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Human immunodeficiency virus (HIV-1)-infected patients frequently harbour hepatitis B and C viruses (HBV and HCV, respectively). Possible modifications of the natural history of hepatitis B may occur. The aim of this study was to characterise HBV diversity and evolutionary and mutational viral genome profiles in HIV-1/HBV coinfections. HIV-1 and HBV markers determinations (Roche, FRG; Abbott, USA) and HBV genome-length retrospective analysis were performed in follow-up isolates from patients who were either stably HBsAg-negative with a low level of HBV DNA (occult hepatitis B infection, OBI) or HBsAg-positive with a high level of HBV DNA. Phylogenetic analysis (maximum likelihood method, MEGA5), statistical analysis and evolutionary rates calculation (d S/d N) were applied. Positive selection pressures in the PreS/S region and a significantly higher number of mutations in this region including the major hydrophilic region (MHR) and the “a” determinant were shown in HBsAg-negative (possibly OBI) compared to stably HBsAg-positive HIV-1/HBV subgenotypes D3/A2 coinfected patients. Mutants previously described in HIV-1/HBV coinfected patients were found. Known mutants Y100C, P127T and P120A associated to Y134H and S143T and new S mutants, which may potentially affect HBsAg expression and secretion and anti-HBs binding, were detected in baseline sera persisting up to the end of 9 years follow-up. Known mutations of BCP, Pre-C, C and X regions were also characterised. Natural mutants strictly known as being involved in diagnostic failure were not detected; however, numerous corresponding sites showed amino acid variations. Evolutionary and genotypic differences observed, particularly in the PreS/S region, between HBsAg-negative (OBI) and HBsAg-positive HIV-1/HBV coinfected patients, may contribute, in association with mutations of other genomic regions, to the HBsAg-negative phenotype.

Studies focused on the understanding of the molecular mechanisms involved in recovery or progression to chronicity of HBV may take advantage of natural and experimental models that mimic its properties. This is also of relevance for associated diseases such as cirrhosis and hepatocellulocarcinoma. The eastern woodchuck (Marmota monax) infected by the hepadnavirus woodchuck Hepatitis B virus (WHV) has been applied as a predictive model to support development of new HBV vaccines, antivirals, immunotherapies and combination therapies. This report summarizes studies carried out by our and other groups, with the application of this model in natural and experimental infections. Using standardized viral inocula in neonate and adult animals and newly established assays, the presence of the specific patterns of markers of acute, chronic and resolved infections and their relationships in the different virus-host interactions have been shown. B and T cell responses and T(H)1 cytokine expression have been shown to play a crucial role in the outcome of infection. The availability of the WHV/Marmota monax model and specific standardized assays may allow evaluation of new formulations of multimodal therapeutic strategies based on antiviral chemotherapy and immunomodulation. These may also include specifically targeted immunocomplexes. Such therapies could constitute new frontiers for the treatment of HBV chronic disease.

The woodchuck hepatitis B virus (WHV), the closest genetically related virus of HBV, and its natural host Marmota monax constitute a well recognized animal model. The application of this model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use, for lamivudine-CHO-PreS/S vaccine therapy and WHV particles coupled with HBV derived woodchuck PreS/S antibodies (IC complex) studies, as shown that the PreS/S-CHO vaccine is the first human vaccine able to elicit non sterilizing protection in the woodchuck model. The very early appearance and selection of the domain B FLLA motif resistant mutant not neutralized by the antibodies produced following vaccination, has confirmed that more potent antivirals and/or multiple targeted options with possible inclusion of immune complexes should be considered.

The objective of this study was to evaluate, by developing one-step real-time PCR, the outcome of superinfection with hepatitis D virus (HDV) genotype I in woodchucks that were chronic carriers of woodchuck hepatitis virus (WHV) and did not show relevant signs of liver damage. Three woodchucks (Marmota monax) chronically infected with WHV were superinfected with a woodchuck HDV inoculum. The evolution of the WHV and HDV infections was monitored by quantifying HDV-RNA, WHV-DNA, and HDV-WHV antigens and antibodies. WHV and HDV sequencing was also performed and liver markers were evaluated. Liver damage was assessed using the Ishak method. All woodchucks showed a high HDV viral load, antigenemia and short survival after superinfection. Histopathological examination of autoptic liver samples showed massive liver necrosis compatible with an acute fatal course of hepatitis. The WHV sequencing showed that the virus population was not substituted by the WHV inoculum. The HDV sequencing performed during superinfection and at autopsy indicated amino acid changes in immune dominant regions of the HDV antigen. The strong correlation between acute infection with HDV genotype I and rapid and fatal liver failure indicates that HDV can be an important factor in the prognosis of HDV-WHV-superinfected woodchucks.

Several studies have shown the importance of evaluating Recent Thymic Emigrants (RTEs) by quantification of T cell receptor-rearrangement excision circles (TRECs), as a measure of de novo T cell generation during human immunodeficiency virus-1 (HIV-1) infection. To determine whether acute viral infection may have an impact on TRECs, cynomolgus monkeys (Macaca fascicularis) were infected intrarectally with simian human immunodeficiency virus (SHIV) 89.6P(cy11) and the number of signal-joint (sj) TRECs was determined in purified CD4+ and CD8+ populations for up to 28 weeks post-infection. Four weeks after infection, TRECs levels significantly decreased in both CD3+ CD4+ and in CD3+ CD8+ T lymphocytes of infected monkeys, whereas they remained unchanged in uninfected animals. This reduction was followed by a progressive TRECs number recovery in CD3+ CD4+ T lymphocytes that positively correlated with changes in the levels of circulating CD3+ CD4+ T cells. In the CD3+ CD8+ T cell subset, TRECs number remained significantly low and inversely correlated with the increase in the percentages of CD3+ CD8+ T cells. These data suggest that SHIV89.6P(cy11) intrarectal infection of cynomolgus monkeys differently affects TRECs content in CD3+ CD4+ and CD3+ CD8+ T cell subsets.

Alpha-defensins have been shown to inhibit HIV-1 replication in vitro and may contribute to the overall control of viral replication in vivo. In the present work, we quantitatively measured the levels of alpha-defensins in the plasma of healthy and experimentally SHIV-infected Macaca fascicularis (cynomolgus monkeys), an animal model of AIDS pathogenesis and vaccine development. Characterization of physiological plasma alpha-defensins levels was performed in 12 healthy monkeys following longitudinal analysis using an alpha-defensins ELISA kit currently validated for macaque use. Subsequently, alpha-defensins levels were quantitatively measured in 23 cynomolgus monkeys during titration protocols following both the mucosal and systemic routes of infection with the pathogenic SHIV89.6P(cy11). A significant increase in plasma alpha-defensins levels was consistently observed at early time points in all infected animals, regardless of the infection route. Moreover, a positive correlation was observed between viral replication and levels of alpha-defensins during the acute phase of infection. Interestingly, in the animals infected through the mucosal route, alpha-defensins levels remained significantly higher at later time points, up to 19 weeks from the infection, while in cynomolgus infected intravenously, alpha-defensins levels returned to baseline levels by 4 weeks from infection, suggesting that the different route of infection may differently activate the innate immune response.

Background Hepatitis C Virus (HCV) infection is associated with chronically evolving disease and development of hepatocellular carcinoma (HCC), albeit the mechanism of HCC induction by HCV is still controversial. The nucleocapsid (core) protein of HCV has been shown to be directly implicated in cellular transformation and immortalization, enhancing the effect of oncogenes and decreasing the one of tumor suppressor genes, as RB1 and its protein product pRB. With the aim of identifying novel molecular mechanisms of hepatocyte transformation by HCV, we examined the effect of HCV core protein on the expression of the whole Retinoblastoma (RB) family of tumor and growth suppressor factors, i.e. pRb, p107 and pRb2/p130. Methods We used a model system consisting of the HuH-7, HCV-free, human hepatocellular carcinoma cell line and of the HuH-7-CORE cells derived from the former and constitutively expressing the HCV core protein. We determined pRb, p107 and pRb2/p130 protein and mRNA amount of the respective genes RB1, RBL1 and RBL2, RBL2 promoter activity and methylation as well as DNA methyltransferase 1 (DNMT1) and 3b (DNMT3b) expression level. The effect of pRb2/p130 over-expression on the HCV core-expressing HuH-7-CORE cells was also evaluated. Results We found that the HCV core protein expression down-regulated pRb2/p130 protein and mRNA levels in HuH-7-CORE cells by inducing promoter hyper-methylation with the concomitant up-regulation of DNMT1 and DNMT3b expression. When pRb2/p130 expression was artificially re-established in HuH-7-CORE cells, cell cycle analysis outlined an accumulation in the G0/G1 phase, as expected. Conclusions HCV core appears indeed able to significantly down-regulate the expression and the function of two out of three RB family tumor and growth suppressor factors, i.e. pRb and pRb2/p130. The functional consequences at the level of cell cycle regulation, and possibly of more complex cell homeostatic processes, may represent a plausible molecular mechanism involved in liver transformation by HCV.

We investigated the protective efficacy of a systemic triple vector (DNA/rSFV/rMVA)-based vaccine against mucosal challenge with pathogenic simian immunodeficiency virus (SIV) in cynomolgus monkeys. Animals were immunized at week 0 with DNA (intradermally), at weeks 8 and 16 with recombinant Semliki Forest virus (rSFV, subcutaneously) and finally, at week 24, with recombinant modified vaccinia virus Ankara strain (rMVA, intramuscularly). Both DNA and recombinant viral vectors expressed a wide range of SIV proteins (Gag, Pol, Tat, Rev, Env and Nef). This immunization strategy elicited cell-mediated rather than humoral responses that were especially increased following the last boost. Upon intrarectal challenge with pathogenic SIVmac251, three of the four vaccinated monkeys dramatically abrogated virus load to undetectable levels up to 41 weeks after challenge. A major contribution to this vaccine effect appeared to be the T-cell-mediated immune response to vaccine antigens (Gag, Rev, Tat, Nef) seen in the early phase of infection in three of the four vaccinated monkeys. Indeed, the frequency of T-cells producing antigen-induced IFN-γmirrored virus clearance in the vaccinated and protected monkeys. These results, reminiscent of the efficacy of live attenuated virus vaccines, suggest that vaccination with a combination of many viral antigens can induce a robust and stable vaccine-induced immunity able to abrogate virus replication.

The Eastern woodchuck (Marmota monax) is a useful experimental model for evaluating antiviral therapy against chronic HBV infection. In the present study, an immunogenic complex (IGC) composed of immune sera containing PreS/S heterologous antibodies (anti-HBs) and serum-derived WHV particles containing 10(7) WHV-DNA copies/50 µl was developed. The IGC was administered to WHV-negative woodchucks and natural chronic WHV carriers, with the final aim of evaluating the outcome of WHV infection in both groups. A control group of three animals, infected experimentally with viral particles only, was also evaluated. Following IGC administration, two WHV-negative woodchucks exhibited persistent infection, with WHV-DNA levels 3-6 logs lower than the WHV-DNA levels of the controls that developed persistent infection. WHeAg seroconversion to anti-WHe was observed in these two woodchucks and in two control woodchucks which developed self-limited infection. In two of the four chronic carriers, the WHV-DNA level decreased significantly (by 4-6 logs) following IGC administration, with no rebound in viral load during follow-up. WHeAg seroconversion to anti-WHe was observed also in these animals. Analyses of the sequences derived from envelope proteins confirmed that IGC did not induce the emergence of resistant viral variants. The results of this study indicate that the IGC could be useful for breaking the tolerance in hepadnaviral infection and for boosting the host's innate and adoptive immune response.

Results Three out of four HBV patients with occult infection, showed reactivation phases of HBV viremia. Different mutations were observed, with differences between preand post-reactivation sera. HBV-DNA remained at low levels during the entire study period also in absence of specific anti-HBV therapy. The phylogenetic analysis showed that, for each patient with HBV reactivation, all the isolates were originated from a unique parental virus. Specific mutations of PreS/S, Core and X regions were observed. Discussion Mutations in the “a” determinant of the S protein could be responsible for the absence of HBsAg detection. The presence of stop codon in the pre-core region and of mutations in the X region could, in part, explain the reactivation of HBV viremia.

Several innate cellular antiviral factors exist in mammalian cells that prevent the replication of retroviruses. Among them, the tripartite motif protein (TRIM)5α has been shown to block human immunodeficiency virus type 1 (HIV-1) infection in several types of Old World monkey cells. Here we report a novel HIV-1 chronically infected monkey B cell line, F6/HIV-1, characterized by very low levels of TRIM5α expression that allows HIV-1 to overcome the restriction. Virus produced by F6/HIV-1 cells fails to infect monkey cells but retains the ability to infect human peripheral blood mononuclear cells (PBMCs) and T cell lines, although with a reduced infectivity compared to the input virus. Ultrastructural analyses revealed the presence of budding virions at the F6/HIV-1 cells plasma membrane characterized by a typical conical core shell. To our knowledge F6/HIV-1 is the first monkey cell line chronically infected by HIV-1 and able to release infectious particles thus representing a useful tool to gain further insights into the molecular mechanisms of HIV-1 pathogenesis. J. Cell. Physiol. 221: 760–765, 2009. © 2009 Wiley-Liss, Inc.

Present efforts of HBV vaccine research are aimed at defining targeted antigen compositions and adjuvancy systems for earlier and broader immune responses and optimization of immunotherapeutic approaches. We have demonstrated the applicability of the WHV/Marmota monax model for the evaluation of immunogenicity and protection of new formulations of HBV vaccines for human use. Protective activity was evaluated following the administrations of HBV CHO-PreS/S and adjuvanted S/Core vaccines. The administration of a complex constituted by HBV derived woodchuck PreS/S antibodies coupled with WHV particles was able to induce inhibition of viral replication. Future studies on treatment of HBV chronic infection should be addressed to the evaluation of therapies combined with antivirals, vaccines and immunomodulatory compounds.

To determine whether the addition of a pre-S/S human vaccine increases the antiviral activity of lamivudine, four woodchucks were treated with a daily dose of 100 mg/kg lamivudine and four 50 microg doses of CHO-derived pre-S/S human vaccine. WHV DNA titres decreased up to two logarithms in three woodchucks. At week 4, in three of the animals, the sequence analysis showed a predominant strain containing a nucleotide change from A to T at position 1696 of domain B of the WHV DNA polymerase. Vaccination did not further suppress WHV DNA, despite anti-HBs production in three animals. The woodchuck remains a useful model for characterising the biology and kinetics of the emergence of drug-resistant variants and could be used for pre-clinical studies of combinations of new antiviral drugs.

prednisolone with tacrolimus (N=4) or cyclosporine (N=1) with or without mycophenolate mofetil. The 4 lung transplant recipients were treated with ribavirin (RBV) and followed up for up to 19 months. Results: HEV infection became apparent 4 [1–9] (median [range]) months after transplantation. All patients had elevated liver enzymes at diagnosis of HEV infection (mean ALT: 79 [62–110] U/l; [range]) and were infected with HEV genotype 3. Liver biopsy revealed presence of cirrhosis in 3 patients (Metavir fibrosis stage 1 and 2 in the remaining two). During RBV treatment ALT values normalized in all, but HEV-RNA became undetectable in only 2 patients. One Patient achieved a SVR, the other relapsed. The kidney-transplanted patient presented with liver failure and died prior to initiation of ribavirin treatment. Conclusions: Chronic HEV infection may have a rapid fibrosis progression and is an important cause of liver related morbidity in patients after transplantation. Treatment with ribavirin results in viral suppression and biochemical normalization, although it might not be successful in all patients on the long run.

The efficacy of a multicomponent vaccination with modified vaccinia Ankara constructs (rMVA) expressing structural and regulatory genes of simian immunodeficiency virus (SIV(mac251/32H/J5)) was investigated in cynomolgus monkeys, following challenge with a pathogenic SIV. Vaccination with rMVA-J5 performed at week 0, 12, and 24 induced a moderate proliferative response to whole SIV, a detectable humoral response to all but Nef SIV antigens, and failed to induce neutralizing antibodies. Two months after the last boost, the monkeys were challenged intravenously with 50 MID50 of SIV(mac251). All control monkeys, previously inoculated with non-recombinant MVA, were infected by week two and seroconverted by weeks four to eight. In contrast a sharp increase of both humoral and proliferative responses at two weeks post-challenge was observed in vaccinated monkeys compared to control monkeys. Although all vaccinated monkeys were infected, vaccination with rMVA-J5 appeared to partially control viral replication during the acute and late phase of infection as judged by cell- and plasma-associated viral load.

Poliovirus type 2 Sabin mutants were selected for drug resistance and dependence by plating on HeLa cell monolayers in the presence of 3(2H)-isoflavene, a compound related to dichloroflavan, which prevents the shut-off of host translation and poliovirus RNA and protein synthesis. The drug-resistant mutants grew equally well in the presence and in the absence of the drug, while the drug-dependent mutants only grew in the presence of the compound. One dependent and one resistant mutant were characterized biologically in more detail. The resistant mutant did not exhibit thermolability. The mild thermolability exhibited by the dependent mutant was not affected by the addition of 3(2H)-isoflavene, indicating that the substance does not bind the poliovirus type 2 Sabin capsid. The translation of viral proteins and the shut-off of host protein translation during cell infection were not inhibited in either mutant. In the absence of the drug, the cleavage of the precursor VPO, a step in virus protein processing, was affected in the dependent mutant. The dependence of the mutant on the drug was due to the inability of 75S empty particles to reach maturation: our results strongly suggest that this phenomenon is strictly dependent on the reduction of RNA synthesis, confirming the existence of a dynamic equilibrium between RNA production and genome encapsidation during the poliovirus replication cycle.

Surveillance of suspected poliomyelitis cases was conducted in Albania from 1980 through 1995. A total of 93 cases were reported, 11 of which were clinically defined as poliomyelitis cases according to WHO criteria. Poliovirus was isolated from six subjects who were defined as contact vaccine-associated cases. Characterization of isolates by both antigenic and molecular methods showed that, in all cases, the disease was associated with type 2 or 3 polioviruses of vaccine origin with retromutations known to be associated with loss of Sabin attenuated phenotype. Infection occurred despite the fact that all patients had records of previous immunization with oral polio vaccine (OPV), suggesting a failure of vaccination. Four of the five patients from which poliovirus could not be isolated were classified as possible recipient vaccine-associated poliomyelitis on the basis of serology data (presence of antibodies against all three polioviruses) and the temporal association between the latest dose of vaccine received and onset of paralysis. Virological investigation on healthy contacts of the poliomyelitic patients yielded the isolation of a further 12 Sabin-like polio revertant viruses, mostly type 2 and 3. A detailed study of the non-polio acute flaccid paralysis (AFP) cases and their healthy contacts revealed the presence of several enteroviruses, namely Echo, coxsackie and, in three cases type 2 or 3 Sabin-like polioviruses. Overall, these data suggest the absence of circulation of wild-type poliovirus in Albania from 1980 to 1995, before the recent outbreak of poliomyelitis in 1996, and emphasize the need for active surveillance of AFP and laboratory characterization of virus isolates to monitor vaccination efficacy.

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.

Synthetic flavans, isoflavans and isoflavenes substituted with chloro, cyano and amidino groups were tested for their in vitro activity against poliovirus type 2, Coxsackie virus B4, echovirus type 6 and enterovirus 71. Plaque-reduction assays showed that substituted 3-(2 H )-isoflavenes, carrying a double bond in the oxygenated ring, possess antiviral activity higher than that of the corresponding isoflavans. The most effective compounds were 4′-chloro-6-cyanoflavan and 6-chloro-4′-cyanoflavan. Studies on the mechanism of action of these two compounds suggested an effect on the early stages of viral replication.

The immunogenicity and the protective efficacy of a new polyvalent triple vector (DNA/SFV/MVA) based vaccine against mucosal challenge with pathogenic SIV m a c 2 5 1 were investigated. Cynomolgus monkeys (Macaca fascicularis) were primed intradermally with DNA, boosted twice subcutaneously with recombinant Semliki Forest virus (rSFV) and finally intramuscularly with recombinant Modified Vaccinia Virus Ankara strain (rMVA). Both DNA and recombinant viral vectors expressed SIV proteins (Gag, Pol, Tat, Rev, Nef and Env). The vaccinated monkeys developed T helper proliferative responses to viral antigens after the second immunization while interferon (IFN)-γ enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) specific responses appeared only after the last boost with rMVA. Upon intrarectal challenge with pathogenic SIV m a c 2 5 1 , three of four vaccinated monkeys were either fully protected or exhibited a dramatic reduction of virus replication up to undetectable level. A major contribution to this protective effect appeared to be the anamnestic T-cell IFN-γ ELISPOT responses to vaccine antigens (Gag, Rev, Tat, Nef) that mirrored the viral clearance. These results underline the efficacy of a multiprotein approach in combination with a triple vector system of antigen delivery.