Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in infants and the elderly. Like many other pH-independent enveloped viruses, RSV is thought to enter at the cell surface, independently of common endocytic pathways. We have used a targeted small interfering RNA (siRNA) library to identify key cellular genes involved in cytoskeletal dynamics and endosome trafficking that are important for RSV infection. Surprisingly, RSV infection was potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis, including clathrin light chain. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. We conclude that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH. These findings raise the possibility that other pH-independent viruses may share a similar dependence on endocytosis for infection and provide a new potential avenue for treatment of infection.

Infection of humans with influenza

No abstract

Influenza virus infection results in altered responses of human mononuclear leukocytes to mitogen and antigen stimulation. We have previously shown (1,2) that depression of such proliferative responses to alternate (nonviral) stimuli was due to decreased monocyte-macrophage accessory cell function, with lymphocyte responsiveness preserved. It has been unclear whether altered influenza virusinfected macrophage accessory cell function was due to (a) inadequate presentation of mitogens, for example, together with class II HLA determinants; (b) inadequate production of essential cofactors, notably IL-1; (c) production of inhibitory factors, such as IL-1 inhibitors; (d) direct cell-cell inhibition; or (e) a combination of such processes. The studies reported herein were undertaken to determine whether the macrophages produce Ik-I or IL-I inhibitors after exposure to the virus.In addition, production of IL-1 and IL-1 inhibitors by respiratory syncytial virus (RSV)l-exposed human macrophages was examined. In contrast to influenza virus, infections with RSV commonly recur, despite serological evidence of immunity of the individual and the lack of clear evidence of antigenic shift or drift of the virus (2-4). RSV infection of human mononuclear leukocytes also results in depressed proliferative responses (2), but RSV differs significantly in ability to induce production of macrophage-derived immunoregulatory factors, such as IFN (2, 5). Materials and MethodsCell Collection and Separation. Mononuclear leukocytes were obtained from the peripheral blood of healthy adult donors by Ficoll-Hypaque sedimentation, and purified monocyte-derived macrophages were obtained by 24-h adherence separation of the cells in plastic culture dishes, as described previously (1, 2). Such macrophage preparations consist of 93-97% monocytes-macrophages by strict criteria for differentiation (1, 6). Unless noted otherwise, macrophages were cultured at 37°C in Eagle's MEM supplemented with 4% FCS, Hepes buffer, and penicillin (100 U/ml).

Immune function was observed for 144 weeks in 643 human immunodeficiency virus (HIV)-infected subjects who (1) had nadir CD4+ cell counts of <50 cells/mm3, followed by a sustained increase to > or =100 cells/mm3 after the initiation of HAART, and (2) were enrolled in a randomized trial of continued azithromycin prophylaxis versus withdrawal for prevention of Mycobacterium avium complex disease. The median CD4+ cell count was 226 cells/mm3 at entry and 358 cells/mm3 at week 144. Anergy (80.2% of patients) and lack of lymphoproliferative response to tetanus toxoid (TT; 73%) after immunization and impaired antibody responses after receipt of hepatitis A (54%) and TT (86%) vaccines were considered to be evidence of impaired immune reconstitution. Receipt of azithromycin did not have an effect on CD4+ cell count but was associated with higher rates of delayed-type hypersensitivity responses to TT (25% of subjects who received azithromycin vs. 15% of those who did not; P=.009) and mumps skin test antigen (29% vs. 17%; P=.001). Although the subjects had only partial responses to immune function testing, the rate of opportunistic infections was very low, and none of the tests was predictive of risk.

This study examined the effects of nitrogen dioxide (NO(2)) exposure on airway inflammation, blood cells, and antiviral respiratory defense. Twenty-one healthy volunteers were exposed on separate occasions to air and 0.6 and 1.5 ppm NO(2) for 3 h with intermittent moderate exercise. Phlebotomy and bronchoscopy were performed 3.5 h after each exposure, and recovered cells were challenged with respiratory viruses in vitro. Blood studies revealed a 4.1% NO(2) dose-related decrease in hematocrit (P = 0.003). Circulating total lymphocytes (P = 0.024) and T lymphocytes (P = 0.049) decreased with NO(2) exposure. Exposure to NO(2) increased the blood lymphocyte CD4(+)-to-CD8(+) ratio from 1.74 +/- 0.11 to 1.85 +/- 0.12 in males but decreased it from 1.88 +/- 0.19 to 1.78 +/- 0.19 in females (P < 0.001 for gender difference). Polymorphonuclear leukocytes in bronchial lavage increased with NO(2) exposure (P = 0.003). Bronchial epithelial cells obtained after exposure to 1.5 ppm NO(2) released 40% more lactate dehydrogenase after challenge with respiratory syncytial virus than with air exposure (P = 0.024). In healthy subjects, exposures to NO(2) at levels found indoors cause mild airway inflammation, effects on blood cells, and increased susceptibility of airway epithelial cells to injury from respiratory viruses.

Monocyte and lymphocyte surface-expressed viral antigens have been demonstrated after exposure of unseparated human mononuclear leukocytes to influenza virus in vitro. The current studies, using [35S]methionine pulse-labeled purified preparations of virus-exposed macrophages, depleted of lymphocytes, demonstrate that the presence of these viral proteins does represent new synthesis. However, purified lymphocytes, depleted of monocytes-macrophages and exposed to influenza virus, showed no detectable viral protein synthesis. In further experiments, unseparated mononuclear leukocytes were exposed to virus and subsequently separated by countercurrent centrifugal elutriation. Both macrophages and lymphocytes were then shown to synthesize influenza proteins. Cell-free control or influenza virus-infected macrophage-derived supernatant fluids did not facilitate influenza virus infection of the lymphocytes. The data suggest that macrophages are required for influenza virus infection of human lymphocytes, and raise the possibility that macrophage facilitation of an abortive infection of lymphocytes plays a role in the generation of effective immunity to viral antigens.

Recurrent infections with respiratory syncytial virus (RSV) have been well documented despite serological evidence of prior exposure of the host and the absence of clear evidence of antigenic variation of the virus. Therefore, human mononuclear leukocytes, as well as purified lymphocytes and monocytes-macrophages, were exposed to RSV in vitro and examined for expression of viral antigens by using indirect immunofluorescence with monoclonal antibodies to RSV. RSV infected both human monocytes-macrophages and lymphocytes in vitro. RSV infection resulted in both a decrease in the number of T helper phenotype cells and an increase in T suppressor phenotype cells. RSV proteins were disproportionately expressed by atypical or lymphoblastoid cells, many of which were of the T suppressor phenotype. Circulating mononuclear leukocytes obtained from symptomatic children infected with RSV frequently expressed viral antigens. Viral antigens appeared to be detected more frequently in cells from the younger subjects. The findings suggest that initial or early RSV infections in children include infection of circulating immunocompetent cells. It remains to be determined whether the described RSV-induced alterations in lymphocyte subpopulations contribute to recovery from and/or recurrence of RSV infections.