Use of Single-Cycle Infectious Lymphocytic Choriomeningitis Virus To Study Hemorrhagic Fever Arenaviruses

Abstract

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMV⌬GP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMV⌬GP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMV⌬GP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMV⌬GP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

“…1A, lane 4), which correlated with the relative intracellular viral RNA levels in the HCV-and LCMV-infected cells (Table 1). Interestingly, similar amounts of IFN-␣ were produced in pDC cocultures with cells infected with a single-cycle recombinant LCMV (scrLCMV⌬GP/GFP [33]) that cannot produce infectious virus (Fig. 1A, lane 6), suggesting that production of LCMV infectious progeny was not required to trigger IFN-␣ production by the pDCs.…”

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

“…MDCK cells constitutively expressing HA (MDCK-HA) from the pandemic H1N1 virus A/California/4_NYICE_E3/2009 (pH1N1/E3) were previously described (4). MDCK-HA cells stably expressing HA from influenza virus A/Puerto Rico/8/1934 (H1N1; PR8), B/Lee, influenza virus B/Victoria/1/1987 (B/ Vic), and influenza virus B/Yamagata/16/1988 (B/Yam) were generated by cotransfecting each pCAGGS HA plasmid and pCB7 (3:1 ratio) for eukaryotic expression of HA and hygromycin B resistance, respectively (4,26,29). HA-expressing cells were grown in DMEM-10% FBS-1% PSG supplemented with 200 g/ml hygromycin B (Corning).…”

Inhibition of Arenavirus by A3, a Pyrimidine Biosynthesis Inhibitor

“…At the indicated times postinfection, viral titers in TCS were determined. For LCMV and Candid#1, titers were determined based on immunofocus centers on Vero cells as described previously (37). For r3LCMV/CAT-GFP, r3LCMV/GFP-Gluc, and r3Candid#1/CAT-GFP, virus titers were determined on Vero cells based on green fluorescent protein (GFP) expression, which was directly visualized by fluorescence microscopy at 48 h.p.i (36).…”

“…Uracil and orotic acid stocks were prepared at 50 mM in 0.1 M NaOH. Ribavirin was prepared at 4 mM in distilled water, and A3 was prepared at 100 mM in dimethyl sulfoxide (DMSO) (32,37).…”