Kinetics of early HIV-1 gene expression in infected H9 cells assessed by PCR.

The time course of viral gene expression in H9 cells acutely infected with HIV-1 was analysed by the polymerase chain reaction (PCR). Virus-specific sequences were first detected in genomic DNA of H9 cells 1-2 h after infection. RNA for the regulatory genes such as the tat and nef appeared 2-3 h post-infection and RNA for the gag and env at 3 h. The results demonstrate that viral DNA synthesis occurs rapidly after infection of target cells followed by synthesis of viral RNA. Cell-associated reverse transcriptase activity increased after 24 h, while culture supernatant enzyme activity increased later, between 1-5 days. The delay in virus release after rapid integration and transcriptional activity suggests the involvement of additional factors, perhaps both cellular and viral, that control the formation and budding of mature virions.

Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis.

We have used the polymerase chain reaction (PCR) to detect by co-amplification, multiple regions of the HIV-1 genome in infected cells. Genomic RNA and DNA from productively infected H9 cells were independently extracted and amplified in reactions with and without reverse transcriptase respectively using primer pairs to the gag, env, tat and nef regions of the viral genome in the same reaction mixture. PCR-products were analysed by liquid hybridization with end labelled oligonucleotide probes followed by gel-electrophoresis (oligomer hybridization). The primer pairs were capable of detecting as few as 10 copies of RNA and 10-20 copies of integrated proviral DNA. The ability to co-amplify multiple target regions in the same incubation mixture provides a method for detecting and confirming the presence of HIV-1 in samples for which limited nucleic acid is available. In addition, in reconstitution experiments, the same method was used to detect HIV-1 and HTLV-I simultaneously with comparable sensitivity (20-40 gene copies each). This offers the possibility of simultaneous diagnosis of multiple viral infections, such as those that occur in AIDS, on the same sample preparation.

Interferon-gamma inhibits HIV-induced invasiveness of monocytes.

HIV-infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92-kd metalloproteinase (MMP-9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)-infected monocytes with interferon-gamma at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP-9 produced by HIV-infected monocytes treated with interferon-gamma 1 day prior to infection with HIV as compared with that of untreated HIV-infected monocytes. Monocytes treated with interferon-gamma 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP-9 were substantially lower than those produced by monocytes inoculated with live HIV, heat-inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat-inactivated HIV did not result in increased invasiveness or high MMP-9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4-gp120 interactions and required active virus infection. Furthermore, addition of interferon-gamma to monocytes on day 10 after infection inhibited MMP-9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon-gamma-induced down-regulation of MMP-9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN-gamma in monocytes. Down-regulation of MMP-9 in HIV-infected monocytes by interferon-gamma may play an important role in the control of HIV pathogenesis.

Identification and characterization of a highly pathogenic H5N1 avian influenza A virus during an outbreak in vaccinated chickens in Egypt.

Highly pathogenic avian influenza A (HPAI) H5N1 viruses continue to be a major veterinary and public health problem in Egypt. Continued surveillance of these viruses is necessary to devise strategies to control the spread of the virus and to monitor its evolutionary patterns. This is a report of the identification of a variant strain of HPAI H5N1 virus during an outbreak in 2010 in vaccinated chicken flocks in a poultry farm in Assiut, Egypt. Vaccination of chickens with an oil-emulsified inactivated A/chicken/Mexico/232/94 (H5N2) vaccine induced high levels of hemagglutination inhibition (HI) antibody titers reaching up to 9 log2. However, all flocks irrespective of the number of vaccine doses and the resultant HI titer levels came down with severe influenza infections. The qRT-PCR and rapid antigen test confirmed the influenza virus to be from H5N1 subtype. Sequencing of the hemagglutinin (HA) gene fragment from ten independent samples demonstrated that a single H5N1 strain was involved. This strain belonged to clade 2.2.1 and had several mutations in the receptor-binding site of the HA protein, thereby producing a variant strain of HPAI H5N1 virus which was antigenically different from the parent clade 2.2.1 virus circulating in Egypt at that time. In order to define the variability in HPAI H5N1 viruses over time in Egypt, we sequenced another H5N1 virus that was causing infections in chickens in 2014. Phylogenetic analysis revealed that both viruses had further distanced from the parent virus circulating during 2010. This study highlights that the antigenic mutations in HPAI H5N1 viruses represent a definitive challenge for the development of an effective vaccine for poultry. Overall, the results emphasize the need for continued surveillance of H5N1 outbreaks and extensive characterization of virus isolates from vaccinated and non-vaccinated poultry populations to better understand genetic changes and their implications.

Impaired antigen presentation to CD4+ T-cells by HIV-infected monocytes is related to down-modulation of CD4 expression on helper T-cells: possible involvement of HIV-induced cellular factors.

Defective antigen presentation by HIV-infected monocytes is related to severe immune dysfunction in patients with AIDS, although the mechanism by which this process occurs is not well defined. Here we report that reduced capacity by HIV-infected monocytes to stimulate or present antigen to CD4+ T-cells was mediated by cellular factors associated with the plasma membranes of HIV-infected monocytes. In contrast, soluble factors secreted by HIV-infected monocytes had little or no effect on T-cell stimulation. Reduced T-cell stimulation by HIV-infected monocytes was related to down-modulation of CD4 expression on helper T-cells and was not affected by the inclusion of anti-HIV-gpl20 Ab, indicating the involvement of soluble or cell-associated viral envelope protein to be less likely. Exposure of CD4+ T-cells, that had been in co-culture with HIV-infected monocytes, to uninfected monocytes partially restored impaired T-cell stimulation. Thus, for the first time we report that altered capacity of HIV-infected monocytes to stimulate and present antigen to CD4+ T-cells is related to down-modulation of CD4 expression on T-cells, and appears to occur via membrane-associated cellular factors on HIV-infected monocytes.

Assessment by gene amplification and serological markers of transmission of HIV-1 from hemophiliacs to their sexual partners and secondarily to their children.

The transmission of HIV-1 infection from men with hemophilia A to their female sex partners and secondarily to their children was studied by serological markers including antibody, antigen, and HIV genome as detected by the polymerase chain reaction (PCR). Among 27 sex partners of 26 seropositive hemophiliacs, 5 were seropositive-PCR positive (active), 11 were seronegative-PCR positive (latent), and 11 were negative for both. These results were confirmed by testing serial serum samples and paired samples of DNA from peripheral blood mononuclear cells (PBMCs) and serum from seronegative women. PCR negativity in exposed women was correlated with the use of condoms (p less than 0.01). Eight children from five couples were seronegative. However, HIV-1 infection as detected by PCR was transmitted to 60% of exposed children, including one from a seronegative-PCR positive mother.

Poor sensitivity, specificity, and reproducibility of detection of HIV-1 DNA in serum by polymerase chain reaction. The Transfusion Safety Study Group.

A series of recent studies have reported detection by the polymerase chain reaction (PCR) of cell-free human immunodeficiency virus type 1 (HIV-1) DNA (as opposed to virion RNA) in serum from both seropositive and seronegative persons. To evaluate the sensitivity, specificity, and reproducibility of PCR detection of cell-free HIV-1 DNA, we distributed coded panels containing 98 serum specimens obtained from well-characterized, infected individuals and control blood donors to the two laboratories with reported experience with this technique. Positive results were reported with HIV-1 gag primers (SK38/39) for 48 of 188 separate PCR determinations on DNA extracts from 44 serum samples from seropositive patients (25.5% sensitivity). HIV-1 gag signal was also reported for 28 of 151 PCR determinations on 34 samples from noninfected blood donors (18.5% false-positive rate). PCR for HIV-1 env DNA performed in one laboratory was negative on all specimens from seropositive and seronegative patients. Results for cell-free HIV-1 gag and human genomic (beta-globin or HLA DQ-alpha) DNA were inconsistent on replicate and serial specimens evaluated within each laboratory and between laboratories. These results indicate that current techniques for detecting cell-free HIV-1 DNA in serum lack adequate sensitivity, specificity, and reproducibility for widespread clinical applications.

Rubella virus replication: effect of interferons and actinomycin D.

The effect of alpha and gamma interferon (IFN alpha, IFN gamma) and actinomycin D on the expression of wild type rubella virus in African green monkey kidney cells (Vero 76) was studied. Viral protein synthesis in the infected cells was significantly reduced upon treatment of the cells with IFN alpha or IFN gamma, which is accompanied by the reduction in the level of both the (+) stranded and the (-) stranded viral RNAs. The residual rubella viral RNA from interferon-treated cells, however, was structurally intact as judged by Northern blot analysis and in vitro translation. These results suggest that the effect of IFN alpha and IFN gamma on rubella viral protein synthesis is both at the transcriptional and the translational level. The effect of actinomycin D on rubella virus replication was found to be time-dependent. It is much more pronounced during the eclipse phase of the viral growth (first 4 h) than after 8 h at which time actinomycin D had lesser effect. A similar effect on rubella virus replication was observed when alpha-amanitin was used instead of actinomycin D. These results were taken to indicate that during the viral infection, host cell DNA directs the synthesis of a cellular factor(s) which is essential for the viral replication. When the synthesis of this cellular factor(s) is terminated at an early stage of viral infection by actinomycin D or by alpha-amanitin, viral replication is impaired.

HIV-1 infects and alters immune function of a monocyte subset expressing low CD14 surface phenotype.

Monocytes represent a leukocyte subset that express high levels of CD14 on their surface (CD14-high). These cells play a critical role in the pathogenesis of HIV-1 infection. In the present study, we have identified a monocyte subset expressing an extremely low level of CD14 (CD14-low), and examined their susceptibility to HIV-1 infection. Phenotypic analysis by flow cytometry of these cells revealed a low level of CD4, but the absence of CD3, CD14, CD19, and CD83 surface markers. Both CD14-low and CD14-high cell populations expressed CD13 and CD33 markers on their surface, suggesting these cells to be of myeloid origin. Morphologically, CD14-low cells were indistinguishable from CD14-high cells. CD14-low cells were susceptible to infection with a monocytotropic strain of HIV-1 (HIVADA). However, like CD14-high monocytes, CD14-low cells could not be productively infected with a T cell tropic strain of HIV-1 (H9/HTLV(IIIB)). Similar to CD14-high monocytes, CD14-low cells were capable of inducing antigen-stimulated CD4+ T-cell proliferation. HIV-1 infection substantially reduced their ability to induce antigen-stimulated T-cell proliferation. These data indicate that CD14-low cells belong to the monocyte lineage and may play an important role in the immunopathogenesis of HIV-1 infection.

Study of viral replication in HIV-1-infected CEM T-cell subclones which are reduced in their ability to form syncytia.

The derivation of ethyl-methanesulfonate (EMS) mutagenized subclones from the CEM T-cell line has been described. These clones expressed CD4 and bound soluble gp120, however, two of the generated clones were markedly reduced in their ability to form syncytia after infection with either gp160-vaccinia vector or cell-free human immunodeficiency virus type 1 (HIV-1). Here, we asked at what stage(s) viral infection is blocked in these cells. Polymerase chain reaction (PCR) analysis revealed that at 6 and 72 h postinfection with HIV-1, cells of the syncytia-deficient clones expressed markedly reduced amounts of viral-specific DNA compared with cells of the parental line or the syncytia-positive clones. Long-term cultures revealed a marked delay in the appearance of reverse transcriptase (RT) activity in the supernatants of these subclones when compared with the parental line and viral replication did not lead to massive cell death. Syncytia formation in HIV-1-infected cultures of the syncytia-deficient subclones was enhanced by tumor necrosis factor alpha (TNF alpha) when added 24 h postinfection. In contrast, pretreatment with TNF alpha for 48 h followed by washing and infection of the cells with HIV-1 augmented syncytia formation of the syncytia-positive subclones, but not of the syncytia-negative subclones. Thus, the EMS-mutagenized subclones may provide a tool to study host cell factors required for the establishment of a productive HIV-1 infection and responsiveness to TNF alpha.

Evidence of HIV-2 infection in Equatorial Guinea (central Africa): partial genetic analysis of a B subtype virus.

PIP: A case of HIV-2 infection is described in a 35-year-old woman born in Malabo, Equatorial Guinea, who migrated to Madrid, Spain, in 1995. It was determined through questioning the woman that her only HIV-associated risk factor was unprotected heterosexual contacts in her country of origin. Her ex-husband, to whom she had been married for 18 years, was also from Equatorial Guinea and was known to have had multiple sex partners. The woman reported having had heterosexual encounters with five other partners in her native country, although less frequently than with her ex-husband. She was unaware of her sex partners' HIV serostatus and denied having had any sexual relations in Spain during her one-year current residency. The woman was in good health and presented with a CD4+ T cell count of 486 cubic millimeters. HIV-2 infection was identified using the Pepti-LAV immunoassay and Western blot. Isolation of the infecting virus was attempted by coculturing the patient's peripheral blood mononuclear cells (PBMCs) with PBMCs from an HIV-seronegative donor according to standard protocols for HIV isolation. Nested polymerase chain reaction was later conducted on some DNA from the woman's uncultured PBMCs. Phylogenetic analysis showed that the sequence data obtained clustered with the B subtype of HIV-2.^ieng

Co-amplification of specific sequences of HCV and HIV-1 genomes by using the polymerase chain reaction assay: a potential tool for the simultaneous detection of HCV and HIV-1.

A rapid and simple method using the polymerase chain reaction (PCR) was devised for the co-amplification and simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) specific sequences in the same serum sample. Genomic RNA was extracted from 13 blood donor sera that were reactive in ELISA for both anti-HCV and anti-HIV-1. The extracted RNA was reverse transcribed into cDNA and amplified using nested primer pairs (SN01 and SN04; SN02 and SN03) based on the HCV prototype sequence of clones 37b and 81, and SK 38/39 for HIV-1 simultaneously. PCR products were analyzed by liquid hybridization or Southern blot hybridization with 32P end-labeled oligonucleotide probes from the regions between the primer pairs, excluding the primer sequences. HCV-RNA was detected in all 13 (100%) samples tested; HIV-RNA was detected in 11 (85%) samples. The ability to co-amplify specific sequences from two different viral genomes in the same reaction mixture offers the possibility of simultaneous detection and diagnosis of more than one viral agent in serum samples of infected individuals.

Enhanced diagnostic efficiency of the polymerase chain reaction by co-amplification of multiple regions of HIV-1 and HIV-2.

A method for co-amplification of multiple viral sequences of HIV-1 and HIV-2 by polymerase chain reaction was designed. The technique resulted in the specific detection of each type of virus and allowed the amplification of as few as two copies of target DNA. The amplification of multiple regions of the viral genome offers the advantage of detecting multiple target sequences, which may be essential for some viruses, such as HIV, that exhibit a high degree of variability in their gene sequences. In addition, the method permitted the detection of both virus types in the same reaction, allowing for greater utility in geographic areas where coinfections with both viruses occur and cross-reactivity in Western blots is observed. This method was applied successfully to the detection of viral DNA in clinical specimens.