Levels Of Viral Antigen Research Articles

Pathogenesis of transplacental virus infection: pestivirus replication in the placenta and fetus following respiratory infection

Although transplacental virus infections account for considerable morbidity and mortality in both animals and humans, very little is so far known about the pathways whereby virus reaches the conceptus, the subsequent virus–host interactions in the early phases of the infections, and the establishment of persistent non-lethal infection. Using a natural animal model we recently demonstrated that bovine pestivirus can spread from the site of infection to the ovine fetus within 72 h, despite the expression of interferon in the reproductive tract [1]. In the present study we demonstrate that pestivirus first establishes infection and spread within the allantoic and amniotic membranes and then the fetus, followed several days later by infection of the uterine glands. However, virus replication and spread within the fetus is, at least in part, controlled by fetal developmental factors. In fetuses less than 25 days of gestational age, the virus remains restricted to the bulbis cordis, the first brachial pouch and occasionally the aorta. Over the next few days the virus spreads to multiple tissues, in addition to becoming more widespread and pronounced within the initially infected tissues. A potential role for the binucleated cells of the allantochorion in the spread of the virus from the fetal to the maternal tissues was also found. These cells expressed high levels of viral antigen just prior to and during the time period in which virus antigen became detectable in the epithelial cells of the uterine glands, in endothelial cells of uterine vessels and in scattered macrophage-like cells in the uterine stroma. Most likely this relatively late virus transfer is inconsequential for the mother, since it occurs at a time when a maternal virus-specific antibody response is becoming measurable. This is in contrast to the fetus, where the infection will have established itself widely prior to the development of lymphoid tissues and a functional immune response, thus setting the scenario for development of specific tolerance to the persisting virus.

Down-modulation of TCR/CD3 surface complexes after HIV-1 infection is associated with differential expression of the viral regulatory genes.

We have investigated the mechanism(s) involved in progressive abrogation of CD3-gamma gene expression after HIV-1 or HIV-2 infection. A comparison of intracellular virus expression with T cell receptor surface density, revealed both high and low levels of viral p24 antigen in the TCR/CD3(hi), TCR/CD3(lo), and TCR/CD3(-) cells. Furthermore, in non-productively infected cells expressing the multiply spliced, virally encoded tat, rev, and nef regulatory gene transcripts, the same progressive loss of surface TCR/CD3 complexes was observed. We treated HIV-1-infected cells with antisense (AS) phosphorothioate oligodeoxynucleotides (P-OdN) targeted to the viral regulatory genes. All of the HIV-1 sequence-specific AS-P-OdN's inhibited intracellular p24 antigen expression in a time- and dose-dependent manner; although, blocking p24 expression alone was not sufficient to modulate TCR/CD3 surface density. Only Tat-AS and Nef-AS were able to delay TCR/CD3 down-modulation on receptor-positive cells or drive receptor up-regulation on receptor-negative cells. In contrast, Rev-AS accelerated TCR/CD3 loss on receptor-positive cells. RT-PCR revealed that Tat-AS and Nef-AS reduce the level of tat, nef, and rev transcripts, while Rev-AS increases the level of tat and nef transcripts in infected cells. Thus, when intracellular conditions favor expression of tat and/or nef in the absence of rev, CD3-gamma gene transcripts and TCR/CD3 surface density are down-modulated.

Mechanisms for adaptation of simian immunodeficiency virus to replication in alveolar macrophages.

In contrast to the simian immunodeficiency virus SIVmac239, which replicates poorly in rhesus monkey alveolar macrophages, a variant with nine amino acid changes in envelope (SIVmac239/316E) replicates efficiently and to high titer in these same cells. We examined levels of viral DNA, RNA, antigen, and infectious virus to identify the nature of the block to SIVmac239 replication in these cells. Low levels of viral antigen (0.1 to 1.0 ng of p27 per ml) and infectious virus (100 to 1,000 infectious units per ml) were produced in the supernatant 1 to 4 days after SIVmac239 infection, but these levels did not increase subsequently. SIVmac239 DNA was synthesized in these macrophage cultures during the initial 24 h after infection, but the levels did not increase subsequently. Quantitation of the numbers of infectious cells in cultures over time and the results of experiments in which cells were reexposed to SIVmac239 after the initial exposure indicated that only a small proportion of cells were susceptible to SIVmac239 infection in these alveolar macrophage cultures and that the vast majority (>95%) of cells were refractory to SIVmac239 infection. In contrast to the results with SIVmac239, the levels of viral antigen, infectious virus, and viral DNA increased exponentially 2 to 7 days after infection by SIVmac239/316E, reaching levels greater than 100 ng of p27 per ml and 100,000 infectious units per ml. Since SIVmac239/316E has previously been described as a virus capable of infecting cells in a relatively CD4-independent fashion, we examined the levels of CD4 expression on the surface of fresh and cultured alveolar macrophages from rhesus monkeys. The levels of CD4 expression were extremely low, below the limit of detection by flow cytometry, on greater than 99% of the macrophages. CCR5(+) cells were profoundly depleted only from alveolar macrophage cultures infected with SIVmac239/316E. High concentrations of an antibody to CD4 delayed but did not block replication of SIVmac239/316E. The results suggest that the adaptation of SIVmac316 to efficient replication in alveolar macrophages results from its ability to infect these cells in a CD4-independent fashion or in a CD4-dependent fashion even at extremely low levels of surface CD4 expression. Since resident macrophages in brains and lungs of humans also express little or no CD4, our findings predict the presence of human immunodeficiency virus type 1 that is relatively CD4 independent in the lung and brain compartments of infected people.

Drug resistance and drug combination features of the human immunodeficiency virus inhibitor, BCH-10652 [(+/-)-2'-deoxy-3'-oxa-4'-thiocytidine, dOTC

The heterosubstituted nucleoside analogue dOTC [( )-2'-deoxy-3'-oxa-4'-thiocytidine, BCH-10652] is a racemic compound structurally related to 3TC (lamivudine), but has the oxygen and sulphur in the furanosyl ring transposed. Both the enantiomers (-)dOTC (BCH-10618) and (+)dOTC (BCH-10619) had equivalent activity against wild-type strains of HIV-1 in C8166 T-cells (EC50 1.0-10.0 microM) and in PBMCs (EC50 0.1-3.0 microM). Investigation of the activity of dOTC and its enantiomers against laboratory strains of HIV-1 with defined resistance to 3TC, AZT (zidovudine), ddl (didanosine), PMEA (adefovir), nevirapine and saquinavir indicated that sensitivity was maintained (<3-fold change in EC50) in all cases, with the exception of HIV-1RF 3TC-resistant viruses. The degree of resistance recorded for dOTC (four- to sevenfold), (-)dOTC (five- to eightfold) and (+)dOTC (five- to >18-fold) against these M1841 or M184V mutants, was significantly less than that recorded for 3TC (>100-fold). In addition, the inhibitory effect of the compounds against clinical isolates of HIV-1 recovered from patients with suspected resistance to 3TC and AZT was investigated. Clinical isolates were genotyped using the Murex Line Probe Assay (LiPA) and subgrouped into wild-type, 3TC-resistant and dual 3TC/AZT-resistant, as well as undefined or mixed genotype populations. Compared with the mean EC50 values obtained with genotypically and phenotypically wild-type clinical isolates, the mean EC50 values calculated for isolates phenotypically resistant to 3TC or 3TC and AZT were only 2.6-, 1.6- and 8.2-fold higher for dOTC, (-)dOTC and (+)dOTC, respectively. When the rate of emergence of virus resistant to dOTC and its enantiomers in vitro was investigated, virus resistant to (+)dOTC was readily selected for (<10 passages), and a methionine (ATG) to isoleucine (ATA) amino acid change at codon 184 was identified. In contrast, virus resistant to dOTC and (-)dOTC took longer to appear (15-20 passages), with a methionine (ATG) to valine (GTG) amino acid change at position 184 identified in both cases. In addition, virus passaged 20 times in the presence of dOTC also had a partial lysine (AAA) to arginine (AGA) exchange at position 65. These viruses showed only low-level resistance to dOTC and its enantiomers, but were highly resistant to 3TC. The antiviral effects of dOTC in combination with the nucleoside RT inhibitors AZT, 3TC, d4T (stavudine) and ddl, the non-nucleoside RT inhibitor nevirapine and the protease inhibitors saquinavir, ritonavir and indinavir was investigated. Two-way drug combination assays were carried out in peripheral blood mononuclear cell (PBMC) cultures by measuring the reduction in p24 viral antigen levels, and data was analysed using the MacSynergy II program. dOTC in combination with 3TC or d4T showed a moderate synergistic effect while all other combinations had an additive interaction.

High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis.

AIDS dementia and encephalitis are complications of AIDS occurring most frequently in patients who are immunosuppressed. The simian immunodeficiency virus (SIV) model used in this study was designed to reproducibly induce AIDS in macaques in order to examine the effects of a neurovirulent virus in this context. Pigtailed macaques (Macaca nemestrina) were coinoculated with an immunosuppressive virus (SIV/DeltaB670) and a neurovirulent molecularly cloned virus (SIV/17E-Fr), and more than 90% of the animals developed moderate to severe encephalitis within 6 months of inoculation. Viral load in plasma and cerebrospinal fluid (CSF) was examined longitudinally to onset of AIDS, and viral load was measured in brain tissue at necropsy to examine the relationship of systemic and central nervous system (CNS) viral replication to the development of encephalitis. In all animals, plasma viral load peaked at 10 to 14 days postinfection and remained high throughout infection with no correlation found between plasma viremia and SIV encephalitis. In contrast, persistent high levels of CSF viral RNA after the acute phase of infection correlated with the development of encephalitis. Although high levels of viral RNA were found in the CSF of all macaques (six of six) during the acute phase, this high level was maintained only in macaques developing SIV encephalitis (five of six). Furthermore, the level of both viral RNA and antigen in the brain correlated with the severity of the CNS lesions. The single animal in this group that did not have CNS lesions had no detectable viral RNA in any of the regions of the brain. The results substantiate the use of CSF viral load measurements in the postacute phase of SIV infection as a marker for encephalitis and CNS viral replication.

Increased Neurovirulence of Polytropic Mouse Retroviruses Delivered by Inoculation of Brain with Infected Neural Stem Cells

Following intraperitoneal (IP) inoculation of neonatal mice, the polytropic recombinant murine leukemia virus (MuLV), Fr98, induces a severe brain disease characterized by ataxia, seizures and death. In contrast, no apparent clinical neurological disease is seen after IP infection with Fr54, a polytropic MuLV differing from Fr98 in its envelope gene sequences. In the brain both Fr98 and Fr54 infect primarily capillary endothelial cells and microglia. However, the level of microglial infection by Fr98 is twofold higher than by Fr54, which might account for the difference in neurovirulence. In the present study, in order to test directly whether an increase in the number of microglia infected by Fr54 would be sufficient to induce clinical disease, we attempted to increase the level of Fr54 in the brain by changing the route of infection. After intraventricular inoculation with Fr54-infected neural stem cells (clone C17.2), a well-established vehicle for delivery of viruses and genes to the brain, mice became ataxic and died 4 weeks postinfection. In these mice induction of brain disease was correlated with a higher level of viral antigen in the cerebrum and an increase in the number of infected microglial cells in all brain regions examined compared with mice inoculated IP. In contrast, mice inoculated with neural stem cells infected with an ecotropic nonneurovirulent murine leukemia virus, FB29, developed no clinical disease in spite of evidence for widespread infection of microglia in brain. Since the main differences between Fr54 and FB29 are in the SU (gp70) region of the envelope gene, this region is most likely to account for the differences in induction of CNS disease seen in the current experiments.

Sin Nombre virus pathogenesis in Peromyscus maniculatus.

Sin Nombre virus (SNV), a member of the Hantavirus genus, causes acute viral pneumonia in humans and is thought to persistently infect mice. The deer mouse, Peromyscus maniculatus, has been identified as the primary reservoir host for SNV. To understand SNV infection of P. maniculatus, we examined wild deer mice for localization of viral antigens and nucleic acid. Morphologic examination consistently revealed septal edema within lung tissue and mononuclear cell infiltrates in portal areas of the liver. Immunohistochemical analysis of SNV-infected deer mice identified viral antigens within lung, liver, kidney, and spleen. The lungs consistently presented with the highest levels of viral antigen by immunohistochemistry and with the highest levels of nucleic acid by reverse transcriptase (RT) PCR. The mononuclear cell infiltrates surrounding liver portal triads were positive for SNV antigens in addition to resident macrophages in liver sinuses. Spleen tissue contained antigens in both the red pulp and the periartereolar region of the white pulp. The kidney presented with no gross pathology, although antigens could be localized to glomeruli. Virus antigen levels within the kidney were highest in deer mice that did not have antibodies to SNV but contained viral nucleic acid detectable by RT PCR. Since transmission is thought to occur via urine, our results suggest that virus transmission may be highest in the early stages of infection. In addition, these results indicate that SNV does cause some pathology within its reservoir host.

Rhesus macaques that become systemically infected with pathogenic SHIV 89.6-PD after intravenous, rectal, or vaginal inoculation and fail to make an antiviral antibody response rapidly develop AIDS.

A new simian-human immunodeficiency virus (SHIV) stock (SHIV 89.6-PD), derived from plasma of a rhesus macaque used for in vivo serial passage of virulence-attenuated SHIV 89.6, produces systemic infection after intravenous, intravaginal, or intrarectal inoculation of rhesus macaques. Infection with this virus results in high levels of viral antigen in plasma, a precipitous decline in CD4+ T-cell counts, and a disease syndrome that is characteristic of AIDS. Rapid progression to disease was associated with failure to seroconvert to viral antigens, whereas longer survival was associated with production of antiviral antibodies. In intravenously inoculated animals, peak antigenemia occurred at 7 days postinjection (PI) and severe CD4+ depletion occurred at 14 days PI. In mucosally infected animals, peak antigenemia occurred at 14 days PI and severe CD4+ depletion was not evident until 21 days PI. The 1-week delay in both viral antigenemia and CD4+ T-cell decline in mucosally infected animals is consistent with the hypothesis that, following vaginal inoculation, virus dissemination proceeds in a stepwise manner from the mucosal surface to the draining lymph nodes and subsequently to the bloodstream. This animal model can be used to test the ability of HIV-1 envelope-based vaccines to prevent infection or disease after challenge by the three major routes of HIV transmission.

Generation of endogenous tumour necrosis factor-alpha in MOLT-4 cells during the acute replication phase of human immunodeficiency virus type 1 determines the subsequent latent infection.

We have characterized the mechanism for human immunodeficiency virus type 1 (HIV-1) latent infection in a human T cell line MOLT-4 subclone no. 8 (MOLT-#8). The inocula used were HIV-1 recovered from MT-4 during the acute (NL-A) and persistent (NL-P) phases after HIV-1 infection. On infection of MOLT-#8 with NL-A, viral antigens first appeared in almost 100% of the cells whereafter the numbers of viable antigen-positive cells declined. In contrast, following infection with NL-P the expression of viral antigens was maintained in almost 100% of the cells. In fact, limiting dilution of NL-P-infected cells allowed us to isolate 43 subclones, all of which were positive for viral antigen expression in almost 100% of the cells (type I). In sharp contrast, only two of 41 subclones from NL-A-infected cells were of type I. Seven subclones were latently infected with HIV-1; latent HIV-1 in six subclones (type II), but not in one type III subclone, was activated by tumour necrosis factor (TNF)-alpha or phorbol 12-myristate 13-acetate. The remaining subclones were negative for the viral genome. Of particular note is the effect of endogenous TNF-alpha generated during the acute phase of virus replication which shifted the virus phenotype. Thus, the presence of TNF-alpha during the acute phase of virus replication seems to play a key role in the selective destruction of cells expressing higher levels of viral antigens and in subsequent establishment of latent infection in host T cells.

Level of viral antigen in blood leucocytes from cattle acutely infected with bovine viral diarrhoea virus.

Blood samples from 24 calves undergoing experimental acute infection with a non-cytopathogenic bovine viral diarrhoea virus (BVDV) were examined for viral antigen in peripheral leucocytes with an enzyme-linked immunosorbent assay (ELISA), and for presence of virus in blood plasma in a cell culture assay. With the antigen ELISA, low positive values were detected in leucocytes sampled on days 3-4 from two of eight animals inoculated intranasally, and on days 11-13 from three of 16 animals inoculated intramuscularly. From 22 of the animals, low titres of BVDV were detected in blood plasma obtained 2-9 days after inoculation. All other samples, drawn between 2 and 21 days after inoculation, were negative for viral antigen. All animals seroconverted 3-4 weeks after inoculation, some after having shown mild and transient signs of disease.

Kinetics study of human retrovirus antigens expression in T lymphocytic cell lines by indirect immunofluorescence assay.

Indirect immunofluorescence assay (IFA) is a well-accepted assay for the confirmation of human retrovirus infection. Fluctuations in HIV-1 antigen expression in infected E-B2 cells depending on several factors have been reported. Cells kept in log phase expressed the highest levels of viral antigen. Thus, we studied the time kinetics of IFA positivity in MT-2 (HTLV-I), MO-T (HTLV-II), CEM, and H9 (HIV-1) cell lines. Uninfected T cell line, HT, was used as nonspecific control. Reference HTLV-I/II and HIV-1 serum panels from the Centers for Disease Control and Prevention (CDC) were tested by conventional IFA procedure on slides of each cell line made on different days. On the second day after subculture, HTLV-I strongly positive sera reacted on MT-2 and MO-T cells with a bright pericytoplasmic fluorescence pattern. Weakly positive sera showed a faint staining from the fifth day on, when all the sera showed the highest degree of fluorescence. With HIV-1 cell lines, sera predominantly reacted with a diffuse cytoplasmic pattern, although some sera showed a granular and pericytoplasmic capping staining. The highest degree of fluorescence was found at 3-5 days after subculture. We demonstrated that the sensitivity of the IFA for the detection of antibodies against human retroviruses depended on the day when the slides were assayed and on the serum antibody titer. The fifth day was the most appropriate for HTLV-I/II and HIV-1/H9 systems, whereas for HIV-1/CEM, the fourth day was better. Furthermore, the intensity of the immunofluorescence pattern differed with the antibody titers and the level of antigens expressed on the four cell lines studied. The IFA, improved in our laboratory, proved to be very sensitive, specific, and rapid and could be used as a supplementary/confirmatory assay for retrovirus infection.

Interferon Treatment Inhibits Virus Replication in HIV-1- and SIV-Infected CD4+ T-Cell Lines by Distinct Mechanisms: Evidence for Decreased Stability and Aberrant Processing of HIV-1 Proteins

We have examined the effects of interferon (IFN)-α/β on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse–chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.

Zidovudine treatment prolongs survival and decreases virus load in the central nervous system of rhesus macaques infected perinatally with simian immunodeficiency virus.

To assess the potential therapeutic effects of zidovudine, rhesus macaques were inoculated with simian immunodeficiency virus (SIV) strain SMM/B670 at birth and infused either continuously or intermittently with zidovudine for 6-7 months. Zidovudine did not prevent infection but did significantly increase survival time, which was associated with lower serum p26 viral core antigen levels, a lower virus burden in the cerebrospinal fluid (CSF), and lower CSF quinolinic acid levels than in untreated monkeys. Two of 5 infected, untreated monkeys developed motor impairment within 6 months following infection, whereas motor impairments did not occur in infected, zidovudine-treated monkeys until after the drug was discontinued. Zidovudine treatment was well tolerated by rhesus infants with minimal, transient side effects. These results demonstrate that zidovudine treatment significantly decreases virus load within the central nervous system (CNS) and delays the onset of CNS dysfunction and immune disease in rhesus monkeys perinatally infected with SIV.

Levels of viral glycoprotein reflect enhanced effectiveness of combined drug treatments

A chemosensitivity assay with small replicate Mm5mt/cl C3H mammary tumor cell cultures was developed to determine whether charges in viral antigen expression and release into culture fluids could be utilized as an in vitro measure of single and combined drug effect. The measurement of drug concentrations required for identical 50% reductions in viral antigen levels (ED 50s) allowed the dosage-dependence of the same drug to be compared alone and in combination drug treatments. The 52,000 MW viral envelope glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) was measured in culture fluids of control and drug-treated cultures while cell density was simultaneously determined by cell staining and OD 664 mu determination. While extracellular gp52 levels and cell density increased over 72 hours in control cultures, dose-dependent declines in both parameters provided dual measures of effect for combination CAF treatment [cyclophosphamide: doxorubicin (Adriamycin): 5-fluorouracil], combination CMF treatment [cyclophosphamide: methotrexate: 5-fluorouracil] and single component treatments of these combinations. Comparison of ED 50s for gp52 reduction revealed that each drug's ED 50 concentration was lower in combination treatment than it was in single treatment. In combined drug treatments, the ED 50 of doxorubicin was lowered from 5.17 μM to 0.36 μM, that of 5-fluorouracil was lowered from 0.69 μM to 0.22 μM and that of methotrexate was lowered from 15 nM to 1.65 nM. Reductions of ED 50 in combined drug treatments argued for drug synergisms in these combinations. When drug concentrations, rather than gp52 reductions, were fixed at identical levels in single and combined treatments, additional declines in gp52 levels reflected the enhanced effects of CAF and CMF combinations over single drug treatments. When the anti-estrogen, tamoxifen, was added to CAF and CMF treatments, further declines in gp52 levels (below CAF and CMF levels) reflected the fact that these hormone-augmented treatments were more effective than either hormone or combination chemotherapy alone. These results indicate that decreases in extracellular levels of MMTV gp52 can be utilized in this in vitro assay an an alternative measure of chemotherapeutic effect for evaluating the synergistic of antagonistic nature of drug or hormone interactions and as a tool for in vitro optimization of combined drug effect.

Central nervous system pathology in pediatric AIDS.

Children with AIDS frequently have neurological manifestations due to complications of immunodeficiency or intrinsic effects of human immunodeficiency virus type 1 (HIV-1) on the central nervous system (CNS). The most common neurological disorders not directly related to HIV-1 infection include cerebrovascular disease and lymphoma. Global anoxic-ischemic and necrotizing encephalopathies are frequent, while CNS hemorrhages and arteriopathies are less frequent. Opportunistic CNS infections are uncommon, limited predominantly to monilial and cytomegaloviral encephalitides. Only a few cases of CNS toxoplasmosis have been reported in children. CNS lymphomas often occur in the setting of systemic polymorphous, polyclonal B-cell proliferations that have been associated with Epstein-Barr virus infection. Intrinsic effects of HIV-1 on the CNS include microcephaly, diffuse gliosis, basal ganglia mineralization, HIV encephalitis, and corticospinal tract degeneration. Although viral antigens can be detected in microglia and multinucleated cells in HIV encephalitis, most of the CNS effects of HIV-1 infection cannot be attributed to detectable levels of viral antigen, suggesting that the pediatric CNS is unusually susceptible to low-level HIV-1 infection or to systemic effects of HIV-1 infection, possibly mediated by soluble factors, including the inflammatory cytokines, interleukin-1 beta, and tumor necrosis factor-alpha, which have been shown to be increased in serum and cerebrospinal fluid of children with AIDS.

Human cortical neuronal cell line: a model for HIV-1 infection in an immature neuronal system.

HCN-1A is a human cerebral cortical neuronal cell line having properties consistent with cells of immature neuronal origin. This article details evidence for productive low-level infection of HCN-1A cells with human immunodeficiency virus type 1 (HIV-1). In vitro exposure to HCN-1A monolayers to a high titer of either LAV/HTLV-IIIB or HTLV-IIIMN resulted in HIV-1 p24 antigen production and a moderate increase in reverse transcriptase activity in cell-free supernatants. The cells in both LAV/HTLV-IIIB- and HTLV-IIIMN-infected cultures were passaged and proliferated as long as 5 weeks while continuing to express low levels of viral antigen. Virus-positive cells were detected by indirect immunofluorescence, using serum from an individual with acquired immune deficiency syndrome (AIDS) as well as with a gp120 monoclonal antibody. Confirmation of HCN-1A infection was provided by polymerase chain reaction analyses of both nuclear and cytoplasmic DNA and by de novo synthesis of viral proteins as shown by metabolic labeling and immunoprecipitation. Virus in cell-free supernatants from infected HCN-1A cultures was passaged to a permissive human T cell line (A3.01). HCN-1A cells had no detectable surface CD4 protein or CD4 message. However, the cells expressed the membrane glycolipids, galactocerebroside and sulfatide, possible receptors for gp120 on cells of neuronal origin. Undifferentiated HCN-1A cells provide an in vitro model for investigating potential interactions of HIV-1 with a homogeneous population of immature cortical neurons.

Pathogenesis of hemorrhagic enteritis virus infection in turkeys.

The pathogenesis of hemorrhagic enteritis was investigated in 4-week-old specific-pathogen-free (SPF) turkeys after oral administration of hemorrhagic enteritis virus. The virus antigen was detected and quantified in tissues at various days post-infection (DPI) by an avidin-biotin-enhanced enzyme immunoassay and was located by a monoclonal antibody-based immunoperoxidase (IP) staining technique. In the intestinal tract, low levels of viral antigen were detected from 1 to 3 and 9 to 15 DPI, whereas high antigen levels were found from 4 to 7 DPI. The bursa had viral antigen from 2 to 7 DPI. The plasma fraction of blood was positive for the antigen at day 1 PI and the cellular fraction of blood on day 3 PI. Antigen was first detected in the spleen at 2 DPI and reached a peak on day 6 PI. Initially, the viral antigen was present in a few reticular cells of the spleen and an increase in IP positive cells occurred with time. The maximum number of inclusion bodies in the spleen were found on day 6 PI. Following splenomegaly, viremia resulted in high levels of the virus appearing in the lamina propria of the small intestine. The lamina propria had numerous lymphoreticular cells positive for intranuclear viral inclusions from 5-7 DPI. It was at this time that intestinal congestion and hemorrhage were seen. The results suggest that HEV replicates first in the lymphoid cells of intestinal tract including the bursa, and then in those of the spleen with consequent HEV antigen widely distributed in the body. The time course of the high levels of HEV (mainly 4-7 DPI) in the lymphoid organs (cells), and occurrence of hemorrhagic enteritis (congestion, hyperemia) from 5 to 7 DPI and intestinal hemorrhage (5-8 DPI) appear to suggest that the intestinal lesion may be an immune-mediated response.

Clinical Pharmacology of 2',3'-Dideoxyinosine in Human Immunodeficiency Virus-Infected Children

The pharmacokinetics of intravenous and oral 2',3'-dideoxyinosine (ddI) and the relationships between pharmacokinetic parameters and measures of response were studied in 48 human immunodeficiency virus-infected children. Disappearance of ddI from plasma after the intravenous dose was rapid and biexponential, with half-lives of 12 min and 1.0 h and a total clearance of 510 +/- 180 ml/min/m2. After oral administration, ddI absorption was limited and variable (mean bioavailability, 19% +/- 17%). A plasma ddI concentration-response relationship was observed for both decline in viral p24 antigen levels and improvement in intelligence quotient score. A limited sampling model was developed that accurately predicts the area under the ddI plasma concentration-time curve from one to three plasma samples. Although this pharmacokinetic study was done in children, the results also have relevance to adults and suggest that individualization of dose and schedule through therapeutic drug monitoring may be necessary to achieve optimal response.

HIV-1 sensitivity to zidovudine and clinical outcome in children

In adults with the acquired immunodeficiency syndrome, long-term monotherapy with zidovudine selects for human immunodeficiency virus type 1 (HIV-1) strains with substantially reduced in-vitro susceptibility to the drug. We have assessed the relation between in-vitro resistance to zidovudine and clinical outcome in children, in whom disease progression is more rapid than in adults. We studied 23 children with symptoms of HIV-1 disease during extended monotherapy with zidovudine. An in-vitro assay was used to determine the concentration of zidovudine required to inhibit by 50% the replication of viral isolates (IC 50) obtained after 9 to 39 months of treatment. Viral stocks of high enough titre to yield reproducible results were obtained from 19 of the children. During the following 6 months of therapy, 9 children were stable, 7 deteriorated, and 3 died. There was a highly significant relation between decreased zidovudine susceptibility and poor clinical outcome (p<0·001) but no relation between IC 50 and age at start of therapy or length of time on treatment. Age-adjusted CD4 lymphocyte counts were lower at the start of treatment (p=0·02) and at the time of sampling (p=0·01) in children whose viral isolates had an increased zidovudine IC 50. Initial serum p24 antigen levels were not predictive of subsequent emergence of resistant virus, but at the time of sampling for viral sensitivity higher p24 antigen levels were associated with raised IC 50 (p=0·004). The findings suggest that most children who become unresponsive to monotherapy with zidovudine, as judged by clinical criteria, will have changes in in-vitro sensitivity to the drug. In these children, an alternative antiretroviral therapy should be considered.

Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.