Construction of Mouse Adenovirus Type 1 Mutants

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Interleukin-1␤ (IL-1␤), an inflammatory cytokine and IL-1 receptor ligand, has diverse activities in the brain. We examined whether IL-1 signaling contributes to the encephalitis observed in mouse adenovirus type 1 (MAV-1) infection, using mice lacking the IL-1 receptor (Il1r1 Ϫ/Ϫ mice). Il1r1 Ϫ/Ϫ mice demonstrated reduced survival, greater disruption of the blood-brain barrier (BBB), higher brain viral loads, and higher brain inflammatory cytokine and chemokine levels than control C57BL/6J mice. We also examined infections of mice defective in IL-1␤ production (Pycard Ϫ/Ϫ mice) and mice defective in trafficking of Toll-like receptors to the endosome (Unc93b1 Ϫ/Ϫ mice). Pycard Ϫ/Ϫ and Unc93b1 Ϫ/Ϫ mice showed lower survival (similar to Il1r1 Ϫ/Ϫ mice) than control mice but, unlike Il1r1 Ϫ/Ϫ mice, did not have increased brain viral loads or BBB disruption. Based on the brain cytokine levels, MAV-1-infected Unc93b1 Ϫ/Ϫ mice had a very different inflammatory profile from infected Il1r1 Ϫ/Ϫ and Pycard Ϫ/Ϫ mice. Histological examination demonstrated pathological findings consistent with encephalitis in control and knockout mice; however, intranuclear viral inclusions were seen only in Il1r1 Ϫ/Ϫ mice. A time course of infection of control and Il1r1 Ϫ/Ϫ mice evaluating the kinetics of viral replication and cytokine production revealed differences between the mouse strains primarily at 7 to 8 days after infection, when mice began succumbing to MAV-1 infection. In the absence of IL-1 signaling, we noted an increase in the transcription of type I interferon (IFN)-stimulated genes. Together, these results indicate that IL-1 signaling is important during MAV-1 infection and suggest that, in its absence, increased IFN-␤ signaling may result in increased neuroinflammation. IMPORTANCEThe investigation of encephalitis pathogenesis produced by different viruses is needed to characterize virus and host-specific factors that contribute to disease. MAV-1 produces viral encephalitis in its natural host, providing a good model for studying factors involved in encephalitis development. We investigated the role of IL-1 signaling during MAV-1-induced encephalitis. Unexpectedly, the lack of IL-1 signaling increased the mortality and inflammation in mice infected with MAV-1. Also, there was an increase in the transcription of type I IFN-stimulated genes that correlated with the observed increased mortality and inflammation. The findings highlight the complex nature of encephalitis and suggests that IL-1 has a protective effect for the development of MAV-1-induced encephalitis.

Section: Methodsmentioning

confidence: 99%

A Protective Role for Interleukin-1 Signaling during Mouse Adenovirus Type 1-Induced Encephalitis

Castro-Jorge

Pretto

Smith

et al. 2017

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“…Wild-type MAV-1 was propagated and titrated in 3T6 cells (9). The mice used are indicated in Table 1 (11,14,23,24,(29)(30)(31).…”

Section: Methodsmentioning

confidence: 99%

“…Organ homogenates were prepared as described previously (40) or in phosphate-buffered saline (PBS) with 1-mm glass beads (BioSpec Products, Bartlesville, Okla.) in 2 ml/well 96-well plates (Axygen, Union City, Calif.) with a Mini Beadbeater (BioSpec) and the manufacturer's protocol. Virus was titrated by plaque assay as described previously (9). The means of the log titers were compared by a two-tailed t test.…”

Section: Methodsmentioning

confidence: 99%

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Fatal Disseminated Mouse Adenovirus Type 1 Infection in Mice Lacking B Cells or Bruton's Tyrosine Kinase

Moore

McKissic

Brown

et al. 2004

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Human adenoviruses are associated with self-limiting respiratory, conjunctival, and gastrointestinal disease. In immunocompromised people, human adenovirus infection can result in pneumonia, hepatitis, encephalitis, pancreatitis, gastroenteritis, or disseminated disease involving multiple organs (8, 43). Disseminated human adenovirus infection usually results in death. The incidence of disseminated human adenovirus disease is increasing with the increased number of immunocompromised children, and pediatric bone marrow transplant recipients are most at risk (5,12,13,16).Due to species specificity, human adenovirus pathogenesis is poorly understood. Study of mouse adenovirus type 1 (MAV-1) permits the analysis of a replicating adenovirus in vivo. The outcome of infection depends on the virus dose and mouse strain (15,26,33,40). In outbred and C57BL/6 (B6) mouse strains, MAV-1 infects cells of the monocyte-macrophage lineage and endothelial cells (10,20,33). The highest levels of virus are found in the spleen and brain (20,26,39). MAV-1-specific cytotoxic T cells peak at 10 days postinfection (d.p.i.) and then decline (18). T cells cause acute immunopathology and are required for survival 9 to 16 weeks postinfection in MAV-1-induced encephalomyelitis (33). Inbred mouse strains susceptible and resistant to MAV-1 are available, and sublethal irradiation of resistant mice renders them susceptible (40). Mice with a severe combined immunodeficiency (SCID) mutation are susceptible to MAV-1 (10, 35).Here we report findings that survival of acute MAV-1 infection is B-cell dependent and T-cell independent. We postulated that Bruton's tyrosine kinase (Btk) plays a role in protection from MAV-1. Loss of Btk in mice results in the X-linked immunodeficiency (Xid) phenotype (22). Btk Ϫ/Ϫ mice have reductions in serum immunoglobulin (natural antibody), conventional B cells, and peritoneal B-1 cells relative to control mice (22). Here we demonstrate that Btk is required for survival of MAV-1 infection. We present data indicating that early T-cell-independent antiviral immunoglobulin M (IgM) plays a pivotal role in protection against disseminated MAV-1 infection. MATERIALS AND METHODSVirus and mice. Wild-type MAV-1 was propagated and titrated in 3T6 cells (9). The mice used are indicated in Table 1 (11,14,23,24,(29)(30)(31). A ␤ bϪ/Ϫ and Jh mice were purchased from Taconic. C57BL/6NCr (B6) and BALB/CAnNCr mice were purchased from the National Cancer Institute. All other mice were purchased from Jackson Laboratory. The 50% lethal dose (LD 50 ) was determined as described previously (37). Sera were heat inactivated at 57°C for 45 min before passive transfer.Quantitation of virus. Organ homogenates were prepared as described previously (40) or in phosphate-buffered saline (PBS) with 1-mm glass beads (BioSpec Products, Bartlesville, Okla.) in 2 ml/well 96-well plates (Axygen, Union City, Calif.) with a Mini Beadbeater (BioSpec) and the manufacturer's protocol. Virus was titrated by plaque assay as described previously (9). The means of...

“…Sur2 ϩ/ϩ and Sur2 Ϫ/Ϫ MEFs were infected with wild-type MAV-1 at a multiplicity of infection (MOI) of 0.05, 0.1, 1, or 5 or with mouse gammaherpesvirus 68 at an MOI of 0.01. MAV-1 plaque assays were carried out on 3T6 cells as described previously (18). Briefly, cells were harvested at various times post infection by scraping in their medium.…”

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confidence: 99%

Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

Fang

Stevens

Berk

et al. 2004

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Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) encodes a virulence gene in viral infection of mice. To broaden our understanding of the functions of E1A in MAV-1 pathogenesis, an unbiased experimental approach, glutathione S-transferase (GST) pulldown, was used to screen for cellular proteins that interact with E1A protein. We identified mouse Sur2, a subunit of Mediator complex, as a protein that binds to MAV-1 E1A. The interaction between Sur2 and MAV-1 E1A was confirmed in virus-infected cells. Conserved region 3 (CR3) of MAV-1 E1A was mapped as the region required for Sur2-E1A interaction, as is the case for human adenovirus E1A. Although it has been proposed that human adenovirus E1A recruits the Mediator complex to transactivate transcription of viral early genes, Sur2 function in adenovirus replication has not been directly tested previously. Studies on the functions of Sur2 with mouse embryonic fibroblasts (MEFs) showed that there was a multiplicity-dependent growth defect of MAV-1 in Sur2 ؊/؊ MEFs compared to Sur2 ؉/؉ MEFs. Comparison of the viral DNA and viral mRNA levels in Sur2 ؉/؉ and Sur2 ؊/؊ MEFs confirmed that Sur2 was important for efficient viral replication. The viral replication defects in Sur2 ؊/؊ MEFs appeared to be due at least in part to a defect in viral early gene transcription.