HCV is a major public health problem, infecting more than 170 million people worldwide. Most cases of HCV infection become persistent and may eventually lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma. Although hepatocytes are the major site of viral replication, a broad clinical spectrum of extrahepatic complications and diseases are associated with chronic HCV infection, including mixed cryoglobulinemia, non-Hodgkins lymphoma, cutaneous vasculitis, glomerulonephritis, neuropathy, and lymphoproliferative disorders. The existence of extrahepatic reservoirs of HCV replication, particularly in PBMCs, remains highly controversial. It is unclear how B cells become dysregulated during the course of chronic HCV infection. Our group has demonstrated that free HCV is opsonized by complement and binds to CR1 on erythrocytes (ECR1); the presence of HCVspecific antibody significantly increased this binding. Whether binding of free HCV to erythrocytes is related to HCV clearance or pathogenesis has not been investigated. Mixed cryoglobulinemia (type II) is among the most common IC diseases associated with chronic HCV infection. Whereas nearly half of patients with HCV have detectable cryoglobulins, less than 10% develop clinically apparent disease. We have demonstrated that HCVIC binds to erythrocytes, and it is therefore plausible that factors affecting HCVIC/erythrocyte interaction could influence the expression of clinically evident ICrelated disease. (Hepatology 2018). To further explore the pathogenesis of HCV-related immune complex disease, we tested the levels of complement-activated immune complexes (IC), rheumatoid factors, and several chemokines and cytokines in plasma samples from chronic HCV patients and matched healthy control in order to elucidate the possible biological responses and consequences of HCVIC/erythrocyte interaction. We also investigate the genetic polymorphism of complement receptor 1 (CR1/CD35) gene associated with CR1 expression on erythrocytes from both groups of individuals, and how this CR1 expression level correlates with IC level. We have found that several chemokine levels including CXCL10, CXCL12, and BAFF in blood from patients with chronic HCV infection are significantly elevated when compared to those from healthy controls. Similar elevated levels of circulating immune complexes (CIC) and rheumatoid factors are also found in patients with chronic HCV infection. In addition, the levels of CIC in blood from chronic HCV patients with HH genotype in CR1 gene are significantly lower than those from chronic HCV patients with HL genotype. We have a manuscript in preparation. We have also demonstrated complement activation upon HCV infection (Hepatology 2016). We observed that the association of HCV with CD19+ B cells is mediated by the complement system. In addition, using antibodies against cell surface markers, we showed that the binding complex mainly involved CD21 (complement receptor 2), CD19, CD20, and CD81. In human B cells, CD21 is known to form a costimulatory complex with CD19 and CD81. Co-ligation of the B cell antigen receptor (BCR) with this costimulatory complex can lower the threshold required for BCR-mediated B cell activation and proliferation. Epidemiological studies have demonstrated an increased risk of developing B-cell non-Hodgkin lymphoma in patients with chronic HCV infection. Both the regression of HCV-associated lymphoma with antiviral treatment and the beneficial effects of antiviral treatment on overall survival of patients with HCV-related lymphoma have strengthened the aetiological link between HCV infection and NHL presumed from epidemiological, clinical and pathophysiological studies. Our goal is to move to the next step and elucidate the pathways through which complement mediated HCV attachment/infection might lead to malignant transformation of B-cells. Since B-cells proliferation, in response to antigenic stimulation or polyclonal activation, may predispose to genetic aberrations (mutation, gene translocation, gene fusion, chromosomal amplification or deletion) we are using RNA-sequencing, to obtain both mutational and gene expression data from B-cells obtained from patients who are HCV+, HBV+, HDV+ and B-NHL+, and compare to patients who are HCV-, HBV-, HDV-, NHL+; HCV+, HBV+, HDV+, NHL-, and healthy controls. We will validate/ confirm the results on paired Paraffin Embedded lymphatic tissues then. The adoption of RNA-sequencing has been proven useful in clinical oncology as most of the clinically relevant somatic mutations are also expressed on the RNA level. It is a promising technology to sequence genes for the identification of mutational status, while simultaneously obtaining information on structural variations and gene expression perturbations-related diseases. We are recipient of an NIAID-NCI grant: Investigation of HCV and HBV-Mediated B-Cell Malignant Transformation and Prioritization of Treatment for HCV-Related NHL in Mongolia. We are currently processing more than 200 samples received from Mongolia. We are also conducting in vitro studies to address whether the up-regulation of several genes in BCR signaling pathway found in B cells from chronic HCV patients is mediated by the chemokines associated with HCV infection. Preliminary results indicate there is significant up-regulation (20% to 30% increase) of BCR signaling genes in chemokine-treated B cells from healthy blood donor. We are now processing data form more donors to confirm this observation. Another ongoing study in our group is to assess whether we can demonstrate that complement-opsonized HCV particles can activate BCR signaling pathway on B cells. Once the in vitro system will be finalized and validated, we will assess, using qPCR, the BCR pathway genes expression from B cells cultured with and without complement opsonized JFH1 virus. We will compare the data with HCV patient-derived B cells propagated in vitro.

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Salam, Kazi Abdus; Wang, Richard Y; Grandinetti, Teresa et al. (2018) Binding of Free and Immune Complex-Associated Hepatitis C Virus to Erythrocytes Is Mediated by the Complement System. Hepatology 68:2118-2129
Wang, Richard Y; Bare, Patricia; De Giorgi, Valeria et al. (2016) Preferential association of hepatitis C virus with CD19+ B cells is mediated by complement system. Hepatology 64:1900-1910
Prentoe, Jannick; Verhoye, Lieven; Velázquez Moctezuma, Rodrigo et al. (2015) HVR1-mediated antibody evasion of highly infectious in vivo adapted HCV in humanised mice. Gut :
Bukh, Jens; Engle, Ronald E; Faulk, Kristina et al. (2015) Immunoglobulin with High-Titer In Vitro Cross-Neutralizing Hepatitis C Virus Antibodies Passively Protects Chimpanzees from Homologous, but Not Heterologous, Challenge. J Virol 89:9128-32
Thomas, Jaime M; Pos, Zoltan; Reinboth, Jennifer et al. (2014) Differential responses of plasmacytoid dendritic cells to influenza virus and distinct viral pathogens. J Virol 88:10758-66
Fujiwara, Kei; Allison, Robert D; Wang, Richard Y et al. (2013) Investigation of residual hepatitis C virus in presumed recovered subjects. Hepatology 57:483-91
Xu, Chenyu; Wang, Richard Y; Schechterly, Cathy A et al. (2013) An assessment of hepatitis E virus (HEV) in US blood donors and recipients: no detectable HEV RNA in 1939 donors tested and no evidence for HEV transmission to 362 prospectively followed recipients. Transfusion 53:2505-11
Zhang, Lumin; Alter, Harvey J; Wang, Haiping et al. (2013) The modulation of hepatitis C virus 1a replication by PKR is dependent on NF-kB mediated interferon beta response in Huh7.5.1 cells. Virology 438:28-36
Allison, Robert D; Conry-Cantilena, Cathy; Koziol, Deloris et al. (2012) A 25-year study of the clinical and histologic outcomes of hepatitis C virus infection and its modes of transmission in a cohort of initially asymptomatic blood donors. J Infect Dis 206:654-61
Pripuzova, Natalia; Wang, Richard; Tsai, Shien et al. (2012) Development of real-time PCR array for simultaneous detection of eight human blood-borne viral pathogens. PLoS One 7:e43246

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