Primary Human Macrophages Serve as Vehicles for Vaccinia Virus Replication and Dissemination

Abstract

Human monocytic and professional antigen-presenting cells have been reported only to exhibit abortive infections with vaccinia virus (VACV). We found that monocyte-derived macrophages (MDMs), including granulocyte macrophage colony-stimulating factor (GM-CSF)-polarized M1 and macrophage colony-stimulating factor (M-CSF)-polarized M2, but not human AB serumderived cells, were permissive to VACV replication. The titers of infectious virions in both cell-free supernatants and cellular lysates of infected M1 and M2 markedly increased in a time-dependent manner. The majority of virions produced in permissive MDMs were extracellular enveloped virions (EEV), a secreted form of VACV associated with long-range virus dissemination, and were mainly found in the culture supernatant. Infected MDMs formed VACV factories, actin tails, virion-associated branching structures, and cell linkages, indicating that MDMs are able to initiate de novo synthesis of viral DNA and promote virus release. VACV replication was sensitive to inhibitors against the Akt and Erk1/2 pathways that can be activated by VACV infection and M-CSF stimulation. Classical activation of MDMs by lipopolysaccharide (LPS) plus gamma interferon (IFN-␥) stimulation caused no effect on VACV replication, while alternative activation of MDMs by interleukin-10 (IL-10) or LPS-plus-IL-1␤ treatment significantly decreased VACV production. The IL-10-mediated suppression of VACV replication was largely due to Stat3 activation, as a Stat3 inhibitor restored virus production to levels observed without IL-10 stimulation. In conclusion, our data demonstrate that primary human macrophages are permissive to VACV replication. After infection, these cells produce EEV for long-range dissemination and also form structures associated with virions which may contribute to cell-cell spread. O rthopoxvirus, a genus of the subfamily Chordopoxvirinae of the family Poxviridae, is composed of large DNA viruses that infect a wide array of mammalian species. The most famous member of the genus is the human-specific variola virus, which causes smallpox. In nature, variola virus has a strict human-specific tropism, and nonhuman reservoirs of the virus have never been found. Variola virus is generally transmitted via inhalation, with subsequent infection and replication in epithelial cells of the oral and respiratory mucosa. The next stage of infection involves viral infiltration of lymphoid organs accompanied by strong viremia and skin lesions. Recent studies using high doses of variola virus to infect Cynomolgus macaques in an attempt to develop an animal model of smallpox have demonstrated that infected animals develop systemic infection and hemorrhagic symptoms (1, 2). These symptoms were correlated with monocyte/macrophage-mediated viremia and dissemination (1, 2). In mice, macrophages are crucial to control the infection of the orthopoxvirus ectromelia virus (ECTV) (3, 4). However, ECTV replicates in macrophages (5) and directly contributes to dissemination within the host (6). Giv...

IL-1R Type 1–Deficient Mice Demonstrate an Impaired Host Immune Response against Cutaneous Vaccinia Virus Infection

“…In primary human macrophages in vitro , the cells are permissive to VACV infection and can be a vehicle for VACV replication and dissemination. These macrophages can produce extracellular enveloped virions for long-range VACV dissemination and form virion-associated structures contributing to cell-cell spread (36). Although the co-expression of CD11b and F4/80 has been often been used to identify macrophage-lineage cells, macrophages cannot be accurately distinguished from conventional DCs (CD11b + DC) and mo-DCs in skin.…”

Analysis of MHC Class I Processing Pathways That Generate a Response to Vaccinia Virus Late Proteins

“…Viruses VACV (strain WR) was grown and titrated using 143B TK 2 cells, screened regularly for the presence of adventitious pathogens, as previously described (36). Recombinant VACV (rVACV) expressing GFP driven by the early/late p7.5 promoter (VACV-p7.5 GFP) or the late p11 promoter (VACV-p11 GFP) were described previously (31). For titrations of VACV from infected BMDC, cells were lysed at the time points indicated and titrated on 142B TK 2 cells as above.…”

“…The inability of late VACV promoter-driven Ag to stimulate T CD8+ responses has been correlated to an abortive in vitro infection of pAPC, in which late Ags are not produced and so direct presentation cannot occur (27)(28)(29)(30). However, some publications have indicated that VACV can replicate and spread following infection of APC (31), indicating either that all late genes required for productive replication can be produced within infected APC populations or that some APC populations may allow late VACV gene transcription. Therefore, the possibility exists that immunogenic late VACV Ags are primed by VACV-infected DC that directly present Ag.…”

Glycosylphosphatidylinositol Anchor Deficiency Attenuates the Production of Infectious HIV-1 and Renders Virions Sensitive to Complement Attack

“…Signals were sequentially collected using single fluorescence excitation and acquisition settings to avoid crossover. [45][46][47] Images were processed and analyzed using FV10-ASW 3.0 Viewer software (Olympus America, Center Valley, PA) or ImageJ 1.44p (NIH, Bethesda, MD).…”