Exosome-mediated transmission of hepatitis C virus between human hepatoma Huh7.5 cells

HCV transmission by hepatic exosomes establishes a productive infection

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease with an estimated global prevalence of >120 million individuals (1). Of those who are exposed to the virus, an estimated 50–80% will develop chronic infection that can ultimately lead to hepatic fibrosis, hepatocellular carcinoma, and cirrhosis. As the virus is most effectively transmitted via blood, the majority of prevalent infections can be attributable to injection drug use or blood transfusions administered before 1990. Among incident HCV infections in the United States in 2005, the most frequently reported risk factors were injection drug use (50%), multiple sex partners (23%), surgery (14%) and percutaneous injury (10%) (2). Interestingly, no identifiable risk factors were reported by 26% of the participants. In at least some of the HCV-infected individuals without identifiable risk factors, noninjection drug use might be the mechanism of viral acquisition. As 8% of the US population engages in illicit substance use monthly (3), HCV transmission via noninjection drug use may be a common, yet under-appreciated, public health problem. Although most incident HCV infections occur among injection drug users, noninjection drug use is increasingly recognized as an emerging risk factor. Several recent studies have reported that the prevalence of HCV among noninjection drug users (NIDUs), estimated to range from 2 to 35%, is greater than that observed in the general population (4). The wide variability in the prevalence estimates among NIDUs results from differences in the primary aims and in the quality of the data obtained in individual studies. In the majority of these studies, HCV risk factor assessment among NIDUs was a secondary aim as the investigation of HCV transmission among injection drug users (IDUs) was their primary objective. Additional weaknesses of these studies include limited data regarding the likely time of HCV exposure and possible false-negative HCV antibody measurements due to waning serological markers, as HCV antibodies have been shown to disappear in a significant percentage of individuals 20 years after spontaneous resolution of the infection. Additionally, several studies did not use the recombinant immunoblot assay or HCV RNA quantitation to confirm positive serologic results. Misclassification and recall bias is another potential limitation of studies of both noninjection as well as injection drug use, as these studies largely rely on risk factor assessment based upon patient self-report. Misclassification might occur if prior injectors are erroneously classified as NIDUs. Recall bias, the inability to remember episodes of high-risk behavior, may have occurred from the effect of mind-altering substances or patient reluctance to report specific instances. Consequently, the specific behaviors among NIDUs that result in increased HCV seroprevalence and their relative importance toward the establishment of HCV infection remain unclear. How might NIDU behaviors result in HCV transmission? Most likely, two factors are required, exposure to HCV RNA in biological fluids and mucous membrane disruptions permissive for blood–virus interaction. HCV RNA has been detected in saliva from chimpanzees (5) and in up to 52% of humans with chronic HCV infection (6, 7). Injection of saliva derived from an HCV-infected chimpanzee into a second chimpanzee resulted in HCV infection (5). Similar observations have been noted in humans where a skin bite from an HCV-infected individual resulted in productive HCV infection (8). Other body fluids besides saliva also harbor HCV and may be vehicles of viral transmission. For example, HCV RNA has been detected in 59% of gingival crevicular fluid (GCF) specimens, and the detection of HCV RNA in GCF required a minimum plasma level of 100 000 copies/ml (7). In this study, only patients with measurable HCV RNA in GCF had detectable HCV RNA in saliva suggesting that GCF may be a potential viral reservoir. Additionally, secretions from other body compartments may also transit HCV. As the virus has also been detected in nasal sections of an NIDU (9), intranasal cocaine use could lead to HCV infection. Thus, sharing of inhalational equipment contaminated by either intranasal or oral fluids could permit viral transfer among individuals. The other factor required for the development of HCV infection is direct interaction between the virus and the host's blood. Among NIDUs, chronic substance abuse can lead to mucous membrane disruptions. For example, through its vasoconstricting effects as well as irritation from substances with which the drug is diluted (i.e. talc), cocaine can be locally irritating to the thin respiratory epithelium of the nasal cavity resulting in nasal septum perforations of both the cartilaginous and bony tissues. In the oral cavity, persistent cocaine use can result in ischaemic mucosal ulceration, rapid gingival recession, and dental erosions (10). In addition, individuals with chronic substance abuse display high rates of dental abnormalities, including decay and periodontal disease, which could further facilitate interaction between the virus and the host. Consequently, blood or saliva on inhalation equipment in combination with cocaine-induced lesions of oral or nasal mucous membranes is a mechanism by which noninjection drug use could result in productive HCV infection. In this issue of Liver International, Macias et al. (11) assessed risk factors for HCV acquisition in 182 NIDUs. Study participants were recruited from a drug abuse treatment center, and detailed information on previous drug addiction behaviors was available. Subjects underwent a structured interview that included detailed questions on potential routes of exposure, substance abuse patterns, and behaviors that increase the likelihood of HCV transmission. A study physician subsequently examined all subjects for signs of skin perforation such as evidence of recent venipuncture, tattoos and body piercings. Serological testing for human immunodeficiency virus, hepatitis B virus and HCV as well as measurement of HCV RNA levels was subsequently performed on each individual. The authors report an HCV prevalence of 12.6%, and they observed that age ≥34 years, the presence of tattoos, and sharing of crack cocaine inhalation equipment were independently associated with HCV infection in NIDUs. These results, which are corroborated by those of prior studies (12–17) (Table 1), suggest that HCV transmission can occur via shared crack cocaine inhalation equipment. The authors hypothesize that blood (from oral ulcerations) or saliva contamination of inhalational equipment could transmit quantities of virus sufficient to surpass the critical threshold, estimated to be 20 viral copies/ml in chimpanzees (18), necessary for productive infection. Macias et al. also found that tattoos were significantly associated with HCV transmission. Tattooing, especially when performed by a friend or relative, has been reported to be an independent risk factor for HCV transmission among high-risk individuals (12–14, 16, 17) (Table 1). Unprofessional tattooing, especially among incarcerated individuals, can be performed using a paperclip, staple or other sharp objects to break the surface of the skin, which is then pigmented with ink commonly from a ballpoint pen. HCV transmission might occur as a consequence of the same object being used on more than one individual and the fact that these implements are infrequently sterilized between insertions. Besides noninjection drug use and tattooing, other risk factors for HCV include incarceration (13, 19), altercations that result in traumatic injury (20), and sharing the same electric shears among many prisoners (20). How does this study rate in comparison with other studies that have assessed risk factors for HCV acquisition among NIDUs? A recent meta-analysis of 28 studies of NIDUs reported mean and median scores of 7.11 and 7.00, respectively, on an objective quality measurement scale (4). Using these criteria, we calculated a score of 10 for the Macias et al. study. Strengths of the present study are the inclusion of a relatively large sample, the fact that physicians experienced in drug addiction management conducted patient interviews and physical examinations, the fact that a comprehensive ascertainment of prior noninjection drug use behaviors was performed, and the analysis of the data using multivariate techniques. Of note is the fact that study participants did not receive payment for their involvement. The exclusion of IDUs from this study is an additional advantage. Recall bias, especially because patients may have been reluctant to report or did not recall specific high-risk behaviors, is a potential limitation of the study as it is with all studies that rely on patient self-report. This important study by Macias et al. identifies potential modes of HCV transmission in NIDUs, and it underscores the importance of noninjection drug use as a potential mechanism of viral acquisition. The exclusion of IDUs may permit detection of the weaker association between NIDU behaviors and HCV. Several factors concerning the mechanism of viral transmission, such as the relative contribution of blood versus salivary contamination and the minimum viral quantity required for human oral transmission, remain to be investigated. With an enhanced understanding of the factors that promote viral transmission in NIDUs, effective infection control interventions may be implemented.

Asian Pacific Association for the Study of the Liver consensus statements on the diagnosis, management and treatment of hepatitis C virus infection

Hepatitis C Virus Triggers Apoptosis of a Newly Developed Hepatoma Cell Line Through Antiviral Defense System

Liver transplantation for hepatitis C virus (HCV) non-viremic recipients with HCV viremic donors.

Transmission of Hepatitis C Virus Associated with Surgical Procedures—New Jersey 2010 and Wisconsin 2011: February 27, 2015 / 64(07);165-170

Preventing hepatitis C virus infection in injection drug users: risk reduction is not enough

HCV Activates Protein Kinase R and Attenuates Interferon-Induced MHC Class I Expression to Circumvent CD8+ T-Cell Responses

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread.

Acute and chronic hepatitis: Working Group Report of the First World Congress of Pediatric Gastroenterology, Hepatology, and Nutrition.

Ultra-short duration direct acting antiviral prophylaxis to prevent virus transmission from hepatitis C viremic donors to hepatitis C negative kidney transplant recipients.

With the development of effective therapies against human immunodeficiency virus (HIV), hepatitis C virus (HCV) infection has become a major cause of morbidity and mortality among patients with both infections (coinfection). In addition to the high prevalence of chronic HCV, particularly among HIV-infected injection drug users, the rate of incident HIV infections is increasing among HIV-infected men who have sex with men, leading to recommendations for education and screening for HCV in this population. Liver disease is the second leading and, in some cases, a preventable cause of death among coinfected patients. Those at risk for liver disease progression are usually treated with a combination of interferon (IFN) and ribavirin (RBV), which is not highly effective; it has low rates of sustained virologic response (SVR), especially for coinfected patients with HCV genotype 1 and those of African descent. Direct-acting antivirals might overcome factors such as immunodeficiency that can reduce the efficacy of IFN. However, for now it remains challenging to treat coinfected patients due to interactions among drugs, additive drug toxicities, and the continued need for combination therapies that include pegylated IFN. Recently developed HCV protease inhibitors such as telaprevir and boceprevir, given in combination with pegylated IFN and RBV, could increase the rate of SVR with manageable toxicity and drug interactions. We review the latest developments and obstacles to treating coinfected patients.

EASL Clinical Practice Guidelines: Management of hepatitis C virus infection

Hepatitis viruses and human immunodeficiency virus co-infection: pathogenisis and treatment

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.