Dynamics of Persistent TT Virus Infection, as Determined in Patients Treated with Alpha Interferon for Concomitant Hepatitis C Virus Infection

TT virus (TTV) is a recently identified widespread DNA virus of humans that produces persistent viremia in the absence of overt clinical manifestations. In an attempt to shed light on the dynamics of chronic infection, we measured the levels of TTV in the plasma of 25 persistently infected patients during the first 3 months of alpha interferon (IFN-alpha) treatment for concomitant hepatitis C virus (HCV) infection. The first significant decline of TTV loads was observed at day 3 versus day 1 for HCV. Subsequently, the loads of TTV became progressively lower in most patients, but some initial responders relapsed before the end of the follow-up, suggesting that at least in some subjects the effects of IFN on TTV can be very short-lived. No correlation between the responses of TTV and HCV to therapy was found. Fitting the viremia data obtained during the first week of treatment into previously developed mathematical models showed that TTV sustains very active chronic infections, with over 90% of the virions in plasma cleared and replenished daily and a minimum of approximately 3.8 x 10(10) virions generated per day. Low levels of TTV were occasionally detected in the peripheral blood mononuclear cells of patients who had cleared plasma viremia, thus corroborating previous results showing that these cells may support TTV replication and/or persistence.     

Co-infections with hepatitis G and TT virus in patients with chronic hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection. Seventy-seven patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.     

Prevalence of TT virus in patients with hepatitis B,C and hepatitis of unknown etiology

Discovery of TT virus in 1997 gave raise to intensive subsequent studies to learn about its structure, features and, what is the most important, about its role in pathogenesis of liver disease. The aim of the work was to analyze prevalence of TTV DNA in patients with diagnosed hepatitis B, C, that of unknown etiology and in healthy blood donors as well. Additionally the divergence of TTV sequence was estimated in selected cases. TTV DNA was detected by PCR technique using specific oligonucleotide primers for coding regions. TT virus has been detected in 25.6% (32/125) HBsAg positive patients and in 23.9% (51/213) HCV infected patients. In healthy blood donors the frequency of TTV was 24.3% (34/140) similarly to that found in HCV and HBV infected patients. The frequency of TTV DNA among patients with hepatitis of unknown etiology was 9.1%. This result was statistically significant lower than in the other groups. When detected sequences have been compared to these from NCBI base the homology result was 71% to 95%, and among different patients and groups of patients identity was 46% to 73%. On the basis of the obtained results it can be concluded that it is very unlikely that TTV coinfection plays any significant role in HCV or HBV infection. The hypothetical role of TTV infection in the etiopathogenesis of cryptogenic chronic hepatitis has not been confirmed. The results obtained in the small group of patients with hepatitis of unknown etiology are not conclusive and should be taken with some precaution. The final conclusion is the TTV coinfection does not contribute to the liver pathology. The divergence of TTV sequences may explain the various frequency of TTV viremia reported by other authors.     

Human immunodeficiency virus type 1-hepatitis C virus coinfection: intraindividual comparison of cellular immune responses against two persistent viruses

Both human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lead to chronic infection in a high percentage of persons, and an expanding epidemic of HIV-1-HCV coinfection has recently been identified. These individuals provide an opportunity for simultaneous assessment of immune responses to two viral infections associated with chronic plasma viremia. In this study we analyzed the breadth and magnitude of the CD8(+)- and CD4(+)-T-lymphocyte responses in 22 individuals infected with both HIV-1 and HCV. A CD8(+)-T-lymphocyte response against HIV-1 was readily detected in all subjects over a broad range of viral loads. In marked contrast, HCV-specific CD8(+)-T-lymphocyte responses were rarely detected, despite viral loads in plasma that were on average 1,000-fold higher. The few HCV-specific responses that were observed were relatively weak and limited in breadth. CD4-proliferative responses against HIV-1 were detected in about half of the coinfected subjects tested, but no proliferative response against any HCV protein was found in these coinfected persons. These data demonstrate a major discordance in immune responses to two persistent RNA viruses. In addition, they show a consistent and profound impairment in cellular immune responses to HCV compared to HIV-1 in HIV-1-HCV-coinfected persons.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

TT virus in the nasal secretions of children with acute respiratory diseases: relations to viremia and disease severity

The natural history and pathogenic potential of the recently identified TT virus (TTV) are currently a matter of intensive investigation. In an attempt to shed some light on these issues, nasal and blood specimens of 1- to 24-month-old children hospitalized with a clinical diagnosis of acute respiratory disease (ARD) were examined for the presence, load, and genetic characteristics of TTV. The results have indicated that at least in young children, the respiratory tract not only represents a route by which abundant TTV can be shed into the environment but also may be a site of primary infection and continual replication. Although we found no compelling evidence that TTV was the direct cause of ARD in some of the children studied, the average loads of TTV were considerably higher in patients with bronchopneumonia (BP) than in those with milder ARD, raising interesting questions about the pathophysiological significance of TTV at this site. Furthermore, group 4 TTV was detected almost exclusively in children with BP.     

Hepatitis C virus infection induces the beta interferon signaling pathway in immortalized human hepatocytes

Beta interferon (IFN-beta) expression is triggered by double-stranded RNA, a common intermediate in the replication of many viruses including hepatitis C virus (HCV). The recent development of cell culture-grown HCV allowed us to analyze the IFN signaling pathway following virus infection. In this study, we have examined the IFN-beta signaling pathway following infection of immortalized human hepatocytes (IHH) with HCV genotype 1a (clone H77) or 2a (clone JFH1). We observed that IHH possesses a functional Toll-like receptor 3 pathway. HCV infection in IHH enhanced IFN-beta and IFN-stimulated gene 56 (ISG56) promoter activities; however, poly(I-C)-induced IFN-beta and ISG56 expression levels were modestly inhibited upon HCV infection. IHH infected with HCV (genotype 1a or 2a) exhibited various levels of translocation of IRF-3 into the nucleus. The upregulation of endogenous IFN-beta and 2',5'-oligoadenylate synthetase 1 mRNA expression was also observed in HCV-infected IHH. Subsequent studies suggested that HCV infection in IHH enhanced STAT1 and ISG56 protein expression. A functional antiviral response of HCV-infected IHH was observed by the growth-inhibitory role in vesicular stomatitis virus. Together, our results suggested that HCV infection in IHH induces the IFN signaling pathway, which corroborates observations from natural HCV infection in humans.     

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.     

TT virus loads and lymphocyte subpopulations in children with acute respiratory diseases

TT virus (TTV) produces chronic plasma viremia in around 90% of healthy individuals of all ages and has, therefore, been proposed as a commensal human virus. We recently demonstrated that in children hospitalized for acute respiratory diseases high TTV loads were associated with severe forms of disease. Here, we report that in such children TTV loads showed an inverse correlation with the percentage of circulating total T and helper T cells and a direct correlation with the percentage of B cells. Thus, florid TTV replication might contribute to lymphocyte imbalances and, possibly, immunosuppressive effects, thus resembling related animal viruses.     

Interferons: Success in anti-viral immunotherapy

The interferons (IFNs) are glycoproteins with strong antiviral activities that represent one of the first lines of host defense against invading pathogens. These proteins are classified into three groups, Type I, II and III IFNs, based on the structure of their receptors on the cell surface. Due to their ability to modulate immune responses, they have become attractive therapeutic options to control chronic virus infections. In combination with other drugs, Type I IFNs are considered as “standard of care” in suppressing Hepatitis C (HCV) and Hepatitis B (HBV) infections, while Type III IFN has generated encouraging results as a treatment for HCV infection in phase III clinical trials. However, though effective, using IFNs as a treatment is not without the need for caution. IFNs are such powerful cytokines that affect a wide array of cell types; as a result, patients usually experience unpleasant symptoms, with a percentage of patients suffering system wide effects. Thus, constant monitoring is required for patients treated with IFN in order to reach the treatment goals of suppressing virus infection and maintaining quality of life.     

Different effects of the immunostimulatory drug Stimforte on infections of hepatitis C virus and herpes simplex virus type 1

Stimforte, an immune response-stimulating preparation, is active with respect to hepatitis C virus (HCV) and herpes simplex virus type I (HSV-1). The effects of Stimforte in animals infected with either HCV or HSV-1 are fundamentally different. In mice with acute herpes virus infection, Stimforte administration leads to a higher activity of natural killer cells and cytotoxic lymphocytes, and the amount of interferon (IFN) λ grows. In mice infected with HCV, Stimforte administration results in a significant increase in IFN-β but not IFN-λ in blood and affected organs. Stimforte has been found to affect directly HCV reproduction that causes the infected cell death, but it does not affect HSV-1 reproduction in the Vero cells (V).     

Hepatitis C virus biology

Hepatitis C virus infection represents a major problem of public health with around 350 millions of chronically infected individuals worldwide. The frequent evolution towards severe liver disease and cancer are the main features of HCV chronic infection. Antiviral therapies, mainly based on the combination of IFN and ribavirin can only assure a long term eradication of the virus in less than half of treated patients. The mechanisms underlying HCV pathogenesis and persistence in the host are still largely unknown and the efforts made by researchers in the understanding the viral biology have been hampered by the absence of a reliable in vitro and in vivo system reproducing HCV infection. The present review will mainly focus on viral pathogenetic mechanisms based on the interaction of HCV proteins (especially core, NS3 and NS5A) with host cellular signaling transduction pathways regulating cell growth and viability and on the strategies developed by the virus to persist in the host and escape to antiviral therapy. Past and recent data obtained in this field with different experimental approaches will be discussed.     

Phosphorylation of serine-rich protein encoded by open reading frame 3 of the TT virus genome

TT virus (TTV) is a newly discovered human virus with a single-stranded, circular DNA genome. The TTV DNA sequence includes two major open reading frames (ORFs), ORF1 and ORF2. Recently, spliced TTV mRNAs were detected and revealed two additional coding regions, ORF3 and ORF4. We found sequence similarity between the TTV ORF3 protein and hepatitis C virus (HCV) nonstructural 5A (NS5A) protein, which is a phosphoprotein and is thought to associate with various cellular proteins. To test whether the TTV ORF3 protein is phosphorylated, the state of phosphorylation was analyzed with a transient protein production system. The TTV ORF3 protein was phosphorylated at the serine residues in its C-terminal portion. Furthermore, the TTV ORF3 gene generated two forms of proteins with a different phosphorylation state, similar to the HCV NS5A region, suggesting that TTV ORF3 protein has function(s) similar to phosphorylated viral proteins such as the HCV NS5A protein.     

Elevated levels of interleukin-8 in serum are associated with hepatitis C virus infection and resistance to interferon therapy

Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). We have recently found that the HCV NS5A protein induces expression of the proinflammatory chemokine IL-8 to partially inhibit the antiviral actions of IFN in vitro. To extend these observations, in the present study we examined the relationship between levels of IL-8 in serum, HCV infection, and biochemical response to IFN therapy. Levels of IL-8 were significantly elevated in 132 HCV-infected patients compared to levels in 32 normal healthy subjects and were also significantly higher in patients who did not respond to IFN therapy than in patients who did respond to therapy. This study suggests that HCV-induced changes in levels of chemokine and cytokine expression may be involved in HCV antiviral resistance, persistence, and pathogenesis.     

Immune-based novel therapies for chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection is a great public health problem, with an estimated 200 million chronically infected patients worldwide. No vaccines are currently available for HCV, and only a subset of HCV patients responds to interferon-alpha (IFN-alpha) and Ribavirin treatment. Substantial evidence has emerged recently to support the role of the host immune response in the outcome and pathogenesis of HCV infection. Our aims of this article are to present the immune-based novel therapeutic options for HCV infection and the evidence supporting their use in patients with chronic hepatitis C. There is a growing consensus that acute control of HCV infection is associated with a vigorous intrahepatic antiviral CD4+ and CD8+ T cell response. IFN-gamma was detectable in the livers of the chimpanzees that cleared or controlled the virus, raising the possibility that IFN-gamma might perform antiviral effector functions during HCV infection. Based on these observations, therapeutic induction of intrahepatic IFN-gamma by adoptive immunotherapy might be able to control chronic HCV infection. Immune-based novel therapies appear to hold great promise in treating chronic HCV infection.     

Establishment of interferon alpha-resistant hepatitis C virus using cell culture system

To investigate the molecular mechanisms underlying interferon alpha (IFNα) treatment failure in hepatitis C virus (HCV) patients with chronic hepatitis, we aimed to develop an IFNα-resistant clone of HCV. By treating JFH-1-infected Huh7.5 cells with a prolonged low-dose treatment of IFNα, we selected a clone of HCV that survived against 100 U/ml of IFNα. By genetic analysis of this clone, we found four substitution mutations in the C-terminal coding sequence of non-structural 5A (NS5A). By introducing these four mutations into wild-type JFH-1, we established a new HCV clone that acquired IFNα resistant phenotype. These data suggest that four amino acid substitutions in NS5A are involved in IFNα resistance and thus this newly established HCV may be a useful tool for elucidating the molecular mechanisms of IFNα resistance in HCV patients.     

Hepatitis C virus infection among First Nation and non-First Nation people in Manitoba, Canada: a public health laboratory study

Demographic information and laboratory test results on 136 169 clinical serum specimens submitted to the public health laboratory in Manitoba, Canada, for hepatitis C virus (HCV) testing between January 1995 and December 2003 were analyzed. The difference in the clearance rates of HCV infection, without therapeutic intervention, and the HCV genotypes infecting First Nation and non-First Nation people were studied. The rates of co-infection of HCV-positive individuals with other hepatitis viruses were also compared between the two study groups. The results of the analyses of the data indicated that there was a 4.4-fold increase in the number of specimens tested and a 4.9-fold decrease in HCV antibody (anti-HCV) positive cases during the study period. The proportion of specimens submitted for testing from First Nation individuals was lower than their proportion in the Manitoba population. Our study also indicated that there was a significantly higher proportion of First Nation patients who had self-limiting infection (patients cleared the infection and became HCV RNA negative without anti-HCV treatment) in comparison to non-First Nation patients. The proportion of First Nation females who had self-limiting infection was significantly higher than non-First Nation females. HCV genotype 1 infection represented more than 60% of HCV infection in Manitoba. The rate of individuals positive for the hepatitis A virus antibody in the HCV-positive population was higher among First Nation than non-First Nation individuals. On the other hand, there were more HCV-infected First Nation patients than non-First Nation patients who were not immune to the hepatitis B virus. The data indicate that fewer First Nation patients seek anti-HCV therapy in comparison to non-First Nation. In conclusion, the differences in the rates of HCV self-limiting infection between First Nation and non-First Nation individuals in Manitoba may reflect the genetic differences between the two cohorts, which may consequently affect the immune response to the HCV infection.     

Bile acids promote the expression of hepatitis C virus in replicon-harboring cells

Hepatitis C virus (HCV) is a cause of chronic liver disease, with more than 170 million persistently infected individuals worldwide. Although the combination therapy of alpha interferon (IFN-alpha) and ribavirin is effective for chronic HCV infection, around half of all patients infected with HCV genotype 1 fail to show sustained virologic responses and remain chronically infected. Previously, we demonstrated that bile acids were essential for growth of porcine enteric calicivirus in cell culture in association with down-regulation of IFN responses. Because hepatocytes are exposed to high concentrations of bile acids in the liver, we hypothesized that bile acids have similar effects on HCV replication. We incubated HCV replicon-harboring cells (genotype 1b, Con1) in the presence of various bile acids and monitored the expression of HCV RNA and protein (NS5B). The addition of an individual bile acid (deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, or glycochenodeoxycholic acid) in the medium increased the levels of HCV RNA and proteins up to fivefold at 48 h of incubation. An antagonist of bile acid receptor farnesoid X receptor (FXR), Z-guggulsterone, reduced the bile acid-mediated increase of HCV RNA. When IFN (alpha or gamma) and each bile acid were incubated together, we observed that bile acid significantly reduced the anti-HCV effect of IFN. These results indicated that bile acids are factors in the failure of IFN treatment for certain patients infected with HCV genotype 1. Our finding may also contribute to the establishment of better regimens for treatment of chronic HCV infections by including agents altering the bile acid-mediated FXR pathway.