Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Abstract

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

Duplex One-Step RT-qPCR Assays for Simultaneous Detection of Genomic and Subgenomic RNAs of SARS-CoV-2 Variants

“…Passage 3 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Coronaviridae: Sarbecovirus ) Washington strain (SARS-CoV-2/human/USA/WA-CDC-02982586-001/2020; GenBank #MN985325) was obtained from the CDC, Atlanta, GA, USA. SARS-CoV-2 Washington strain was passaged once at the Integrated Research Facility at Fort Detrick (IRF-Frederick) in Vero cells for 72 h. The resulting virus stock (SARS-CoV-2/human/USA/WA-CDC-WA1/2020 [IRF-0394]; GenBank #MW161259) was titrated by plaque assay using Vero E6 cells (ATCC #CRL-1586) cultured in DMEM media with 10% FBS, as previously described [ 38 ]. Subsequently, under identical growth conditions and media, Vero cells were exposed to the IRF-0394 virus stock at a multiplicity of infection (MOI) of 0.01 to generate working stocks (SARS-CoV-2/human/USA/WA-CDC-WA1/2020 [IRF-0399]; GenBank #MT952134 and SARS-CoV-2/human/USA/WA-CDC-WA1/2020 [IRF-0449]; GenBank #OK091601).…”

“…Plaques were then counted on a lightbox. Viral titers were calculated as plaque-forming units (PFU) per mL [ 38 ].…”

Quantification of virus-infected cells using RNA FISH-Flow

“… 3 N/A Recombinant DNA Wild-type recombinant SHFV (rSHFV)-encoding cDNA-launch plasmid Cai et al. 6 N/A rSHFV expressing enhanced green fluorescent protein (rSHFV-eGFP)-encoding cDNA-launch plasmid Cai et al. 6 N/A Chemicals, peptides, and recombinant proteins Eagle’s minimum essential medium (EMEM) ATCC Cat# 30-2003 10% fetal bovine serum (FBS) MilliporeSigma Cat# TMS-013-B 1× penicillin-streptomycin solution (Pen Strep) Avantor Cat# 45000-652 32% paraformaldehyde (PFA) VWR Cat# 100504-858 100% ice-cold ethanol (EtOH) Thermo Fisher Scientific Cat# 04-355-223 5-propargylamino-2′,3′-dideoxyuridine-5′-triphosphate (ddUTP) ATTO-633 Jena Bioscience Cat# NU-1619-633 5-propargylamino-2′,3′-dideoxyuridine-5′-triphosphate (ddUTP) ATTO-550 Jena Bioscience Cat# NU-1619-550 5-propargylamino-2′,3′-dideoxyuridine-5′-triphosphate (ddUTP) ATTO-488 Jena Bioscience Cat# NU-1619-488 Phosphate-buffered saline (PBS) Caisson Labs Cat# PBL01-6X500ML Terminal deoxynucleotidyl transferase (TdT) (20 U/μL) Thermo Fisher Scientific Cat# EP0161 5× Reaction buffer Thermo Fisher Scientific Cat# EP0161 Sodium acetate 3M (CH₃COONa; NaOAc) Amresco Cat# E498-200ML Dextran sulfate 50% solution MilliporeSigma Cat# S4030 Deionized formamide MilliporeSigma Cat# S4117 20× nuclease-free saline-sodium citrate (SSC) Thermo Fisher Scientific Cat# AM9770 Trypsin ethylenediaminetetraacetic acid (EDTA) Corning Incorporated Cat# 25-052-Cl <...…”

“…To achieve this, we first transfected cells with a recombinant SHFV expressing eGFP (rSHFV-eGFP)-encoding cDNA-launch plasmid. 6 Then we measured the percent of vRNA-expressing cells by RNA FISH-Flow and compared that to the percent of eGFP-positive cells enumerated by flow cytometry ( Figure 6 A). In rSHFV-eGFP transfected “producer” cells, there were no differences between the percentages of eGFP or vRNA-positive cells ( Figure 6 B).…”

“…From these data, we conclude that RNA FISH-Flow is a suitable alternative to fluorescent reporter viruses in rapidly measuring infection kinetics by flow cytometry.

Figure 6 Comparison of RNA fluorescence in situ hybridization flow cytometry (RNA FISH-Flow) and standard flow cytometry using a recombinant infectious SHFV reporter virus clone (A) Schematic of the experimental setup to compare virus-encoded eGFP and RNA FISH-Flow measurements using cells transfected with a recombinant simian hemorrhagic fever virus expressing eGFP (rSHFV-eGFP)-encoding cDNA launch plasmid 6 (producer cells) and cells infected with rSHFV-eGFP (infected cells). (B and C) (B) MA-104 cells were transfected with rSHFV-eGFP-encoding plasmid or (C) exposed to rSHFV-eGFP and then processed for RNA FISH-Flow 72 h after plasmid transfection or 24 h after virus exposure.

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