Immunobiology of the Torque teno viruses and other anelloviruses.

Many features of the Torque teno virus and the other anelloviruses (AVs) that have been identified after this virus was discovered in 1997 remain elusive. The immunobiology of the AVs is no exception. However, evidence is progressively accumulating that at least some AVs have an interesting interplay with cells and soluble factors known to contribute to the homeostasis of innate and adaptive immunity. Evidence is also accumulating that this interplay can have a significant impact on how effectively an infected host can deal with superimposed infectious and non-infectious noxae. This review article discusses the scanty information available on these aspects and highlights the ones that would be more urgent to precisely understand in order to get an adequate assessment of how important for human health these extremely ubiquitous and pervasive viruses really are.

Effectiveness of nanofiltration in removing small non-enveloped viruses from three different plasma-derived products.

The objective of this study was to assess the ability of nanofiltration of albumin solution, prothrombin complex (PTC) and factor IX (FIX) to remove two small, non-enveloped DNA viruses, parvovirus B19 (B19V) and torque teno virus (TTV). Virus removal was investigated with down-scale experiments performed with sequential steps of 35-nm and 15-nm nanofiltrations of products spiked with virus DNA-positive sera. Viral loads were determined by real-time PCRs. The 15-nm nanofiltration removed more than 4.0 B19V log from all the products, TTV was reduced of more than 3.0 log from albumin solution and FIX by 35-nm and 15-nm nanofiltrations, respectively, being viral DNA undetectable after these treatments. Traces of TTV were still found in PTC after the 15-nm nanofiltration. In conclusion, nanofiltration can be efficacious in removing small naked viruses but, since viruses with similar features can differently respond to the treatment, a careful monitoring of large-scale nanofiltration should be performed.

Reversal of leukemia virus-induced immunosuppression in vitro by peritoneal macrophages.

When spleen cells from mice infected with Rowson-Parr virus (RPV) were cultivated with sheep red blood cells (SRBC), antibody plaque responses were markedly lower than those in similarly cultivated spleen cells from normal mice. Addition of as few as 10(3) spleen cells from RPV-infected mice to cultures of normal aplenocytes markedly depressed the expected immune response. Although RPV-infected mice showed maximum immunodpression in vivo only during the first week after infection, their spleen cells, obtained later in the course of infection, depressed the immunologic responsiveness of normal splenocytes in vitro. Increased doses of SRBC or addition of bacterial lipopolysaccharide to cultures of spleen cells from immunodepressed, RPV-infected mice stimulated antibody formation, and near-normal numbers of antibody-producing cells were evident. Peritoneal exudate (PE) cells, but not thymus, bone marrow, or unfractioned spleen cells, restored immunocompetence to cultures of spleen cells from RPV-infected mice but did not affect the suppressive properties of the infected cells on normal splenocytes. The function of PE cell macrophages in restoring immunocompetence to infected spleen cells in cultures seemed related to a possible antigen-focusing activity of the cells; antibody-producing cell precursors in infected cultures seemed to be preferentially affected by the presence of normal PE cells.

Suppression of natural killer cell activity by Friend murine leukemia virus.

BALB/c mice infected with Friend murine leukemia virus (F-MuLV) evinced a decreased natural killer (NK) cell activity to susceptible target cells. This suppression increased as the interval between infection and assay was lengthened. The decrease in NK activity due to F-MuLV infection was partially reversible when spleen cells were pretreated with interferon before the cytolytic assay. The ability of F-MuLV-infected splenocytes to bind to target cells was unaltered, indicating that the defect was in the lytic phase of NK cytolysis. When mixed with uninfected spleen cells, F-MuLV-infected splenocytes suppressed their NK cell activity. This suppression was associated with a nylon wool-adherent cell population in the F-MuLV-infected spleens.

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis.

Rapid changes in hepatitis C virus quasispecies produced by a single dose of IFN-alpha in chronically infected patients.

The effects of a single dose of 3 international megaunits of interferon-alpha2b (IFN-alpha2b) on hepatitis C virus (HCV) load and quasispecies were examined 24 h after administration in 12 previously untreated, chronically infected patients. All patients had viremia loads appreciably reduced relative to baseline values, thus confirming that the viral load is rapidly affected by IFN-alpha2b. Five patients also exhibited changes in plasma HCV quasispecies composition that were clearly evident by single-strand conformation polymorphism, analysis, thus indicating that one dose of IFN-alpha2b may suffice to produce rapid perturbations in the genetic heterogeneity of circulating HCV. Prior to IFN-alpha2b administration, 3 patients exhibited viral quasispecies differences between plasma and peripheral blood mononuclear cells (PBMC). Interestingly, in 2 such patients, the viral quasispecies found in the 24-h plasma resembled that in the pre-IFN PBMC. The latter finding raises the possibility that in these patients, the differences in quasispecies composition between pre-IFN and post-IFN plasma resulted from increased representation of lymphoid tissue-originated variants in the latter sample, possibly because of poor sensitivity to IFN-alpha2b of HCV replication in the lymphoid compartment.

Alterations of T-cell functions during Friend leukemia complex infection: defective signal transduction?

Proliferative and interleukin responses to T-cell mitogens such as concanavalin A (Con A) were rapidly and progressively reduced in BALB/c mice infected with the Friend leukemia complex (FLC) or its helper, Friend murine leukemia virus (F-MuLV). In contrast, a combination of the protein kinase C activator phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and the Ca++ ionophore A23187 elicited a normal lymphoproliferative response up to 8 days postinfection (p.i.) and normal interleukin-2 (IL-2) and interferon-gamma responses up to day 14 p.i. Exogenous IL-2 failed to restore the lymphoproliferative response of infected cells regardless of the stimulation used. These results showed that the T-cell deficits may be at least partly attributable to a derangement of the signal transduction pathway leading to activation. Spleen cells passed through nylon wool columns reacquired a normal responsiveness to Con A +/- TPA up to 14 days p.i. The latter finding suggests that the alterations in signal transduction are not caused by primary defect of the responder-T cells but may result from an extrinsic suppressive mechanism.

Role of interferon in lethality and lymphoid atrophy induced by Coxsackievirus B3 infection in mice.

To assess the importance of interferon (IFN) in the pathology of coxsackievirus B3 (CVB-3) infection, we evaluated both mortality rate and lymphoid involution in young adult BALB/C mice infected with lethal doses of the virus and treated either with anti-IFN antibody or with murine IFN-alpha/beta. Administration of antibody to IFN caused a profound worsening of the pathology and an increase in the mortality rate in infected animals. Treatment with murine IFN exerted a significant ameliorative effect on lethality when administered concomitantly with or soon after virus infection. The extent of this protection was correlated with the plasma levels of exogenous or endogenous IFN at 6 h postinfection, whereas no correlation with IFN titers was found later. The effects of IFN apparently were not directly mediated by antiviral effects, because at the times studied, no relation was found between IFN levels and virus titers, at least in the plasma of the infected animals. Lymphoid atrophy, assessed by measuring spleen weight, was only partially reversed by early IFN treatment. These data suggest that IFN production is critical during the early phases of infection, whereas it does not seem to play a significant protective role at later stages.

Effect of enzymatic deglycosylation on feline immunodeficiency virus sensitivity to antibody-mediated neutralization.

We have investigated the effect of deglycosylation with peptide-N-glycosidase F (PNGase F) on the sensitivity to inhibition by immune sera of two variants of the Petaluma strain of feline immunodeficiency virus (FIV-Pet), one sensitive to antibody-mediated neutralization because tissue culture adapted and the other, obtained by passaging the previous one in vivo, resistant to neutralization. The partial deglycosylation achieved did not appreciably affect FIV-Pet infectivity for T lymphoid cell cultures and did not increase the susceptibility to serum neutralization of the resistant variant but totally prevented neutralization of the sensitive variant. These finding suggest that the epitopes involved in neutralization of tissue culture-adapted FIV-Pet are effectively recognized by antibody only when the viral surface is properly glycosylated.

Detection of B epitopes on the p24 gag protein of feline immunodeficiency virus by monoclonal antibodies.

Seven monoclonal antibodies were obtained after immunization of mice with purified sodium dodecyl sulfate (SDS)-disrupted feline immunodeficiency virus (FIV). Six antibodies specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and reacted with FIV p24 gag gene product in immunoblots. One reacted positively with virus-infected cells, but failed to recognize FIV structural proteins by immunoblotting. Using competition binding studies, the anti-p24 monoclonals were shown to detect four distinct B-cell epitopes. Competition with sera of FIV-infected cats showed that such epitopes are immunogenic also in the natural host species.

Acid sensitivity of cell-free and cell-associated HIV-1: clinical implications.

Infectivity of free and cell-associated human immunodeficiency virus type 1 (HIV-1) treated in vitro at pH 7.4 to 4.9 for 2 hours was assessed on susceptible CEM-ss cells. Viral activity was monitored by cytopathology and production of reverse transcriptase and p24 antigen. The infectivity of cell-free virus was gradually inactivated and at pH 5.4 was completely lost, with or without subsequent adjustment of pH to neutral. Virus-producing cells also gradually lost their ability to infect as the pH decreased; however, restoration of neutral pH resulted in regained infectivity. Since the pH values used in the study are similar to those found at various entry sites of the human body, the data may be relevant to the mode of transmittal of HIV.

Feline immunodeficiency virus infection of macrophages: in vitro and in vivo inhibition by dideoxycytidine-5'-triphosphate-loaded erythrocytes.

Although HIV-1 and other mammalian lentiviruses infect macrophages, they are not cytopathic. Consequently, these infected long-lived cells serve as major virus reservoirs with a key role in the propagation of the virus throughout the body as well as in the pathogenesis of AIDS. Furthermore, well-differentiated macrophages possess low abilities to phosphorylate the most common reverse transcriptase inhibitors of the nucleoside analog family. In an attempt to overcome these problems we have evaluated in vitro and in vivo in a feline immunodeficiency animal model whether it is possible to protect macrophages from FIV infection by direct administration of dideoxycytidine-5'-triphosphate (ddCTP). Because the cell membranes are impermeable to phosphorylated drugs we have encapsulated ddCTP into autologous erythrocytes. The drug-loaded erythrocyte membranes were then modified to target these carrier cells to macrophages. ddCTP-loaded erythrocytes were able to reduce FIV production by macrophages infected in vitro or obtained from naturally or experimentally infected cats. Furthermore, the administration of ddCTP-loaded erythrocytes protected the majority of peritoneal macrophages during a 7-month experimental FIV infection and reduced the percentage of circulating lymphocytes stained by an anti-p24 antibody. These results suggest that the administration of nucleoside analogs in phosphorylate form is feasible and their targeting to macrophages reduces FIV infection both in vitro and in vivo.

Most potential linear B cell epitopes of Env glycoproteins of feline immunodeficiency virus are immunogenically silent in infected cats.

A battery of sixty-six 20- to 23-amino acid synthetic peptides, partially overlapping by 10-12 amino acids, spanning the entire sequence of the envelope (Env) glycoproteins of the Petaluma isolate of feline immunodeficiency virus (FIV-Pet), has been used to map Env linear B cell epitopes. By screening FIV-infected cat sera for anti-peptide reactivity, the existence of two immunodominat domains, namely the V3 region of the surface (SU) glycoprotein and the domain including the highly conserved sequence QNQFF of the transmembrane (TM) glycoprotein, was detected; antibody-binding sites were also mapped in the domain overlapping the cleavage site between SU and TM encompassing the V6 variable region. Moreover, at least two novel linear B epitopes, the former spanning residues 427M-H446 and the latter spanning residues 737N-N756 and likely representing a "type-specific" determinant, have been revealed. The battery of synthetic peptides was then used to immunize outbred Swiss mice in the attempt to reveal other potential sites of immunogenicity of the Env glycoproteins. Analysis of peptide-immunized mouse sera for anti-peptide reactivity revealed more numerous B cell epitopes, generally mapping in different peptides, as compared with those defined in the feline system. None of the mouse anti-peptide sera, however, proved neutralizing for FIV-Pet.

Defective natural killer cell cytotoxic activity in feline immunodeficiency virus-infected cats.

Flow cytometry has been employed to study NK cell cytotoxic activity in cats infected with feline immunodeficiency virus. The results show that animals infected for 12 months or more have decreased levels of NK cell cytotoxic activity in their blood. The impairment could not be overcome by in vitro treatment of effector cells with interleukin 2. Additional results suggest that the NK cells of infected cats are defective, in that they are still able to bind to target cells but have a reduced ability to kill them.

Reversal of immunosuppression induced by murine leukemia viruses.

BALB/c mice infected with Rowson-Parr virus, a lymphatic leukemia virus isolated from the Friend complex, undergo a rapid depression of antibody response. Spleen cells from these mice in culture show a similar deficit in the response to stimulation with sheep red cells and inhibit the reactivity of normal splenocytes. In an attempt to reverse this immunosuppression, near normal responses were obtained in vitro from infected splenocytes by increasing antigen dose, by adding E. coli lipopolysaccharide, or, more effectively, by cocultivating with small numbers of unfractionated or T cell-depleted peritoneal exudate cells (PC), whereas other manipulations proved ineffective. PC did not prevent the inhibition of normal splenocytes by infected spleen cells, but exhibited substantial restorative activity in vivo. In similar experiments, the immunosuppression exerted by the entire Friend complex could be reversed by PC in vitro but not in vivo. These results indicate that a functional deficit of macrophages may be partially responsible for the immunological impairment induced by leukemia viruses and suggest rational approaches to evaluate the relevance of this impairment to oncogenesis.

Quantitation of feline immunodeficiency proviruses in doubly infected cats using competitive PCR and a fluorescence-based RFLP.

A nested polymerase chain reaction assay, which amplifies a region of the gag gene, was developed for the direct detection of feline immunodeficiency virus (FIV) DNA sequences in the blood of infected cats. This method detects as few as ten copies of a plasmid containing the whole genome of the FIV-Pet isolate on agarose gel. To distinguish two FIV isolates in double infected cats, we devised an RFLP analysis on PCR amplified products exploiting sequence differences in the gag gene of the two strains. To quantitate the two strains, a fluorescent inner-sense primer was used in the second amplification step. Amplicons were subsequently digested, heat-denatured and loaded on a polyacrylamide gel in an automated DNA sequencer. The proportion of the two isolates was determined using the laser-excited fluorescence of labelled strain specific fragments. These data were used to extrapolate the numbers of proviral genomes from the total viral load as estimated by using a competitive PCR assay. These sensitive and specific assays complement virological detection of FIV and enable superinfection studies to be evaluated; a prerequisite for the testing of live attenuated immunodeficiency virus vaccines.

Comparative evaluation of five rapid methods for identifying subtype 1b and 2c hepatitis C virus isolates.

A panel of 61 HCV isolates belonging to five different subtypes were used to evaluate five methods for rapid typing of HCV RNA: an in-house type-specific polymerase chain reaction based on the core region (type-specific PCR), a commercial amplification of the core region followed by hybridisation to probe coated wells (DEIA), a commercial amplification of the 5'-UTR region followed by hybridisation to probes immobilised on strips (LiPA), an in-house restriction fragment polymorphism analysis of the 5'UTR (RFLP), and a commercial serological method using synthetic peptides from the NS4 region (serotyping). The correct viral type was identified in 90% of cases by DEIA, in 82% of cases by type-specific PCR, in 80% of cases by LiPA and RFLP, and in 67% of cases by serotyping. Correct identification of the virus subtype was much less frequent and was beyond the performance characteristics of some assays. Major problems were found in the identification of isolates belonging to type 2. This was probably at least partly due to the fact that all type 2 isolates in the viral panel were of subtype 2c, which has been considered rare until recently.

Detection of feline immunodeficiency proviral sequences in lymphoid tissues and the central nervous system by in situ gene amplification.

The availability of sensitive methods for detecting and localising the feline immunodeficiency virus (FIV) may help shed light on its role in generating tissue damage observed during infection. As immunohistochemical and in situ hybridisation techniques might not be sufficiently sensitive for this type of study, we adapted to FIV PCR-in situ hybridisation (PCR-ISH) that combine the extreme sensitivity of PCR with the precise localisation provided by ISH. The steps important for the success of PCR-ISH, such as sample preparation, permeabilisation, amplification profile, type of labels, and hybridisation conditions were optimised using paraformaldehyde-fixed and formalin-fixed paraffin-embedded sections of cells infected in vitro with FIV. As controls for amplification, the feline tumor necrosis factor-alpha gene (TNF-alpha) and the non-related EBNA-1 gene of the human Epstein-Barr virus were used. Once the method proved sufficiently sensitive and specific with these cells, the PCR-ISH assay was applied to paraffin sections of the lymph nodes, spleen and central nervous system of a 2-year FIV infected cat that, at the time of challenge, harboured low copy numbers of proviral genomes. Comparison of the results of PCR-ISH, competitive PCR and immunohistochemical analysis are described.

A colorimetric reverse transcriptase assay optimized for Moloney murine leukemia virus, and its use for characterization of reverse transcriptases of unknown identity.

A non-radioactive reverse transcriptase (RT) assay, reported as useful for lentivirus RTs, was optimized for the measurement of Moloney murine leukemia virus (MMuLV) RT. The optimized assay could detect 0.3 microU of MMuLV RT. The specificities of the MMuLV and lenti RT assays were demonstrated using the RTs of human immunodeficiency virus type 1, simian immunodeficiency virus, feline immunodeficiency virus (FIV), visna virus, human T-cell lymphotropic virus type 1, MMuLV and feline leukemia virus (FeLV). An RT activity blocking antibody (RTb-ab) assay was standardized for Mn2+ dependent MuLV-related RTs. The assay was used to demonstrate the distinct antigenic properties of RTs from mammalian MuLV-related retroviruses and lentiviruses. Cross-reactivity between MMuLV RTb-ab and FeLV RT but not between MMuLV RTb-ab and e.g. FIV RT was demonstrated. An RT activity found in the murine myeloma cell line SP2/0 was found to have similar assay preferences as MMuLV RT, and the MMuLV-RT hyperimmune sera reacted strongly against this RT, indicating the RT to be of MuLV-related etiology. The use of the RT and RTb-ab assays for detection and characterization of RTs of known or unknown identity is discussed.

Susceptibility of human and non-human cell lines to HCV infection as determined by the centrifugation-facilitated method.

The centrifugation-facilitated inoculation method was used to test 51 human and non-human cell lines for ability to support HCV replication. As determined by nested RT-PCR, one fifth of the cell lines tested were virus positive 15 days post inoculation suggesting that the centrifugation-facilitated inoculation is an efficient method for cell infection with HCV. However, virus production by infected cultures remained of low grade, thus showing that the unknown factors which limit HCV replication in vitro are not overcome by the procedure.