Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus (SARS-CoV) strain. Analyses of T cell repertoires in health care workers who survived SARS-CoV infection during the 2003 outbreak revealed that their effector memory V gamma 9V delta 2 T cell populations were selectively expanded ~3 months after the onset of disease. No such expansion of their alpha beta T cell pools was detected. The expansion of the V gamma 9V delta 2 T cell population was associated with higher anti-SARS-CoV immunoglobulin G titers. In addition, in vitro experiments demonstrated that stimulated V gamma 9V delta 2 T cells display an interferon- gamma -dependent anti-SARS-CoV activity and are able to directly kill SARS-CoV-infected target cells. These findings are compatible with the possibility that V gamma 9V delta 2 T cells play a protective role during SARS.

Molecular beacons are dual-labelled probes that are typically used in real-time PCR assays, but have also been conjugated with solid matrices for use in microarrays or biosensors. We have developed a fluid array system using microsphere-conjugated molecular beacons and the flow cytometer for the specific, multiplexed detection of unlabelled nucleic acids in solution. For this array system, molecular beacons were conjugated with microspheres using a biotin-streptavidin linkage. A bridged conjugation method using streptavidin increased the signal-to-noise ratio, allowing for further discrimination of target quantitation. Using beads of different sizes and molecular beacons in two fluorophore colours, synthetic nucleic acid control sequences were specifically detected for three respiratory pathogens, including the SARS coronavirus in proof-of-concept experiments. Considering that routine flow cytometers are able to detect up to four fluorescent channels, this novel assay may allow for the specific multiplex detection of a nucleic acid panel in a single tube.

Lymphocytic choriomeningitis virus (LCMV) induces type I interferon (alpha and beta interferon [IFN-␣ and IFN-␤]) upon infection and yet is sensitive to the addition of type II interferon (gamma interferon[Arenaviruses can replicate without significantly impacting the host or causing cytopathic effects. The arenavirus replication complex contains the viral genomic single-stranded RNA segments, nucleocapsid protein (NP), an RNA-dependent RNA polymerase (RdRp or L protein), and a small zinc-binding protein (Z) (17). Cellular proteins are also involved in viral replication (3,4,12). Here we describe the inhibitory influence of the promyelocytic leukemia protein (PML) that coprecipitates and colocalizes with cell-associated arenavirus complexes (2). PML is an oncoprotein that is expressed primarily in myeloid, epithelial, and endothelial cells, all infectable by arenaviruses and important in the pathogenesis of arenaviral hemorrhagic fevers. PML is induced by the alpha/beta interferons (IFN-␣/␤) acting on the ISRE and GAS promoter response elements (5,13,20). Interferons IFN-␣ and IFN-␤ are produced by many cell types upon viral infection, and IFN-␥ is produced in T lymphocytes or natural killer cells in response to antigens (16). IFNs are known for their inhibitory effects on cellular proliferation, and PML, as an effector of this function, is capable of suppressing cell proliferation (11,22,24).IFNs are also known for their antiviral effects. There are 50 to 100 IFN-inducible genes and several of them have antiviral activity, e.g., the p68 protein kinase, the 2Ј,5Ј-oligoadenylate synthetase (OAS), and certain Mx family proteins (19,20,23). The IFN-inducible PML has also recently been shown to have antiviral activity. In the absence of IFN, overexpression of PML diminishes infection by vesicular stomatitis virus (VSV) and influenza A virus, without affecting infection by encephalomyocarditis virus (EMCV), a virus known to be IFN resistant (6).Coimmunoprecipitation studies show specific interaction between PML and Z proteins of LCMV and Lassa fever virus, a related arenavirus. Genetically engineered mutations in PML were used to show that the Z protein binds the N-terminal region of PML, and this domain of PML, unlike the PML RING or the nuclear localization signal, is essential for colocalization of Z and PML (2). The work presented here demonstrates that PML expression diminishes LCMV expression, possibly through its interaction with the LCMV Z protein.PML and LCMV affect proliferation of MEF. The effects of PML expression on cell proliferation were examined in earlypassage mouse embryonic fibroblasts (MEFs) (22; this study). Fibroblasts lacking PML (PML Ϫ/Ϫ) grew faster and achieved higher cell densities than wild-type (PML ϩ/ϩ) cells and yet their cultures were morphologically indistinguishable. IFN treatment, which increases PML expression, reduces cell growth rates even more in both PML ϩ/ϩ and Ϫ/Ϫ fibroblasts. Infection with LCMV shortens the life of both MEF cultures approximately twofold (P Ͻ 0.05) (Fig. 1)...