Comparison of hepatitis C virus subtyping by ns5b sequencing with 5'utr based methods.

AIM: We compared Hepatitis C virus (HCV) genotyping by direct sequencing of the non-structural 5b region (NS5b) and a commercial PCR/hybridization method based on the conserved 5´-untranslated region (5'UTR). METHODS: One hundred twenty HCV containing plasma samples were analyzed by NS5b sequencing with focus on samples with undetermined results or 1b subtype identification in the used combination of Cobas® AmpliPrep/Cobas® TaqMan96® PCR and subsequent Versant® HCV Genotype 2.0 Assay (LiPA). RESULTS: There was 100% concordance between the two methods for genotyping but only 83% for subtyping. Seventeen samples were designated 1b by hybridization but subtype 1a by NS5b sequencing. This is a general 5'UTR problem as the discordant results were additionally confirmed by 5'UTR sequencing. Thus our routine combination not only misclassified 38.6% of subtype 1a isolates as 1b but in contrast to NS5b sequencing was unable to discriminate between subtypes 2a/c, or 4a/c/d and also failed on a newly described subtype (10a/3k). [corrected]. CONCLUSIONS: [corrected] The applied 5'UTR methods allow the rapid determination of HCV genotypes but failed to correctly identify the subtype in many samples. This has implications for epidemiological studies or forensic evaluation of chains of infection and NS5b sequencing therefore is our method of choice under those circumstances.

Intracellular hepatitis B virus nucleocapsids survive cytotoxic T-lymphocyte-induced apoptosis.

Following antigen recognition, hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTL) induce a necroinflammatory liver disease in HBV-transgenic mice. An early event in this process is CTL-dependent activation of apoptosis in a small fraction of HBV-positive hepatocytes. Here we show that cytoplasmic HBV nucleocapsids and their cargo of replicative DNA intermediates survive CTL-induced apoptosis of hepatocytes in vitro. These results suggest that destruction of infected cells per se is not sufficient to destroy the replicating HBV genome in infected tissue and that other events in addition to this process are required for viral clearance to occur.

Host-virus interactions during malaria infection in hepatitis B virus transgenic mice.

We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. We now report that intrahepatic HBV replication is also inhibited in mice infected by the malaria species Plasmodium yoelii 17X NL. P. yoelii infection triggers an intrahepatic inflammatory response characterized by the influx of natural killer cells, macrophages, and T cells. During this process, interferon (IFN)-gamma and IFN-alpha/beta suppress HBV gene expression and replication in the liver. Collectively, the data suggest that malaria infection might influence the course and pathogenesis of HBV infection in coinfected humans.

Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen.

Antibody(Ab)-mediated inhibition of sporozoite invasion of hepatocytes is a mechanism that has been clearly demonstrated to act upon Plasmodium falciparum pre-erythrocytic stages in humans. Consequently we have analyzed the Ab response to a recently identified P. falciparum sporozoite surface protein, STARP, in malaria-exposed individuals and tested the inhibitory effect of these Ab upon hepatocyte invasion in vitro. STARP-specific IgG were detected in 90 and 61% of sera from regions where individuals were exposed to 100 and 1-5 infectious bites per year, respectively. These IgG were predominantly of the cytophilic IgG1 or IgG3 type. STARP and the major sporozoite surface protein, CS, elicited equivalent IgG levels in adults. When affinity purified from either African immune sera or the serum of an individual experimentally protected by irradiated sporozoite immunization, STARP-specific Ab prevented up to 90% of sporozoites from invading human hepatocytes. The dose-dependent and reproducible inhibition was more pronounced than that observed with human CS-specific Ab affinity purified under identical conditions. Substantial reduction of sporozoite invasion was also observed with Ab induced by artificial immunization with recombinant STARP protein and reactive with the native protein. Taken together with recent findings of human cytotoxic T lymphocytes specific for this antigen, these results promote the interest of studying the efficacy of STARP as a target for immune effector mechanisms operating upon pre-erythrocytic stages.

Pathogenetic effector function of CD4-positive T helper 1 cells in hepatitis B virus transgenic mice.

The pathogenetic effector functions of hepatitis B virus (HBV)-specific CD4+, Th1 cells were analyzed in two inbred lineages of HBV transgenic mice, one of which overexpresses the HBV large envelope protein rendering the hepatocytes hypersensitive to the cytopathic effects of IFN-gamma, and another that expresses all of the HBV proteins and replicates the virus in the liver. Transfer of HBV envelope-specific Th1 cells resulted in recognition of viral Ag expressed by hepatic nonparenchymal cells, cytokine release, and a transient necroinflammatory liver disease in both lineages. The liver disease was very severe in the IFN-gamma-sensitive lineage, and it was less severe in the lineage that replicates the HBV genome; nonetheless, in this lineage the Th1 cytokines produced by these cells suppressed viral replication in the liver. These results demonstrate that CD4+ T cells with a Th1 functional phenotype can perform pathogenetic and antiviral effector functions in vivo. This suggests that CD4+ T cells can contribute directly to disease pathogenesis and inhibit viral replication during HBV infection.

Differential target cell sensitivity to CTL-activated death pathways in hepatitis B virus transgenic mice.

The current study was designed to explore the relative contribution of Fas ligand (FasL), perforin, IFN-gamma, and TNF-alpha-induced death pathways in the pathogenesis of CTL-induced liver disease. Hepatitis B virus-specific CTL that are genetically unable to produce either FasL, perforin, or IFN-gamma were injected into Fas-competent and Fas-deficient hepatitis B virus transgenic mice that are either sensitive or resistant to the cytopathic effects of IFN-gamma based on the extent to which their hepatocytes retain hepatitis B surface Ag (HBsAg). The results of these experiments indicate that FasL- and perforin-dependent signals are primarily responsible for the induction of liver disease in the absence of HBsAg retention, but both signaling pathways must be activated simultaneously by the CTL in order to kill the hepatocyte in vivo. In contrast, neither FasL nor perforin are required to kill hepatocytes that retain HBsAg as long as the CTL secrete IFN-gamma on antigen recognition. Finally the results indicate that, irrespective of their HBsAg content, hepatocytes are much less sensitive to destruction by TNF-alpha than by the other death pathways. While all of these death pathways appear to be operative during a normal CTL response, the current experiments suggest that the target cell determines which pathway is dominant and selects its mode of execution.

Human Plasmodium liver stages in SCID mice: a feasible model?

In a recent issue of Parasitology Today, Stanley and Virgin have stressed the potential of B- and T-cell deficient mice, among which severe combined immunodeficiency (SCID) mice are most frequently used, as models for the study of parasites. One of the most tantalizing prospects has been in the development of liver stages (LS) of human Plasmodium.

In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria blood parasites or adjuvants.

The mechanisms responsible for malarial immunity induced by repetitive injections of X-irradiated sporozoites have not been fully established. We demonstrate here that a single injection of irradiated sporozoites induced, as soon as 24 h after, a non-permissive state to hepatocyte reinfection with sporozoites in vitro. The same effect was observed when malarial blood forms, irradiated promastigotes of Leishmania infantum, adjuvants (muramyl dipeptide, poly acidylic uridylic) or interferon-gamma was injected. Activation of the nitric oxide (NO) pathway in the hepatocyte by these factors was found to be responsible for hepatocyte refractory status. Additionally, this metabolic pathway is involved in protection given by repeated injections of irradiated sporozoites since protection could be reversed by treating mice at the time of sporozoite challenge with a competitive inhibitor (NG-monomethyl-L-arginine) of the NO pathway. These results suggest that, in view of an antisporozoite vaccine, further studies are needed to find out how to activate specifically a long-lasting nonspecific immune response.