ABSTRACT Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

This report describes the fabrication and successful use of the ion channel switch biosensor (ICSB) for rapid point-of-care detection of influenza A in different types of respiratory specimens. Virus culture – regarded as the “gold standard” – and an immunochromatographic rapid point-of-care test for influenza A virus were compared with the biosensor. The ICSB rapid test provided an objective readout within 10min of specimen inoculation into the ICSB chamber wells, without the need for chemical or other pretreatments. Construction of the ICSB with specific antibodies also enables rapid detection and identification of appropriate influenza A subtypes.

The establishment of reservoirs of latently infected cells is thought to contribute to the persistence of HIV-1 infection in the host. Studies so far have mainly focused on the long-lived reservoir of HIV-infected resting CD4+ T cells. A discrete population of HIV-infected CD4-/CD8- double negative (DN) T cells has recently been shown to exist and may also play a role in HIV-1 persistence. DN T cells are CD3 positive, either TCRalphabeta or TCRgammadelta positive, but lack both CD4 and CD8 surface markers. We developed a novel, magnetic bead column-based cell fractionation procedure for isolating >99% pure DN T cells. CD4+, CD8+, and DN T cells were purified from 23 samples of a cohort of 18 HIV-1-infected patients. Each cell fraction was analyzed for levels of total and integrated HIV-1 DNA. A correlation was observed between the presence of HIV-1 DNA in the DN T cell fraction and plasma viral load (VL). Using a micrococulture technique, we saw an initial release of virus from DN T cells of a patient with high VL. Analysis of env and nef sequence data suggested that the HIV-1 present in CD4+ and DN T cells originated from a common infecting strain. Different from the published literature, we have demonstrated the presence of HIV-1 DNA in DN T cells only in patients who are experiencing HAART failure. While these cells may have a limited role in viral persistence in high VL patients, our results suggest DN T cells are unlikely to be a major reservoir in patients on HAART with clinically undetectable plasma viral RNA.

Vif is air HIV accessory protein whose primary function is to negate the action of APOBEC3G, a naturally occurring cellular inhibitor of HIV replication. Vif acts by binding to APOBEC3G, inducing its protein degradation within infected cells and reducing its levels in progeny virions. Interventions that interfere with the Vif-APOBEC3G interaction, raise intracellular or virion associated levels of APOBEC3G, or reduce intracellular levels of Vif, all could hold promise as potential therapeutic approaches aimed at enhancing the cells innate antiviral activity. Levels of APOBEC3G might be increased or Vif levels decreased, by strategies targeting protein synthesis, protein degradation or cellular localisation and function, and properties of APOBEC3G and Vif relevant to these strategies are discussed. Recent data have suggested that Vif may have other mechanisms of action apart from the above activities against APOBEC3G, including effects against other anti-viral mechanisms independent of APOBEO3G cytidine deaminase activity. In addition to interaction with APOBEO3G, Vif may have other accessory functions, which are discussed in relation to potential therapies that may affect multiple stages of the HIV life cycle. Future development of strategies that combine enhancement of APBOEC3G functional with inhibition of multiple Vif functions may become useful tools for HIV therapy.

Astrocytes persistently infected with HIV-1 can transmit virus to CD4+ cells, suggesting that astrocytes may be a source of viral persistence and dissemination in the brain. In the present study, we investigated the fate of HIV-1 upon infection of astrocytes. HIV-1 was observed in vesicle-like structures. Unspliced genomic RNA and extrachromosomal HIV-1 DNA were detected in astrocytes, with levels declining over time. The extrachromosomal viral DNA was not de novo reverse transcribed in astrocytes but most likely the products of intravirion reverse transcription present in the virus inoculum. Integrated HIV-1 DNA was not detected in assays sensitive to detect 2 integrated copies of provirus. However, the majority of astrocyte cultures released infectious virus that could be transmitted to CD4+ cells. Our findings suggest a novel pathway of HIV-1 uptake and release in astrocytes that does not necessarily require virus replication, which may contribute to persistence and spread of HIV-1 in the brain.

Reverse transcription (RTn) in HIV-infected cells occurs in a nucleoprotein complex termed the reverse transcription complex (RTC). RTCs containing RT activity and integrase (IN) were shown to be heterogeneous in size and density on sucrose velocity and equilibrium gradients. WT and Vif-deficient (Δvif) RTCs produced by infection with virus from permissive cells displayed similar sedimentation characteristics, while RTCs from Δvif virus produced in non-permissive cells demonstrated a reduction in the major RTC form and more of the RTn products in rapidly sedimenting structures. APOBEC3G derived from virions did not co-sediment with RTCs, but RTCs from Δvif infections showed elevated levels of mutations in RTn products, consistent with APOBEC3G and other mutational mechanisms. The most mutated transcripts were present within rapidly sedimenting RTCs. Thus, virus without functional vif, produced from non-permissive cells, forms abnormal RTCs that contain increased mutation of RTC-associated RTn products in newly infected target cells.

ABSTRACT Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.i.). Resolution of DHBV infection occurred in 13 out of 15 ducks by 1-month p.i., defined as clearance of DHBV surface antigen-positive hepatocytes from the liver and development of anti-DHBV surface antibodies. At 9 months p.i., residual DHBV DNA was detected using nested PCR in 10/11 liver, 7/11 spleen, 2/11 kidney, 1/11 heart, and 1/11 adrenal samples. Residual DHBV DNA was not detected in serum or peripheral blood mononuclear cells. Within the liver, levels of residual DHBV DNA were 0.0024 to 0.016 copies per cell, 40 to 80% of which were identified as covalently closed circular viral DNA by quantitative PCR assay. This result, which was confirmed by Southern blot hybridization, is consistent with suppressed viral replication or inactive infection. Samples of liver and spleen cells from recovered animals did not transmit DHBV infection when inoculated into 1- to 2-day-old ducklings, and immunosuppressive treatment of ducks with cyclosporine and dexamethasone for 4 weeks did not alter levels of residual DHBV DNA in the liver. These findings further characterize a second form of hepadnavirus persistence in a suppressed or inactive state, quite distinct from the classical chronic carrier state.

The ability of dengue virus-infected human monocyte-derived macrophages to induce permeability changes in primary human umbilical vein endothelial cells was investigated. Supernatants from dengue virus type 2-infected monocyte-derived macrophages increased permeability in human umbilical vein endothelial cell monolayers without inducing endothelial cell infection. Production of permeabilising activity from monocyte-derived macrophages occurred after the peak of progeny virus release. TNF-alpha, a known inducer of endothelial cell permeability, was released from dengue virus infected monocyte-derived macrophages but its release did not coincide with release of endothelial cell permeabilising activity. Permeability induction was enhanced by pre-incubation with supernatants from infected monocyte-derived macrophages harvested at the time of peak release of TNF-alpha and infectious virus. Thus, supernatants from dengue virus-infected monocyte-derived macrophages contain factors that increase human umbilical vein endothelial cell permeability, but this is not accompanied by endothelial cell infection or directly correlated with release of dengue virus or TNF-alpha from monocyte-derived macrophages. This model system can be used for further in vitro analysis of mechanisms that may relate to capillary leakage and the development of dengue haemorrhagic fever/dengue shock syndrome.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i) their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii) the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii) the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.

Integration of HIV-1 DNA is essential both for productive viral replication and for viral persistence in patients. Methods to measure specifically proviral HIV DNA are required for investigating the mechanisms of HIV integration, for screening novel integrase inhibitors in cell culture and for monitoring levels of persistent integrated viral DNA in patients. In this report, the linker primer polymerase chain reaction (LP-PCR) and Alu-PCR methods for the quantitation of integrated HIV-1 DNA have been modified and evaluated. Each of the two modified assays allowed the quantitative detection of 4 copies of integrated HIV DNA in presence of 2×105 cell-equivalents of human chromosomal DNA. The results show that proper DNA isolation procedures and the inclusion of appropriate controls in these assays are important for the accurate quantitation of integrated HIV DNA. With further improvements, it should be possible to use these methods as diagnostic tools to monitor closely the efficacy of antiretroviral therapy.

The genome of an Australian strain of duck hepatitis B virus (AusDHBV) was cloned from a pool of congenitally DHBV-infected-duck serum, fully sequenced and found by phylogenetic analyses to belong to the 'Chinese' DHBV branch of the avian hepadnaviruses. Sequencing of the Pre-S/S gene of four additional AusDHBV clones demonstrated that the original clone (pBL4.8) was representative of the virus present in the pool, and a head-to-tail dimer of the clone was infectious when inoculated into newly hatched ducks. When the published sequences of 20 avian hepadnaviruses were compared, substitutions or deletions in the polymerase (POL) gene were most frequent in the 500 nt segment encoding the 'spacer' domain that overlaps with the Pre-S domain of the Pre-S/S gene in a different reading frame. In contrast, substitutions and deletions were rare within the adjacent segment that encodes the reverse transcriptase domain of the POL protein and the S domain of the envelope protein, presumably because they are more often deleterious.

ABSTRACT We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 × 10 5 cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for ∼10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells.

A one-step cell-to-cell transmission model of human immunodeficiency virus (HIV) infection was used to study viral DNA integration in the early phase of viral replication. Co-culturing H3B cells as virus donors with CD4+ Hut78 recipient cells in a ratio of 1:4 produced a synchronous, one-step viral infection with de novo synthesis of unintegrated HIV DNA within 4 h p.i., which subsequently integrates in the host genomic DNA to form provirus. To study the kinetics of viral DNA integration, cellular chromosomal DNA was isolated at different times after co-culturing and extensive electrophoresis was used to remove residual unintegrated viral DNA. Removal of contaminating, unintegrated viral DNA in the purified chromosomal DNA fraction was confirmed by various experiments. When purified chromosomal DNA (free of contaminating unintegrated viral DNA)--from the mix of acutely infected cells--was digested with restriction enzymes KpnI, BamHI or PstI and analysed by Southern blot hybridization, integration of viral DNA into chromosomal DNA was first observed at 8 h p.i. and was essentially complete by 72 h p.i. In addition, evidence was found for a relatively stable, partially integrated HIV DNA structure within the chromosomal DNA, that was first detectable at 8 h p.i. and did not become fully integrated until 72 hours post infection.

To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA. The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA. In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays. Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays. This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials.

Macrophages are considered of central importance in cell-to-cell transmission of human immunodeficiency virus (HIV) infection in vivo. In this report, we describe a novel cell-to-cell transmission model using HIV-infected monocyte-derived macrophages (MDMs) as donor cells and peripheral blood lymphocytes (PBLs) as recipients. Virus was transmitted during a 2-h coincubation period from intracellular or tightly cell-associated viral stores in adherent infected MDMs to nonadherent CD3(+) PBLs. Transmission required cell contact, but syncytia formation was not observed. HIV cell-to-cell transmission occurred in both allogeneic and autologous systems, and replication was higher in phytohemagglutinin (PHA)-stimulated than unstimulated recipient PBLs. In contrast, transmission of infection by cell-free virus was barely detectable without PHA stimulation of recipients, suggesting the cell-cell interaction may have provided stimuli to recipient cells in the cell-to-cell system. Viral DNA levels increased 5-24 h postmixing, and this increase was inhibited by pretreatment of cells with the reverse transcription inhibitor azidothymidine, indicating de novo reverse transcription was involved. Cell-to-cell transmission was more efficient than infection with cell-free virus released from donor MDMs, or 0.1 TCID(50)/cell cell-free viral challenge. This model provides a system to further investigate the mechanisms and characteristics of HIV cell-to-cell transmission between relevant primary cells that may be analogous to this important mode of virus spread in vivo.

Macrophages are considered of central importance in cell-to-cell transmission of human immunodeficiency virus (HIV) infection in vivo. In this report, we describe a novel cell-to-cell transmission model using HIV-infected monocyte-derived macrophages (MDMs) as donor cells and peripheral blood lymphocytes (PBLs) as recipients. Virus was transmitted during a 2-h coincubation period from intracellular or tightly cell-associated viral stores in adherent infected MDMs to nonadherent CD3 PBLs. Transmission required cell contact, but syncytia formation was not observed. HIV cell-to-cell transmission occurred in both allogeneic and autologous systems, and replication was higher in phytohemagglutinin (PHA)-stimulated than unstimulated recipient PBLs. In contrast, transmission of infection by cell-free virus was barely detectable without PHA stimulation of recipients, suggesting the cell–cell interaction may have provided stimuli to recipient cells in the cell-to-cell system. Viral DNA levels increased 5–24 h postmixing, and this increase was inhibited by pretreatment of cells with the reverse transcription inhibitor azidothymidine, indicating de novo reverse transcription was involved. Cell-to-cell transmission was more efficient than infection with cell-free virus released from donor MDMs, or 0.1 TCID50/cell cell-free viral challenge. This model provides a system to further investigate the mechanisms and characteristics of HIV cell-to-cell transmission between relevant primary cells that may be analogous to this important mode of virus spread in vivo. © 1999 Academic Press

This paper describes the use of one-step growth conditions to study the kinetics of duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Synchronized infection was achieved using partially purified DHBV virions at an m.o.i. of 640 DHBV DNA-containing virions per cell, and these conditions were shown to produce a single cycle of infection. In this model, input purified DHBV DNA was rapidly internalized by cells at > or = 0.5 h, and localized to the nucleus by 4 h, but both covalently closed circular (CCC) DNA and single-stranded DNA were not detected until 48 h postinoculation (p.i.), suggesting that there was a > or = 40 h delay between DHBV localization to the nucleus and formation of CCC DNA. In contrast, CCC DNA can be first detected in hepatocytes at 6 h p.i. in in vivo infection of ducks with the same DHBV strain. In an analysis of the nuclear transport of the DHBV genome, release of nuclear viral DNA from a particulate form to a soluble nucleoplasmic form was only 50% complete by 48 h p.i. However, this process occurred simultaneously with genome uncoating since all soluble nucleoplasmic DHBV DNA was free of nucleocapsid material; this suggests that nucleocapsid disassembly and genome uncoating may occur at the nuclear membrane and not within the nucleus. Quantitative analysis demonstrated inefficiency in a number of steps including virus uptake and internalization, translocation of nucleocapsid across the nuclear membrane and antigen expression from intranuclear viral DNA.

Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.

Using a one-step model for cell-to-cell transmission of HIV infection we have identified two distinct phases of HIV RNA synthesis. The first phase (4 h–12 h p.i.) was marked by an increase in only the full-length 9 kb RNA, while the second phase (24 h p.i. onwards) comprised a significant increase in the levels of all three species of viral RNA. We now report that while the continual presence of actinomycin D at 50 μg/ml abolished all detectable viral nucleic acid synthesis when virus donor H3B cells were pre-treated with 50 μg/ml of actinomycin D (AmD), washed free of unbound drug (a procedure which inhibited >99% of total cellular RNA transcription) then mixed with untreated recipient Hut78 cells, normal amounts of full length linear unintegrated viral DNA were produced and the first phase of viral RNA transcription was unaffected. Similarly, when both the virus donor cells and recipient cells were arrested in the late G1 phase of the cell cycle by aphidicolin and then mixed, linear unintegrated viral DNA was the major viral DNA species roduced. The appearance of circular viral DNA and progeny virus was inhibited, but the first phase of induced viral RNA synthesis was unaffected. When AZT was added at 2 h or 4 h after cell-cell mixing, the level of 9 kb RNA detected was significantly lower, corresponding to reduction in the level of viral DNA. These and previous results indicate that the template for the first phase of viral RNA synthesis was likely to be newly synthesized, linear unintegrated viral DNA and not pre-existing proviral DNA present in the donor cells. Taken together, our results suggest that there exists a yet to be fully characterized pathway of concurrent viral DNA and RNA synthesis early after cell to cell transmission of HIV infection.