Modulation of indoleamine-2,3-dioxygenase expression and activity by HIV-1 in human macrophages.

Human immunodeficiency virus (HIV) infection is often complicated by the development of acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). Implications of kynurenine pathway (KP) are suggested in ADC and other inflammatories brain diseases. The first and regulatory enzyme of the KP is the indoleamine-2,3-dioxygenase (IDO). IDO activation is known to contribute to the modulation of the immune response during various infectious diseases particularly in AIDS. HIV and viral proteins can activate IDO in immune cells leading to an increase catabolism of tryptophan through the KP; the consequence being the production of immuno-modulative and neuroactive metabolites. This mechanism is likely to favour HIV persistence. The present study analysed concomitantly several parameters involved in IDO regulation and activity associated with HIV-1-infection. We investigated relevant intracellular and extracellular mechanisms involved in the regulation of IDO expression and activity during the HIV infection and replication in human monocyte-derived macrophages (MdM). Using a complementary set of in vitro experiments, we found that HIV-1/Ba-L infection induces IDO expression and increases its activity in MdM. We also showed that IDO activation by HIV-1 is likely to be a direct effect of the infection and seems to be independent of IFN-gamma production.

Increase of monoamine oxidase-B activity in the brain of scrapie-infected hamsters.

In the present study, the purpose is to determine activities of monoamine oxidases (MAO) in the brain of 263K scrapie-infected hamsters during the development of this experimental prion disease. Indeed, MAO activity modifications which have already been related in aging and neurodegenerations is suspected to be involved in the neuron loss process by elevated hydrogen peroxide formation. Monoamine oxidase type A (MAO-A) and B (MAO-B) activities were followed in the brain at different stages of the disease. MAO-A activity did not change significantly during the evolution of the disease. However, concerning the MAO-B activity, a significant increase was observed from 50 days post-infection and through the course of the disease and reached 42.9+/-5.3% at its ultimate stage. Regarding these results, MAO-B could be a potential therapeutic target then we have performed a pre-clinical treatment with irreversible (Selegiline or L-deprenyl) or and reversible (MS-9510) MAO-B inhibitors used alone or in association with an anti-scrapie drug such as MS-8209, an amphotericin B derivative. Our results show that none of the MAO-B inhibitors used was able to delay the onset of the disease. Neither these MAO-B inhibitors nor R-NMDA inhibitors (MK-801) can enhance the effects of MS-8209. The present findings clearly indicate a significant increase of cerebral MAO-B activity in scrapie-infected hamsters. Furthermore, inhibitors of MAO-B do not have any curative or palliative effect on this experimental model indicating that the raise of this activity is probably more a consequence rather than a causal event of the neurodegenerative process.

Comparative effects of two type I interferons, human IFN-alpha and ovine IFN-tau on indoleamine-2,3-dioxygenase in primary cultures of human macrophages.

Type I interferons (IFNs) are widely used to treat viral diseases. Depressive symptoms and suicide attempts are common neuropsychiatric side-effects during treatment with type I IFNs. Activation of indoleamine-2,3-dioxygenase (IDO), the first and rate-limiting enzyme of the kynurenine pathway by IFNs, leads to an increase in tryptophan (Trp) catabolism. Low levels of Trp lead to decrease of serotonin synthesis, which is likely to be related to the depressive symptoms. Ovine type I interferon-tau (IFN-tau) has a more potent antiretroviral effect and is less toxic than human type I IFN-alpha. Effects of IFN-tau and IFN-alpha on IDO expression and activity in primary cultures of human macrophages were compared in parallel to those of IFN-gamma, considered as one of the most potent IDO inducer. We found that both IFN-alpha and IFN-tau were poor inducers of IDO compared with IFN-gamma. However, IDO activation was slightly and significantly lower with ovine IFN-tau than human IFN-alpha, suggesting that ovine IFN-tau might have a lower impact on serotoninergic pathway compared with human IFN-alpha.

Using budding yeast to screen for anti-prion drugs.

Prions are misfolded proteins capable of propagating their altered conformation which are commonly considered as the causative agent of transmissible spongiform encephalopathies, a class of fatal neurodegenerative diseases. Currently, no treatment for prion-based diseases is available. Recently we have developed a rapid, yeast-based, two-step assay to screen for anti-prion drugs [1]. This new method allowed us to identify several compounds that are effective in vivo against budding yeast [PSI+] and [URE3] prions but also able to promote mammalian prion clearance in three different cell culture-based assays. Taken together, these results validate our method as an economic and efficient high-throughput screening approach to identify novel prion inhibitors or to carry on comprehensive structure-activity studies for already isolated anti-mammalian prion drugs. These results suggest furthermore that biochemical pathways controlling prion formation and/or maintenance are conserved from yeast to human and thus amenable to pharmacological and genetic analysis. Finally, it would be very interesting to test active drugs isolated using the yeast-based assay in models for other diseases (neurodegenerative or not) involving amyloid fibers like Huntington's, Parkinson's or Alzheimer's diseases.

Characterization of human monocyte-derived microglia-like cells.

Microglial cells are central to brain immunity and intervene in many human neurological diseases. The aim of this study was to develop a convenient cellular model for human microglial cells, suitable for HIV studies. Microglia derive from the hematogenous myelomonocytic lineage, possibly as a distinct subpopulation but in any case able to invade the CNS, proliferate, and differentiate into ameboid and then ramified microglia in the adult life. We thus attempted to derive microglia-like cells from human monocytes. When cultured with astrocyte-conditioned medium (ACM), monocytes acquired a ramified morphology, typical of microglia. They overexpressed substance P and the calcium binding protein Iba-1 and dimly expressed class II MHC, three characteristics of microglial cells. Moreover, they also expressed a potassium inward rectifier current, another microglia-specific feature. These monocyte-derived microglia-like cells (MDMi) were CD4(+)/CD14(+), evocative of an activated microglia phenotype. When treated with lipopolysaccharide (LPS), MDMi lost their overexpression of substance P, which returned to untreated monocyte-derived macrophage (MDM) level. Compared with MDM, MDMi expressed higher CD4 but lower CCR5 levels; they could be infected by HIV-1(BaL), but produced less virus progeny than MDM did. This model of human microglia may be an interesting alternative to primary microglia for large scale in vitro HIV studies and may help to better understand HIV-associated microgliosis and chronic inflammation in the brain.

Preclinical metabolic changes in mouse prion diseases detected by 1H-nuclear magnetic resonance spectroscopy.

Magnetic resonance spectroscopy studies in animal models of prion disease are very few and concern terminal stages of infection. In order to study earlier stages of the disease, we used in-vivo magnetic resonance spectroscopy in a mouse model of scrapie and, for the first time, in mice infected with a bovine spongiform encephalopathy strain. In bovine spongiform encephalopathy-infected mice, we observed an increase in myo-inositol preceding clinical signs by 20 days, followed by a decrease in N-acetylaspartate at advanced stages. In scrapie-infected mice, changes in N-acetylaspartate and myo-inositol were detected at the beginning of the symptomatic phase. These results show that magnetic resonance spectroscopy is a valuable tool for detecting subtle metabolic changes associated to gliosis and neuronal dysfunction in prion diseases.

Bovine prion is endocytosed by human enterocytes via the 37 kDa/67 kDa laminin receptor.

Some forms of transmissible spongiform encephalopathies result from oral infection. We have thus analyzed the early mechanisms that could account for an uptake of infectious prion particles by enterocytes, the major cell population of the intestinal epithelium. Human Caco-2/TC7 enterocytes cultured on microporous filters were incubated with different prion strains and contaminated brain homogenates in the apical compartment. Internalization of infectious particles was analyzed by Western blotting and immunofluorescence. We observed internalization by enterocytes of prion particles from bovine spongiform encephalopathy brain homogenates but not from mouse-adapted scrapie-strain brain homogenates or purified bovine spongiform encephalopathy scrapie-associated fibrils. Bovine prion particles were internalized via endocytosis within minutes of infection and were associated with subapical vesicular structures related to early endosomes. The endocytosis of the infectious bovine PrP(Sc) was reduced by preincubating the cells with an anti-LRP/LR blocking antibody, identifying the 37 kDa/67 kDa laminin receptor (LRP/LR), which is apically expressed in Caco-2/TC7 cells, as the receptor for the infectious prion protein. Altogether, our results underscore a potential role of enterocytes in the absorption of bovine prions during oral infection through specific LRP/LR-dependent endocytosis.

Lack of IFN-gamma production in response to antigenic stimulation in human IFN-tau-treated lymphocytes.

Interferon-tau (IFN-tau) is a type I IFN responsible for maternal recognition of the fetus in ruminants. In addition to its physiologic role, IFN-tau also inhibits HIV replication in human lymphocytes and macrophages and displays immunomodulatory effects but lacks the toxicity associated with other type I IFNs. Human IFN-alpha promotes a Th1 response, whereas IFN-tau has anti-inflammatory properties, inducing the production of Th2 cytokines in murine models of experimental autoimmune encephalitis (EAE) or fetal loss. We compared the effects of ovine IFN-tau (OvIFN-tau) and human IFN-alpha (HuIFN-alpha) on cytokine mRNA and protein production in human peripheral blood mononuclear cells (PBMCs) activated with a recall antigen, such as purified protein derivative (PPD) of tuberculin or with a proinflammatory stimulus, such as lipopolysaccharide (LPS). In both cases, IFN-alpha increased IFN-gamma production, whereas IFN-tau did not and thereby promoted Th2 cytokine production. This original property renders IFN-tau a potential candidate for therapeutic applications in immune disorders, such as multiple sclerosis (MS), but its therapeutic use in the treatment of HIV infection should be considered with caution.

High incidence of scrapie induced by repeated injections of subinfectious prion doses.

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 x 10(-5) to 2 x 10(-8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 x 10(-4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 x 10(-4), 2 x 10(-5), or 2 x 10(-6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 x 10(-5) or 2 x 10(-6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 x 10(-7) or 2 x 10(-8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease.

Involvement of IL-6 in the anti-human immunodeficiency virus activity of IFN-tau in human macrophages.

IFN-tau is a non-cytotoxic type I IFN responsible for maternal recognition of the foetus in ruminants. IFN-tau has been found to inhibit HIV replication more strongly than human IFN-alpha, particularly in human monocyte-derived macrophages, without associated toxicity. Ovine IFN-tau uses the same anti-viral cellular pathways as human IFN-alpha in human macrophages, principally inhibiting the early steps of the biological cycle of HIV, preventing the integration of HIV DNA into the host-cell genome. In this study, we investigated the immunomodulatory properties of IFN-tau in human macrophages. We found that IFN-tau increased the production of IL-10 and IL-6, but not of IL-1beta or tumour necrosis factor alpha, in unstimulated, LPS-stimulated and HIV-1/Ba-L-infected macrophages. We also found that treatment with IL-6 inhibited HIV replication. Moreover, the neutralization of IL-6 activity in the cell culture supernatants of IFN-tau-treated macrophages led to a decrease in the anti-retroviral effects of IFN-tau, suggesting that IL-6 was involved in the anti-viral activity induced by IFN-tau. By focusing on the very early steps of the biological cycle of HIV, we showed that IL-6 co-operated with IFN-tau to decrease intracellular HIV RNA levels 2 h after infection.

Expression of human IL-1alpha after intramarrow gene transfer into healthy non-human primate by adenoviral vector.

Interleukin-1alpha (IL-1alpha) is a multifunctional cytokine that stimulates myelopoiesis in macaque. However, daily systemic injections of IL-1alpha are associated with severe side effects. We therefore investigated the feasibility of a gene therapy strategy aimed at increasing the IL-1alpha local production in bone marrow with limited release of the vector into the blood circulation. Intra-medullar administration of adenoviral vector containing human IL-1alpha (huIL-1alpha) gene resulted in enhanced neutrophil, monocyte and platelet counts during the two first weeks after injection. The DNA vector, the transgene expression and the huIL-1alpha production was detected in treated bone marrow without significant detection of huIL-1alpha in the peripheral blood. Associated with huIL-1alpha production, we observed concomitant plasma C reactive protein and IL-1Ra peaks in the acellular fraction of treated bone marrow at days 3 and 7. No abnormal clinical side effects were observed in any of the animals following the adenoviral vector injection.

Impact of bone marrow hematopoiesis failure on T-cell generation during pathogenic simian immunodeficiency virus infection in macaques.

Experimental infection of macaques with pathogenic strains of simian immunodeficiency virus (SIV) represents one of the most relevant animal models for studying HIV pathogenesis. In this study, we demonstrated a significant decrease in the generation of CD4+ T cells from bone marrow (BM) CD34+ progenitors in macaques infected with SIVmac251. This decrease appears to result from changes in the clonogenic potential of BM progenitors of both the myeloid and lymphoid lineages. We also demonstrated a significant decrease in the numbers of the most immature long-term culture-initiating cells (LTC-ICs). Hematopoietic failure occurred as early as primary infection, in the absence of CD34+ BM cell infection and was not related to plasma viral load. No major change was observed in the phenotype of BM CD34+ cells from infected macaques, including apoptosis markers such as annexin V staining and BcL-2 expression, but a significantly higher that normal proportion of cells were in the G0/G1 phase. This is the first demonstration that failure of BM hematopoiesis results in impaired T-cell production, which may contribute to the disruption of T-lymphocyte homeostasis characteristic of pathogenic lentiviral infections in primates.

Expression of excitatory amino acid transporter-1 (EAAT-1) in brain macrophages and microglia of patients with prion diseases.

The mechanisms of neuronal apoptosis in prion diseases are unclear. Experimental studies suggest that it may result from 2 associated mechanisms: glutamate-mediated excitotoxicity and oxidative stress. Recent studies showed that activated macrophages/microglia (AMM) express excitatory amino acid transporters (EAATs) in HIV infection, suggesting that they may play a neuroprotective role by clearing extra-cellular glutamate and producing anti-oxidant glutathione. In order to test this hypothesis in prion diseases, samples from cerebral cortex, striatum, thalamus, and cerebellum from 14 patients with Creutzfeldt-Jakob disease (8 sporadic, 2 familial, 2 iatrogenic, and 2 variant), and 4 with fatal familial insomnia (3 homozygous Met/Met at codon 129 of the PRNP gene, 1 heterozygous Met/Val), and 3 controls were immunostained for EAAT-1, GFAP, HLA-DR, CD68, IL-1, caspase 3, and PrP. In prion diseases, EAAT-1 immunopositivity was found in affected areas. Only AMM, interstitial, perivascular, perineuronal (sometimes around apoptotic neurons), or close to reactive astrocytes, expressed EAAT-1. Astrocyte EAAT-1 expression was scarcely detectable in controls and was not detected in prion disease cases. The proportion of AMM expressing EAAT-1 did not correlate with the severity of neuronal apoptosis, spongiosis, astrocytosis, microgliosis, or PrP deposition, but only with disease duration. Occasional EAAT-1 expressing AMM were found in patients with short survival, whereas diffuse EAAT-1 expression by AMM was observed in cases with long survival (24 to 33 months) that most often were heterozygous for Met/Val at codon 129 of the PRNP gene. Our findings suggest that AMM may develop a partial neuroprotective function in long-lasting prion diseases, although it does not seem to efficiently prevent neurological and neuropathological deterioration. Whether this neuroprotective function of microglia is the cause or the effect of longer survival needs to be clarified.

Structure-activity relationships in platelet-activating factor. 12. Synthesis and biological evaluation of platelet-activating factor antagonists with anti-HIV-1 activity.

The HIV-1 central nervous system infection leads to the onset of neurological impairments called AIDS dementia complex (ADC). PAF plays an important role in this pathology, as it is an HIV-1-induced neurotoxin produced by infected or activated macrophages and microglia, in the brain. We previously reported that PAF-antagonists bearing a trisubstituted piperazine presented in vitro anti-HIV-1 activity in human macrophages. To improve the pharmacological activities of our lead compound, 1a, we modified its carbamate function and evaluated both its antiretroviral and anti-PAF activities. One carbamate derivative (10c) demonstrated a similar antiviral activity but a higher anti-PAF potency, whereas 4a, with an ureide function, presents an increased antiviral activity and can be considered as a pure antiretroviral drug, as it does not present PAF-antagonism. Moreover, we measured the ability of 1a to cross the blood-brain barrier, using the in situ mouse brain perfusion method and its plasmatic concentrations after iv and po administration. The transport parameter measured (K(in)) proves that 1a is able to cross this biological barrier, but a pharmacokinetic study reveals its weak bioavailability in rats.

ATP binding cassette multidrug transporters limit the anti-HIV activity of zidovudine and indinavir in infected human macrophages.

OBJECTIVES: To investigate whether P-glycoprotein (P-gp) and multidrug resistance proteins (MRPs), which limit the bioavailability of HIV protease inhibitors (PIs) and nucleoside reverse transcriptase inhibitors (NRTIs), modulate the anti-HIV activity of NRTIs, non-NRTIs and PIs in vitro. DESIGN: We used primary cultures of major HIV target cells: human monocyte-derived macrophages (MDMs) and lymphocytes. METHODS: P-gp and MRP expression in response to long-term zidovudine (3'-azido-3'-deoxythymidine; AZT) or indinavir treatment was quantified by RT-PCR. MDM and lymphocytes were infected in vitro with HIV-1/Ba-L and HIV-1-LAI, respectively, and treated with antiretroviral drugs. We evaluated the activity of these drugs in combination with PSC833, a P-gp inhibitor, and/or probenecid, an MRP1 inhibitor. Intracellular AZT triphosphate derivative (AZT-TP) was quantified by HPLC-MSMS. P-gp ATPase activity was measured with inside-out native membrane vesicles enriched in P-gp. RESULTS: Levels of MDR1, mrp4 and mrp5 mRNA were high following AZT treatment. In infected MDM, PSC833 and probenecid increased the anti-HIV activity of AZT and indinavir. AZT (5 nM) decreased HIV replication by 34% alone and by 72% in combination with P-gp/MRP inhibitors. Indinavir (10 nM) gave 14% inhibition alone and 81% in combination. The increase in anti-HIV activity of AZT was correlated with an increase in intracellular AZT-TP concentration. However, unlike PIs, neither AZT nor its metabolites interacted with P-gp. CONCLUSION: AZT increases the expression of multidrug transporters, thereby decreasing its pharmacological activity. The cellular efflux of AZT probably involves MRP4 or MRP5. In contrast, increases in indinavir anti-HIV activity require the inhibition of both P-gp and MRP1.

Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251.

BACKGROUND: The aim of this study was to evaluate gene therapy for AIDS based on the transduction of circulating lymphocytes with a retroviral vector giving low levels of constitutive macaque interferon beta production in macaques chronically infected with a pathogenic isolate of SIVmac251. RESULTS: Two groups of three animals infected for more than one year with a pathogenic primary isolate of SIVmac251 were included in this study. The macaques received three infusions of their own lymphocytes transduced ex vivo with the construct encoding macaque IFN-beta (MaIFN-beta or with a vector carrying a version of the MaIFN-beta gene with a deletion preventing translation of the mRNA. Cellular or plasma viremia increased transiently following injection in most cases, regardless of the retroviral construct used. Transduced cells were detected only transiently after each infusion, among the peripheral blood mononuclear cells of all the animals, with copy numbers of 10 to 1000 per 106 peripheral mononuclear cells. CONCLUSION: Long-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-beta did not prevent animals from the progressive decrease in CD4+ cell count typical of infection with simian immunodeficiency virus. These results reveal potential pitfalls for future developments of gene therapy strategies of HIV infection.

Hydrolysis of the amyloid prion protein and nonpathogenic meat and bone meal by anaerobic thermophilic prokaryotes and streptomyces subspecies.

Transmissible spongiform encephalopathies are caused by accumulation of highly resistant misfolded amyloid prion protein PrPres and can be initiated by penetration of such pathogen molecules from infected tissue to intact organism. Decontamination of animal meal containing amyloid prion protein is proposed thanks to the use of proteolytic enzymes secreted by thermophilic bacteria Thermoanaerobacter, Thermosipho, and Thermococcus subsp. and mesophilic soil bacteria Streptomyces subsp. Keratins alpha and beta, which resemble amyloid structures, were used as the substrates for the screening for microorganisms able to grow on keratins and producing efficient proteases specific for hydrolysis of beta-sheeted proteic structures, hence amyloids. Secretion of keratin-degrading proteases was evidenced by a zymogram method. Enzymes from thermophilic strains VC13, VC15, and S290 and Streptomyces subsp. S6 were strongly active against amyloid recombinant ovine prion protein and animal meal proteins. The studied proteases displayed broad primary specificities hydrolyzing low molecular mass peptide model substrates. Strong amyloidolytic activity of detected proteases was confirmed by experiments of hydrolysis of PrPres in SAFs produced from brain homogenates of mice infected with the 6PB1 BSE strain. The proteases from Thermoanaerobacter subsp. S290 and Streptomyces subsp. S6 are the best candidates for neutralization/elimination of amyloids in meat and bone meal and other protein-containing substances and materials.

Aluminum hydroxide adjuvant induces macrophage differentiation towards a specialized antigen-presenting cell type.

Aluminum hydroxide (AlOOH) has been used for many years as a vaccine adjuvant, but little is known about its mechanism of action. We investigated in this study the in vitro effect of aluminum hydroxide adjuvant on isolated macrophages. We showed that AlOOH-stimulated macrophages contain large and persistent intracellular crystalline inclusions, a characteristic property of muscle infiltrated macrophages described in animal models of vaccine injection, as well as in the recently described macrophagic myofasciitis (MMF) histological reaction in humans. AlOOH-loaded macrophages exhibited phenotypical and functional modifications, as they expressed the classical markers of myeloid dendritic cells (HLA-DR(high)/CD86(high)/CD83(+)/CD1a(-)/CD14(-)) and displayed potent ability to induce MHC-II-restricted antigen specific memory responses, but kept a macrophage morphology. This suggests a key role of macrophages, in the reaction to AlOOH-adjuvanted vaccines and these mature antigen-presenting macrophages may therefore be of particular importance in the establishment of memory responses and in vaccination mechanisms leading to long-lasting protection.

[Neurotoxicity and neuroprotection, two aspects of microglial activation in human immunodeficiency virus (HIV) infection]. / Neurotoxicité et neuroprotection, les deux facettes de l'activation microgliale au cours de l'infection par le virus de l'immunodéficience humaine (VIH).

Microglial cells and macrophages are the only cells within the central nervous system, in which productive HIV infection has been unquestionably demonstrated. Those cells play a key role in the origin of the neuronal dysfunction underlying HIV-related cognitive disorders. The neurotoxicity of the cells is both direct, related to HIV proteins, and indirect, through the release by activated macrophages and microglial cells (AMM) of multiple neurotoxic factors. The mechanisms of neuronal damage, the final irreversible stage of which is neuronal apoptosis, are only partly understood but appear to involve oxidative stress and glutamate-receptor mediated toxicity. On the other hand, recent experimental in vitro and in vivo studies, and neuropathological studies in HIV infected patients at different stages of the disease, tend to show that AMM express excitatory amino acid transporters (EAAT) suggesting that in addition to their neurotoxic properties, they also have a neuroprotective role by clearing extra-cellular glutamate and producing antioxidant glutathione. This neuroprotective role could counteract, at least in the early stages of the disease, the neurotoxicity of AMM explaining the discrepancy between the conspicuous microglial activation at that stage and the absence of cognitive disorder, neuronal loss and neuronal apoptosis. It could also explain the regression of the cognitive disorders in some patients who received highly active antiretroviral treatment.