Biomedical subjects

Jeffrey Vieira

Publications and source records attributed to Jeffrey Vieira.

8 recordsLinked to original sources

PubMed2006

KSHV/HHV-8 infection of human hematopoietic progenitor (CD34+) cells: persistence of infection during hematopoiesis in vitro and in vivo.

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV, purified HPCs were exposed to KSHV, and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells, and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection, persistence, and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo.

Animals↗

PubMed2006

Sequence-based methods for identifying epidemiologically linked herpes simplex virus type 2 strains.

Traditional methods for confirming the identity of herpes simplex virus (HSV) isolates use restriction fragment length polymorphism (RFLP). However, RFLP is less amenable to high-throughput analyses of many samples, and the extent to which small differences in RFLP patterns distinguish between different viral strains remains unclear. Viral HSV type 2 (HSV-2) DNA isolates from 14 persons experiencing a primary HSV-2 infection and from their sexual partners were analyzed by RFLP and heteroduplex mobility assays. We also compared the HSV-2 sequences from seven regions, including noncoding regions between UL19 and UL20, UL24 and UL25, UL37 and UL38, and UL41 and UL42 and coding segments of the gC, gB, and gG genes. Although the resulting RFLP patterns of the couples were almost identical, minor banding differences existed between the source and susceptible partners in five couples. Heteroduplex mobility assays were unable to distinguish between unrelated strains. Overall, 22 sites of sequence variation were found in 1,482 bp of analyzed sequence. The DNA sequences differentiated between all unrelated infections, and epidemiologically related isolates had identical sequences in all but two pairs. Our results suggest that a multilocus assay based on several DNA sequences has the potential to be an informative tool for identifying epidemiologically related HSV-2 strains.

DNA, Viral↗

PubMed2005

Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice.

Herpes simplex virus (HSV) has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP) 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.

Journal Article↗

PubMed2005

Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation.

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

Cell Differentiation↗

PubMed2004

Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression.

A hallmark of all herpesvirus is the ability to exist in either a latent, or lytic, state of replication, enabling the lifelong infection of its host. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) can efficiently establish a latent infection in a variety of cell types in vitro, making it a valuable model for the study of latency and reactivation. To facilitate the identification of KSHV lytic replication, and allow subsequent experiments with live cells, a recombinant virus, rKSHV.219, was constructed using JSC-1 cells that expresses the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-1alpha promoter, and with the gene for puromycin resistance as a selectable marker. rKSHV.219 from JSC-1 cells was used to infect Vero cells for purification of the recombinant virus. Vero cells were also used for the production of rKSHV.219 at levels of 10(5)-10(6) infectious units (IU) of virus per milliliter using a combination of KSHV/RTA expressed from a baculovirus vector, BacK50, and butyrate. Virus produced from Vero cells was used to infect human fibroblasts (HF), 293, DU145, T24, HaCaT, and HEp-2 cells, and in all cells except 293 cells, only a latent infection was established with GFP expression, but no RFP expression. In 293 cells, 10-15% of cells showed lytic gene expression. Both primary and immortalized microvascular endothelial cells (MVEC) were also infected with rKSHV.219, and reduced spontaneous lytic replication was found in immortalized cells. In all cells used in this study, rKSHV.219 efficiently established latent infections from which the virus could be reactivated to productive lytic replication. This work also demonstrated strong synergy between KSHV/RTA and butyrate for the activation of KSHV lytic replication and the production of infectious virus.

Animals↗

PubMed2004

Reduced levels of neutralizing antibodies to Kaposi sarcoma-associated herpesvirus in persons with a history of Kaposi sarcoma.

Kaposi sarcoma-associated herpesvirus (KSHV) has been identified as the etiologic agent of Kaposi sarcoma (KS). Although KSHV is required for the development of KS, immune dysfunction is a common and important cofactor in the development of KS, as illustrated by the presence of KS in association with HIV infection or immunosuppressive-drug treatment after transplantation. Because neutralizing antibodies (NAb) constitute an important component of an antiviral immune response, we examined the functionality of the humoral immune response associated with KS, by measuring KSHV NAb titers in 3 groups of subjects. Group 1 included subjects who were KSHV positive, KS positive, and human immunodeficiency virus (HIV) positive; group 2 included subjects who were KSHV positive, KS negative, and HIV positive; and group 3 included subjects who were KSHV positive, KS negative, and HIV negative. NAb titers were significantly lower among subjects with KS, compared with subjects who were infected with KSHV but who did not have clinical evidence of KS, in a multivariate model adjusted for HIV infection and CD4 T cell count. The data from the present study suggest that NAb may play a role in the control of KSHV infection and the prevention of progression of KSHV infection to KS.

Adult↗

PubMed2003

Immunodominance among herpes simplex virus-specific CD8 T cells expressing a tissue-specific homing receptor.

The study of immunodominance within microbe-specific CD8 T cell responses has been challenging. We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides. Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen. The specificity of the resultant cytotoxic clones was determined. Clonal frequencies were compared with each other and with the total number of cytotoxic clones. For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides. Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects. Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots. We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.

Antigens, CD↗

PubMed2002

The expression of the cytomegalovirus chemokine receptor homolog US28 sequesters biologically active CC chemokines and alters IL-8 production.

We hypothesized that US28, a cytomegalovirus (CMV) CC chemokine receptor homolog, plays a role in modulating the host antiviral defense. Monocyte chemotaxis was induced by supernatants from fibroblasts infected with a US28 deletion mutant of CMV (CMV Delta US28) due to endogenously produced CC chemokines MCP-1 and RANTES. However, these chemokines were sequestered from the supernatants of CMV-infected cells that did express US28. US28 was also capable of sequestering exogenously added RANTES. Surprisingly, cells infected with CMV Delta US28 transcribed and secreted increased levels IL-8, a CXC chemokine, when compared to CMV-infected cells. Finally, because chemokines are potent mediators of immune cell migration through the endothelium, we characterized the CC chemokine binding potential of CMV-infected endothelial cells. We propose that US28 functions as a 'chemokine sink' by sequestering endogenously and exogenously produced chemokines and alters the production of the CXC chemokine IL-8, suggesting that CMV could significantly alter the inflammatory milieu surrounding infected cells.

Blotting, Northern↗