Paul Lewis

Human immunodeficiency virus infection of cells arrested in the cell cycle.

Cell proliferation is necessary for proviral integration and productive infection of most retroviruses. Nevertheless, the human immunodeficiency virus (HIV) can infect non‐dividing macrophages. This ability to grow in non‐dividing cells is not specific to macrophages because, as we show here, CD4+ HeLa cells arrested at stage G2 of the cell cycle can be infected by HIV‐1. Proliferation is necessary for these same cells to be infected by a murine retrovirus, MuLV. HIV‐1 integrates into the arrested cell DNA and produces viral RNA and protein in a pattern similar to that in normal cells. In addition, our data suggest that the ability to infect non‐dividing cells is due to one of the HIV‐1 core virion proteins. HIV infection of non‐dividing cells distinguishes lentiviruses from other retroviruses and is likely to be important in the natural history of HIV infection.

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes : Expression of archival virus and replication of virus

ABSTRACT Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.

Cell-Free Human Immunodeficiency Virus Type 1 in Breast Milk

Breast-feeding may be an important route of human immunodeficiency virus type 1 (HIV-1) vertical transmission in settings where it is routinely practiced. To define the prevalence and quantity of HIV-1 in cell-free breast milk, samples from HIV-1-seropositive women were analyzed by quantitative competitive reverse transcription-polymerase chain reaction (QC-RT-PCR). HIV-1 RNA was detected in 29 (39%) of 75 specimens tested. Of these 29 specimens, 16 (55%) had levels that were near the detection limit of the assay (240 copies/mL), while 6 (21%) had >900 copies/mL. The maximum concentration of HIV-1 RNA detected was 8100 copies/mL. The prevalence of cell-free HIV-1 was higher in mature milk (47%) than in colostrum (27%, P = 0.1). Because mature milk is consumed in large quantities, these data suggest that cell-free HIV-1 in breast milk may contribute to vertical transmission of HIV-1.

A Community-Based Outbreak of Severe Respiratory Illness Caused by Human Adenovirus Serotype 14

BACKGROUNDnHuman adenoviruses (Ads) typically cause mild illnesses in otherwise healthy hosts. We investigated a community-based outbreak that had substantial morbidity caused primarily by Ad14, an uncommon serotype.nnnMETHODSnWe retrospectively reviewed the medical records of all patients with confirmed cases of Ad infection from 1 November 2006 through 31 July 2007 in Oregon. Isolates were typed by sequencing. We analyzed clinical and laboratory variables to identify risk factors for severe Ad14 disease.nnnRESULTSnAd14 first emerged in Oregon in 2005. Of 67 cases of Ad infection detected during the study period, 40 (60%) involved Ad14. Most of the 38 Ad14-infected patients who had medical records available for review presented with fever and cough; 29 (76%) required hospitalization, 23 (61%) required supplemental oxygen, 18 (47%) required critical care, 9 (24%) required vasopressors, and 7 (18%) died. Lobar infiltrates on chest radiographs suggestive of bacterial pneumonia were common among those needing hospitalization. Older age, chronic underlying condition, low absolute lymphocyte counts, and elevated creatinine levels were associated with severe illness. Except for 1 case of possible hospital transmission, we identified no epidemiological links among patients.nnnCONCLUSIONnAd14 emerged in Oregon in 2005 and became the predominant circulating type by 2007. Infection with this uncommon virus was primarily associated with a community-acquired pneumonia syndrome and caused substantial morbidity and mortality.

Multiple Viral Genetic Analyses Detect Low-Level Human Immunodeficiency Virus Type 1 Replication during Effective Highly Active Antiretroviral Therapy

ABSTRACT To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by ≥0.9 log10, resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.

Studies of human immunodeficiency virus type 1 mucosal viral shedding and transmission in Kenya

If human immunodeficiency virus type 1 (HIV-1) vaccines are to be highly effective, it is essential to understand the virologic factors that contribute to HIV-1 transmission. It is likely that transmission is determined, in part, by the genotype or phenotype (or both) of infectious virus present in the index case, which in turn will influence the quantity of virus that may be exchanged during sexual contact. Transmission may also depend on the fitness of the virus for replication in the exposed individual, which may be influenced by whether a virus encounters a target cell that is susceptible to infection by that specific variant. Of interest, our data suggest that the complexity of the virus that is transmitted may be different in female and male sexual exposures.

Human Immunodeficiency Virus (HIV) Type 1 Vpu Induces the Expression of CD40 in Endothelial Cells and Regulates HIV-Induced Adhesion of B-Lymphoma Cells

ABSTRACT AIDS-related B-cell non-Hodgkins lymphoma (AIDS-NHL) is a significant cause of morbidity and mortality among individuals infected with human immunodeficiency virus type 1 (HIV-1). AIDS-NHL is clinically and histologically heterogeneous, but common features include an aggressive clinical course and frequent extranodal presentation. HIV-1 infection of nonimmune cells that interact with malignant B cells at extranodal sites may influence both the development and the clinical presentation of disease. Our previous studies have shown that coculture of B-lymphoma (BL) cells with HIV-1-infected endothelial cells (EC) leads to contact activation of EC and firm BL-cell adhesion. The key event promoting EC-BL-cell adhesion was HIV-1 upregulation of endothelial CD40, which allowed induction of vascular cell adhesion molecule 1 (VCAM-1) in a CD40-dependent manner. The present study was designed to identify the HIV-1 protein(s) that influence EC-BL-cell adhesion. When HIV-1 proteins were individually expressed in EC by using recombinant adenoviruses, cultured BL cells adhered exclusively to Vpu-transduced EC. As with HIV-infected EC, adhesive properties were linked to the capacity of Vpu to upregulate CD40, which in turn allowed efficient expression of VCAM-1. When EC were infected with an HIV-1 pseudotype lacking the Vpu gene, CD40 upregulation and BL-cell adhesive properties were lost, indicating an essential role for Vpu in EC-BL-cell interactions. Thus, these data reveal a novel function for HIV-1 Vpu and further suggest a role for Vpu in the development of AIDS-NHL at EC-rich extranodal sites.

Correction of deep anterior overbite A report of three cases

The maintenance of deep overbite correction is one of the criteria by which we judge the long-term success of orthodontic treatment. To correct and maintain an excessive overbite, the orthodontist must intrude the overerupted teeth and establish a more ideal interincisal angle. The mechanical means to achieve this have been discussed. Three case reports have been presented to illustrate that attention to these details during treatment is of importance in achieving long term results.

Duration of Shedding and Secondary Household Transmission of Shiga Toxin–Producing Escherichia coli O26 During an Outbreak in a Childcare Center, Oregon, October–December 2010

We assessed shedding duration and secondary household transmission of Shiga toxin 1-positive Escherichia coli O26 during a childcare-associated outbreak. No severe illness was noted. Shedding duration was 15-46 days (median, 29). No secondary transmission to household members was identified. Value of isolating asymptomatic infected children with this low-virulence infection remains uncertain.

Influence of HIV-1 on induced expression of granulopoietic factors: The role of HIV-1 vpr

Abstract IL-1-induced expression of granulopoietic factors, including G-CSF, by human bone marrow stromal cells is suppressed by HIV-1 infection, a phenomenon that may account for the regenerative granulopoietic failure often seen in HIV-1 infected patients. Seeking to identify the HIV-1 gene products that account for stromal cell dysfunction, we used replication incompetent HIV-1 mutants to demonstrate that deletion of vpr abrogates the stromal cell defect. Bone marrow stromal cell cultures were established in primaria dishes containing VEGF and were allowed to grow to near confluence. HIV-1 pseudotyped with VSV-G envelope protein was produced by transient co-transfection of 293T cells with a VSV-G expression vector and HIV-1 proviral plasmids containing: (1) a deletion in the envelope gene (env-) or (2) deletions of both env and vpr. Expression of G-CSF, GM-CSF, and IL-6 by stromal cells expressing the env deleted mutant was reduced by more than 50%, but this inhibitory effect was largely relieved by mutants with deletions of both env and vpr. To confirm the vpr effect, we carried out gain-of-function studies using bone marrow stromal cells transduced with the Moloney based expression vector pSLX-CMV- vpr or vector control pSLX-CMVX. IL-6 and G-CSF expression was reduced in stromal cells expressing vpr. We conclude that blunted responses of HIV-1 infected hematopoietic stromal cells depends, at least in part, upon the expression of HIV-1 vpr and that, in the case of G-CSF and IL-6, vpr is sufficient to account for this effect.