim of this study was to compare the environmental and thermostability of all so far known seven HCV genotypes in vitro and in vivo. Incubation experiments at room temperature revealed that all HCV genotypes showed similar environmental stabilities in suspension with viral infectivity detectable for up to 28 days. The risk of HCV infection may not accurately be reflected by determination of HCV RNA levels. However, viral stability and transmission risks assessed from in vitro experiments correlated with viral infectivity in transgenic mice containing human liver xenografts. A reduced viral stability for up to 2 days was observed at 37 °C with comparable decays for all HCV genotypes confirmed by thermodynamic analysis. These results demonstrate that different HCV genotypes possess comparable stability in the environment and that noninfectious particles after incubation in vitro do not cause infection in an HCV in vivo model. These findings are important for estimation of HCV cross-transmission in the environment and indicate that different HCV genotypes do not display an altered stability or resistance at certain temperatures.

Two pathogen reduction technologies--methylene blue plus light and shortwave ultraviolet light--effectively inactivate hepatitis C virus in blood products.

BACKGROUND: Contamination of blood products with hepatitis C virus (HCV) can cause infections resulting in acute and chronic liver diseases. Pathogen reduction methods such as photodynamic treatment with methylene blue (MB) plus visible light as well as irradiation with shortwave ultraviolet (UVC) light were developed to inactivate viruses and other pathogens in plasma and platelet concentrates (PCs), respectively. So far, their inactivation capacities for HCV have only been tested in inactivation studies using model viruses for HCV. Recently, a HCV infection system for the propagation of infectious HCV in cell culture was developed. STUDY DESIGN AND METHODS: Inactivation studies were performed with cell culture-derived HCV and bovine viral diarrhea virus (BVDV), a model for HCV. Plasma units or PCs were spiked with high titers of cell culture-grown viruses. After treatment of the blood units with MB plus light (Theraflex MB-Plasma system, MacoPharma) or UVC (Theraflex UV-Platelets system, MacoPharma), residual viral infectivity was assessed using sensitive cell culture systems. RESULTS: HCV was sensitive to inactivation by both pathogen reduction procedures. HCV in plasma was efficiently inactivated by MB plus light below the detection limit already by 1/12 of the full light dose. HCV in PCs was inactivated by UVC irradiation with a reduction factor of more than 5 log. BVDV was less sensitive to the two pathogen reduction methods. CONCLUSIONS: Functional assays with human HCV offer an efficient tool to directly assess the inactivation capacity of pathogen reduction procedures. Pathogen reduction technologies such as MB plus light treatment and UVC irradiation have the potential to significantly reduce transfusion-transmitted HCV infections.

Hepatitis C virus induces interferon-λ and interferon-stimulated genes in primary liver cultures.

UNLABELLED: Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture-produced HCV (HCVcc) induced expression of Type III (λ) IFNs and of IFN-stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFNλ and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of λ IFNs. Gene induction was dependent on HCV replication, as ultraviolet light-inactivated virus was not stimulatory and an antiviral drug, 2'-C-methyladenosine, reduced induction of λ IFNs and ISGs. The level of IFNλ protein induced was sufficient to inhibit HCVcc infection of naïve cultures. CONCLUSION: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and λ IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo.

Thermal stability and inactivation of hepatitis C virus grown in cell culture.

BACKGROUND: Hepatitis C virus (HCV) is a blood-borne flavivirus that infects many millions of people worldwide. Relatively little is known, however, concerning the stability of HCV and reliable procedures for inactivating this virus. METHODS: In the current study, the thermostability of cell culture-derived HCV (HCVcc, JFH-1 strain) under different environmental temperatures (37 degrees C, room temperature, and 4 degrees C) and the ability of heat, UVC light irradiation, and aldehyde and detergent treatments to inactivate HCVcc were evaluated. The infectious titers of treated viral samples were determined by focus-forming unit (FFU) assay using an indirect immunofluorescence assay for HCV NS3 in hepatoma Huh7-25-CD81 cells highly permissive for HCVcc infection. MTT cytotoxicity assay was performed to determine the concentrations of aldehydes or detergents at which they were no longer cytotoxic. RESULTS: HCVcc in culture medium was found to survive 37 degrees C and room temperature (RT, 25 +/- 2 degrees C) for 2 and 16 days, respectively, while the virus was relatively stable at 4 degrees C without drastic loss of infectivity for at least 6 weeks. HCVcc in culture medium was sensitive to heat and could be inactivated in 8 and 4 min when incubated at 60 degrees C and 65 degrees C, respectively. However, at 56 degrees C, 40 min were required to eliminate HCVcc infectivity. Addition of normal human serum to HCVcc did not significantly alter viral stability at RT or its susceptibility to heat. UVC light irradiation (wavelength = 253.7 nm) with an intensity of 450 microW/cm2 efficiently inactivated HCVcc within 2 min. Exposures to formaldehyde, glutaraldehyde, ionic or nonionic detergents all destroyed HCVcc infectivity effectively, regardless of whether the treatments were conducted in the presence of cell culture medium or human serum. CONCLUSIONS: The results provide quantitative evidence for the potential use of a variety of approaches for inactivating HCV. The ability of HCVcc to survive ambient temperatures warrants precautions in handling and disposing of objects and materials that may have been contaminated with HCV.

Hepatitis C and human immunodeficiency virus RNA degradation by methylene blue/light treatment of human plasma.

Treatment of human plasma with methylene blue in combination with visible light (MB/light) inactivates several bloodborne viruses such as retro viruses and herpes viruses. The viral nucleic acid is thought to be a critical target for the inactivation procedure. We investigated the effects of photodynamic treatment on the RNA of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) using Amplicor reverse transcriptase polymerase chain reaction (RT-PCR), which detects and quantifies a small fragment of the viral RNA. The detectable HCV RNA load (5-nontranslated region) in infected human plasma declined by 94-97 % within 10 min of illumination in small-scale experiments (1-2 ml vol.). Since the same effect was observed in both anti-HCV positive and negative ("window") samples, it can be concluded that HCV antibodies do not influence virus inactivation by photodynamic treatment. The effect of treatment on RT-PCR signals of HIV-1, which is known to be inactivated rapidly by MB/light treatment, was examined. Plasma was infected with HIV-1 and subjected to RT-PCR, which detected a part of the gag gene. The extent and kinetics of PCR signal reduction induced by MB/light treatment were similar to those observed for HCV. Experiments at production scale where single plasma units (300 ml) were infected with HCV showed reduction rates of PCR signals consistent with those measured in the small-scale experiments. The data support the view that MB/light treatment affects the viral nucleic acids and suggest that HCV is susceptible to the procedure.

Virus inactivation of blood products by phenothiazine dyes and light.

Methylene blue (MB) and its derivatives azure A, B, C and thionine are photoactive and, in principle, are suitable for photodynamic virus inactivation of blood and blood products, such as therapeutic plasma. Methylene blue was selected for plasma decontamination because it is being clinically used and because of its known toxicological and other properties. The standard procedure for photodynamic treatment of single units of fresh plasma involves illumination with visible light at an MB concentration of 1 microM. Polymerase chain reaction analysis revealed that, in addition to model viruses, the bloodborne viruses hepatitis B virus, hepatitis C virus, human immune deficiency virus-1 and probably also the nonenveloped parvovirus B19 are sensitive to MB/light treatment. The procedure is further improved when the fluorescent tubes routinely used for illumination are replaced by more intense light sources, e.g. light-emitting diodes or low-pressure sodium lamps. Surprisingly, the improved virus kill is accompanied by reduced damage to plasma proteins.

Transmission of the hepatitis-C virus by tissue transplantation.

The hepatitis-C virus has been the most prevalent cause of chronic hepatitis in both blood and organ recipients. The introduction of a second-generation immunoassay for antibodies to the hepatitis-C virus (HCV 2.0) provided the opportunity to determine if the hepatitis-C virus can be transmitted through tissue transplantation. Banked sera from tissue donors that had previously been found to be non-reactive to the first-generation hepatitis-C virus antibody assay (HCV 1.0) and non-reactive for antibodies to hepatitis-B core antigen were retested with HCV 2.0. The sera from two donors were reactive; the transplant records of recipients of tissues from these donors were reviewed, and the surgeons or hospitals were contacted. The tissue recipients were tested with HCV 2.0, and positive sera were tested for hepatitis-C virus RNA by polymerase chain reaction. Viral nucleic acids isolated from viremic donors and recipients were analyzed for identity by sequencing of the hepatitis-C virus envelope gene (E2) hypervariable region. There were twenty-one grafts, which had been treated with gamma radiation, from one donor; thirteen had been transplanted to twelve recipients. Serum samples from six of the recipients were tested; one was reactive. This patient had other risk factors for infection with the hepatitis-C virus, and sequence analysis demonstrated non-identity between the donor and recipient hepatitis-C virus isolates. Nine of twelve grafts from a second donor had been transplanted in nine recipients. Serum samples from five patients were tested with HCV 2.0; four were reactive. In three of the four patients, the sera were determined to be positive for the hepatitis-C virus by polymerase chain reaction. E2 sequence analyses of hepatitis-C virus RNA isolates from two of these recipients demonstrated sequence identity with the donor isolate. The results of the present report demonstrate that the hepatitis-C virus can be transmitted by bone, ligament, and tendon allografts. They also support the need for testing of all tissue donors for antibodies to the hepatitis-C virus before the tissue is released for transplantation. The results also suggest that seventeen kilo-gray of gamma radiation may inactivate the hepatitis-C virus in tissue.

Inactivation of viruses in fresh-frozen plasma.

Methylene blue (MB) or solvent/detergent (SD) treatment is used for the inactivation of lipid-enveloped viruses in plasma. One important characteristic of the SD treatment is the necessity to pool plasma from different donors, thus inducing the risk of spreading infectious particles. MB treatment can be applied to single-donor plasma, causing no greater infectious risk than conventional fresh-frozen plasma (FFP). However, the virucidal efficacy of the SD method regarding HIV, HBV and HCV has been significantly better examined and proven than the MB treatment. Most of the therapeutic constituents of both plasma products are well maintained; coagulation factors decrease by roughly 5-20%. SD treatment reduces protein S and alpha 2-antiplasmin by approximately 40%, whereas MB treatment leads to a significant photooxidative alteration of fibrinogen with a disturbance of fibrin polymerization. As current studies show, the use of either plasma product is obviously not limited by acute or chronic toxicity. Several studies are in progress to evaluate the relevance of alterations in FFP quality which may affect the clinical efficacy of virus-inactivated plasma.