Bartonella henselae is usually not viable in lymph nodes of patients with cat scratch disease.

Bartonella henselae, the agent of cat scratch disease (CSD), appears to be a common organism responsible for lymphadenitis in both adults and children. There is a very low isolation rate for B. henselae from lymph nodes of patients with CSD. Our objective was to evaluate B. henselae viability in a large series of lymph nodes from patients with CSD. From January to November 2016, we analyzed lymph node biopsy samples from patients diagnosed with CSD. We used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect B. henselae RNA, as well as cultures, histological analyses, and fluorescence in situ hybridization (FISH). We tested 87 lymph nodes positive for B. henselae DNA but only 8 (9%) presented with B. henselae RNA. We did not find a significant difference for the pap threshold cycle (CT) values between RNA-positive and RNA-negative lymph nodes (p = 0.5). Cultures, histological analyses, and FISH were negative for all the tested samples. We provide evidence that B. henselae are not or are rarely viable in most cases in the lymph nodes of patients with CSD.

New Rickettsia sp. in tsetse flies from Senegal.

Tsetse flies are blood-sucking insects transmitting African trypanosomiasis. They are known to harbor also three intracellular bacteria that play important role in their lifecycle: Wigglesworthia glossinidia, Sodalis glossinidius and Wolbachia sp. We have studied 78 Glossina morsitans submorsitans collected in Senegal. In all studied flies we amplified genes of bacterium phylogenetically close to obligate intracellular pathogen Rickettsia felis, the agent of spotted fever in humans. We also visualized this rickettsia in the cells of tsetse flies by fluorescence in situ hybridization. The role of this probable fourth endosymbiotic bacterium of tsetse flies in Glossina lifecycle and possible pathogenecity for humans should be further investigated.

Tryptophan 95, an amino acid residue of the Caprine arthritis encephalitis virus vif protein which is essential for virus replication.

The Caprine arthritis encephalitis virus (CAEV) vif gene was demonstrated to be essential for efficient virus replication. CAEV Vif deletion mutants demonstrated an attenuated replication phenotype in primary goat cell cultures and resulted in abortive infection when inoculated into goats. In this study, we determined the in vitro replication phenotype of five CAEV Vif point mutant infectious molecular clones and the ability of the corresponding in vitro translated Vif proteins to interact with the CAEV Pr55(gag) in the glutathione S--transferase (GST) binding assay. Here we show that (i) three of the mutants (S170E, S170G, S197G) behaved as the wild-type CAEV according to virus replication and Vif--Gag interactions; (ii) one mutant (Vif 6mut) was replication incompetent and bound weakly to GST-Gag fusion proteins; and (iii) one mutant (Vif RG) was impaired for replication while retaining its interaction properties. This mutant points out the critical importance of the CAEV Vif tryptophan residue at position 95 for efficient virus replication, defining for this lentivirus a functional domain unrelated to the Gag binding region.

Development of a reverse transcriptase PCR-enzyme-linked immunosorbent assay for quantification of human immunodeficiency virus type 1 RNA in plasma: comparison with commercial quantitative assays.

A quantitative reverse transcriptase PCR assay with automated detection by nonradioactive hybridization was developed for the determination of human immunodeficiency virus (HIV) type 1 RNA levels. This assay is based on the use of an external standard curve with an internal standard. The accuracy of quantification was verified by comparison with reference commercial tests, the Chiron branched-DNA and Roche AMPLICOR HIV MONITOR assays. This assay was able to quantify viremia in patients with CD4 cell numbers below and above 500/mm3 and to quantify some HIV strains which could not be titrated by the MONITOR assay.

Induction of neutralizing antibodies against HTLV-I envelope proteins after combined genetic and protein immunizations in mice.

Direct DNA inoculation can induce both protective humoral and cellular responses against several viruses. The HTLV-I envelope glycoproteins are the major antigens recognized by sera of HTLV-I infected patients that generate neutralizing immune responses in vitro and in vivo. We compared immune responses elicited after a single inoculation of two plasmids encoding the complete HTLV-I envelope proteins followed or not by gp62 Baculovirus recombinant protein boosts in BALB/c mice. First, we observe that the coexpression of env and rex genes is not sufficient to raise a detectable specific humoral response after a single DNA inoculation. Protein boosts generated a high antibody response in mice primed with DNA expressing HTLV-I envelope proteins as compared to naive and negative control vector primed groups. This humoral response presented high neutralizing antibody titers. These results suggest that a single inoculation of DNA expressing HTLV-I env gene can stimulate memory B-cell clones that are able to respond effectively to subsequent encounters with HTLV-I envelope proteins and a specific cellular T helper cell response in mice.

Identification of differentially expressed mRNA species during HIV infection by RNA arbitrarily primed PCR.

BACKGROUND: A number of strategies, such as subtractive cDNA libraries and high through-put sequencing, have been devised to assess differential gene expression. Most of these approaches, however, are cumbersome and/or require tremendous technological power. In this paper, we describe a method, RNA fingerprinting using arbitrarily primed polymerase chain reaction (RAP-PCR), that is rapid, less cumbersome and can differentiate low levels of mRNA expression. OBJECTIVES: To identify genes that are differentially expressed following human immunodeficiency virus type 1 (HIV-1) infection in different cell types by RAP-PCR. STUDY DESIGN: RNA was extracted from both HIV-1-infected and uninfected HUT78 cells and peripheral blood mononuclear cells (PBMCs), reverse transcribed, and RAP-PCR amplified using numerous primer sets. RESULTS: Three genes, gamma-actin, the HIV-1 nef and an unknown sequence, were identified as being differentially expressed in HUT78 cells. The level of gamma-actin mRNA expression is increased after HIV infection and, as expected, the nef gene was solely expressed in HIV-infected cells. In contrast, the unknown mRNA is down-regulated by HIV. Northern blot analysis and/or specific PCR confirmed the differential expression of these three genes. RNA fingerprinting using phytohemagglutinin (PHA)-activated PBMCs infected by HIV in vitro, revealed that gamma-actin is still up-regulated by HIV, whereas the unknown product no longer shows down-regulation. CONCLUSIONS: These results illustrate the usefulness of the RAP-PCR method for isolating and identifying differentially expressed genes during HIV-1 infection of primary lymphocytes.

In vivo and in vitro sequence diversity of the V3 env region of HIV type 1 from the Ivory Coast.

PIP: Nine divergent clades of HIV-1 have been detected in Africa, indicating a far greater genetic diversity of the virus on the continent relative to elsewhere in the world. The envelope of HIV-1, especially the V3 loop, which is implicated in viral tropism and in the neutralizing domain, is considered to be a major target for vaccine research. The authors analyzed proviral DNA and RNA viral sequences of the V3 region of the env gene obtained from the plasma and lymphocytes of six people with AIDS originating from the Ivory Coast. The four men and two women exhibited clinical AIDS and pulmonary tuberculosis. The inferred amino acid sequence of the V3 region derived from the DNA sequences obtained in the study were aligned together with the reference sequence of subtype A from the Los Alamos HIV sequence database. The identity of the conserved tetrapeptide motif at the tip of the V3 loop in all the isolates is GPGQ and is consistent with the common motif sequence found in the database for the A subtype. The major changes in the amino acids are localized in the flanking sequences of the V3 loop. The V3 loop of HIV isolates from patient CI-39 presents a change of D to R in position 25, associated with an unusual change in position 24 of G to D. These mutations were found in both viral RNA sequences and in the proviral DNA sequences from uncultured peripheral mononuclear cells (PBMCs), possibly reflecting an infectious viral variant which is actively expressed in vivo and present in plasma. The change of an acidic amino acid to a basic amino acid at position 25 appears to be an important viral tropism determinant.^ieng

The vif gene is essential for efficient replication of caprine arthritis encephalitis virus in goat synovial membrane cells and affects the late steps of the virus replication cycle.

Complex retrovirus genomes contain a variable number of accessory genes, among which is the vif gene. We investigated in vitro the role of the vif gene of caprine arthritis encephalitis virus (CAEV) by studying the phenotype of five vif mutants after infection of primary goat synovial membrane (GSM) cells and blood-derived monocytes/macrophages. Any deletion introduced into the vif gene resulted in slow and low viral replication and production of virions with an infectious titer lower than that of wild-type viral particles. The wild-type phenotype could be restored by the trans expression of the vif gene in a complementation assay. Quantitative PCR and reverse transcription-PCR analyses were performed in order to determine which stage of the replicative cycle was impaired by the vif deletion. Our results demonstrated that CAEV Vif did not act at the level of reverse transcription or transcription but rather at the late stage of virus formation and/or release, as lower amounts of virus were produced after a single replicative cycle. The vif-deleted CAEV produced after 24 h of infection was still able to infect GSM cells, indicating that the vif gene is not essential for virus infectivity but is required for efficient virus production.

Transient adult T-cell leukemia/lymphoma picture during varicella infection in an HTLV-1 carrier.

HTLV-1 (human T-lymphotropic virus type 1) is associated with tropical spastic paraparesis, adult T-cell lymphoma (ATL), and also with opportunistic infections. The risk for developing ATL in HTLV-1 healthy carriers is low, between 1 and 4%. Nothing is known about the events promoting the evolution from the healthy carrier state to symptomatic ATL. We describe the case of a 44-year-old French Caribbean man with a chronic and recurrent strongyloidiasis in which the occurrence of a hemorrhagic and necrotic varicella led to the discovery of an infection by HTLV-1 and an acute form of ATL. All hematological data were normal before the onset of varicella. ATL completely disappeared at the same time as the varicella healed. This leads us to hypothesize that acute infections such as the reactivation of varicella-zoster may act as a promoting factor for the development of ATL in healthy HTLV-1 carriers.

Identification and subcellular localization of the Q gene product of visna virus.

The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.

Subcellular localization of rev-gene product in visna virus-infected cells.

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.

Nucleotide sequence analysis of SA-OMVV, a visna-related ovine lentivirus: phylogenetic history of lentiviruses.

The nucleotide sequence analysis of the visna-related South African Ovine Maedi Visna virus (SA-OMVV) demonstrates extensive genetic polymorphism among ovine lentiviruses. Differences between visna virus and SA-OMVV proteins range from 8.5 to 35% mismatched amino acids. Moreover, there is a new open reading frame (orf W) in the central part of the genome. A phylogenetic history calibrated by the divergence and isolation dates of these two ovine lentiviruses shows that radiation of the lentiviridae family is a recent event. Visna virus and SA-OMVV evolved independent of each other for about 42 years. The inferred molecular clock was used to calculate the minimal time elapsed since the divergence of some lentiviruses: 93 years for ovine and caprine lentiviruses, 430 years for ungulate and primate lentiviruses, and roughly 200 years for HIV-1, HIV-2, and SIVAGM. BRU, ELI, and MAL HIV-1 isolates diverged in the early 1960s.