Vero-E6, BSR-T7/5, and A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum, non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Huh7 cells were cultured in DMEM supplemented with 5% (v/v) fetal calf serum, non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.

LASV strain Josiah (henceforth called Josiah) is an isolate obtained from the serum of a 40-year-old male admitted to Songo hospital (Sebgwema, Sierra Leone) with LF in 1976. LASV strain 812673-LBR-USA-2015 (henceforth called NJ2015) is an isolate obtained from a 55-year-old male who succumbed to LF upon returning to New Jersey, USA, from Liberia in 2015. Infectious recombinant Josiah (rJosiah) was rescued as previously described (Albariño et al., 2011). Similarly, we generated recombinant NJ2015 (rNJ2015) by cloning full-length antigenomic sense copies of the NJ2015 S and L genome segments into T7 transcription plasmids. To create the reassortant recombinant rJos-S/NJ-L and rNJ-S/Jos-L variants, we alternated the S and L segment rescue plasmids used. All recombinant viruses were rescued in BSR-T7/5 cells and passaged twice in Vero-E6 cells. Viral titers were determined in Vero-E6 cells by immunofluorescence assays using anti-LASV polyclonal antibodies (in house, #SPR628), with TCID₅₀ values calculated using the method of Reed and Muench (Reed and Muench, 1938). Sequences for rJosiah (HQ688673.1, HQ688675.1) and rNJ2015 (MG 812650, MG812651) have been deposited in Genbank.

Josiah S segment minigenome assays were performed, as previously described, by replacing the NP and GPC coding sequences (CDS) on the Josiah S segment with those for ZsGreen1 (ZsG) and Gaussia luciferase (gLuc), respectively (Welch et al., 2016). A new Josiah L segment-based minigenome was generated, replacing the L and Z CDS with those for ZsG and gLuc, respectively. Analogous NJ2015 S and L segment-based minigenomes and support plasmids expressing NJ2015 NP and L were also created. The 5′ and 3′ non-coding regions (NCR) and the intergenic regions were unaltered in all constructs. Cells transfected with minigenome segments and support plasmids supplying LASV NP and L proteins produced quantifiable ZsG and gLuc expression. All minigenome reactions were performed in conjunction with a negative control (no polymerase, just pCAGGS empty plasmid) to assess background levels of minigenome activity in the absence of viral transcriptional machinery. ZsG fluorescence or gLuc expression (Renilla Luciferase Assay System, Promega) was quantified 72 or 48 h post-transfection, respectively, on a BioTek Synergy microplate reader.

LASV sequences were obtained from both NCBI Genbank and isolates contained in the Centers for Disease Control and Prevention (CDC) virus collection. Sequencing was performed using TrueSeq reagents and analyzed on the MiniSeq system (both Illumina). Phylogenetic trees were constructed (neighbor-joining method, Jukes-Cantor nucleotide distance measure; bootstrap analysis based on 1,000 replicates) in CLC Genomics Workbench 9. Trees were visualized using FigTree v1.4.3 (University of Edinburgh, http://tree.bio.ed.ac.uk) and rooted using 2 Mopeia virus (MOPV) strains: Mozambique (Genbank DQ328874.1, DQ328875.1) and AN20410 (Genbank AY772170.1, AY772169.1).

Protein lysates were harvested in 2 × Laemmli sample buffer and γ-irradiated (5 × 10⁶ rads). Proteins were denatured for 10 min at 95°C, separated on 4–12% Bis-Tris SDS-PAGE gels, and transferred to nitrocellulose membranes using a semi-dry blotting system (Bio-Rad). LASV NP and GPC/GP1 were detected with mouse monoclonal antibodies generated in-house. Tubulin, used as a loading control, was detected with anti-tubulin antibody at 1:10,000 (Sigma, #T5168). Cellular proteins were detected with anti-STAT1 at 1:500 (BD BioScience, #610120), anti-pSTAT1 at 1:500 (Cell Signaling, #9171), and anti-ISG15 at 1:1000 (ProteinTech, #15981-1-AP) antibodies. Secondary antibodies were detected with Supersignal West Dura Fast Western Blot kits (Thermo Fisher Scientific) and visualized using a Bio-Rad ChemiDoc MP imaging system.

All animal procedures were approved by the CDC Institutional Animal Care and Use Committee (#2833SPEGUIC) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academies, 2011). The CDC is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Procedures conducted with LASV or LASV-infected animals were performed in the CDC biosafety level 4 laboratory.

A total of 22 strain 13/N guinea pigs (males and females aged 6 months to <4 years) were obtained from our breeding colony at the CDC (Atlanta, GA). Groups of 5–6 age- and sex-matched guinea pigs were inoculated subcutaneously with 1 × 10⁴ focus-forming units (FFU; equivalent to ~2 × 10⁴ TCID₅₀) of rJosiah (GenBank: HQ688673.1, HQ688675.1), rNJ2015 (GenBank: MG812679, MG812678), rJos-S/NJ-L, or rNJ-S/Jos-L. FFU, and TCID₅₀ titers were calculated in Vero-E6 cells by immunofluorescence assays using anti-LASV polyclonal antibodies, with the latter calculated using the method of Reed and Muench (Reed and Muench, 1938). All animals were housed individually on deep soft bedding and given daily fresh vegetable enrichment and commercial guinea pig chow and water ad libitum. Animals' health was assessed by experienced CDC veterinarians or animal health technicians. Animals were humanely euthanized with isoflurane vapors and sodium pentobarbital (SomnaSol Euthanasia-III solution; Henry Schein Animal Health) once clinical illness scores (including, but not limited to, piloerection, ocular discharge, weight loss >25% of baseline at −1 dpi, changes in mentation, ataxia, dehydration, dyspnea, and/or hypothermia) indicated that the animal was in the terminal stages of disease, or at the completion of study 41 days post-infection (dpi).

RNA was extracted from blood and homogenized tissue samples using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific) on a 96-well ABI MagMAX extraction platform with a DNase-I treatment step according to manufacturer's instructions. RNA was quantified by a qRT-PCR assay targeting a strain-specific NP gene sequence (primer and probe sequences available on request) and normalized to 18S RNA levels. Viral S segment copy numbers were determined using standards prepared from in vitro-transcribed S segment RNA. Levels of IFN-β, IFN-λ1, ISG56, CCL5, and IL-6 transcripts in infected A549 cells were determined using commercial assays (all Thermo Fisher, #4331182).

Plasma was separated from EDTA-blood by centrifugation at 6,000 rcf and γ-irradiated (5 × 10⁶ rads). Samples were diluted 1:25 in master plate diluent (5% skim milk powder, 0.5% Tween-20, 0.5% Triton X100, and 0.01% thimerosal in PBS). To determine anti-LASV IgG levels, background was first reduced by pre-absorption on plates coated with Vero-E6 lysate. The samples were then further diluted as a 4-fold dilution series (1:100–1:6,400) in 5% skim milk powder and 0.1% Tween-20 in PBS, and incubated for 1 h at 37°C on plates coated with either Vero-E6 lysates or lysates from Josiah-infected Vero-E6 cells. After washing with 0.1% Tween-20 in PBS, bound guinea pig IgG was detected by incubation with an HRP-conjugated secondary antibody for 1 h at 37°C (KPL), washing, and adding ABTS Microwell Peroxidase Substrate (KPL) for 30 min at 37°C to develop absorbance signals. The raw data were first corrected by subtracting the value obtained with unspecific lysates (Vero-E6) from the specific signal (lysates from infected cells). Samples were considered positive for anti-LASV IgG if: (1) individual well signals were >2 standard deviations higher than the average of known negatives; and (2) summed signals from all dilutions for that sample were >2 standard deviations above the average of the known negatives. When 2 repeated measurements gave a positive signal for a sample, the average titer was reported. Otherwise the sample was reported as negative. Total IgM were captured by goat anti-guinea pig IgM antibody (ICL Lab, #GM-60A) and incubated with cell slurries from mock-infected or LASV-infected Vero-E6 cells, and anti-LASV IgM were detected using a polyclonal antibody against LASV (in-house reagent, HMAF #703079) and secondary anti-mouse IgG HRP-conjugate (Pierce, #31446). IgM results were analyzed like IgG results, except that 3 standard deviations were used to determine the cut-off for positive results.

Whole blood samples collected in lithium heparin were analyzed on Piccolo Xpress chemistry analyzers (General Chemistry 13 Panel, Abaxis) within 1 h of collection. Normal values were determined based on samples obtained from in-house colony of strain 13/N guinea pigs aged 6 months to <4 years (n = 101). Range was calculated at mean ±1 standard deviation.

Minigenome and innate immune response qRT-PCR data were analyzed by one-way ANOVA with Dunnett's multiple comparison test. Survival was analyzed by Log-Rank (Mantel-Cox) test. All analyses were performed using GraphPad Prism v7.0 software.

Complete nucleotide CDS of NJ2015 NP, L, and GPC were aligned with those of both historical and recent LASV isolates (Supplementary Table 1). NJ2015 sequences clustered closely with other Liberian isolates within clade IV, but remained distinct from both Josiah and other more recently isolated Sierra Leone strains (Figure 1B). Nucleotide lengths of the 4 CDS were identical between NJ2015 and Josiah, but NCR and intragenic region lengths in both genome segments varied (Table 1). The most striking difference was the minimal L segment 5′-NCR of NJ2015 (52 nt) compared to Josiah (157 nt). Diversity was greatest in the L segment CDS, demonstrating 79.3% (L) and 79.9% (Z) nucleotide identity. Nucleotide identity was 82.6% for NP and 83.7% for GPC (Table 2). Amino acid identity was higher than nucleotide identity in all 4 proteins, ranging from 87.7% (L) to 96.4% (GPC).

Temporal genetic variation, even within clades, was less evident. Although isolated in 1976, Josiah retains remarkable nucleotide and amino acid identity to modern Sierra Leonean strains (Andersen et al., 2015; Table 2). Unfortunately, no extensive database of recent Liberian LASV strains exists for comparison, although NJ2015 clustered closely with 2 recent Liberian isolates included in our analysis. These recent Sierra Leonean and Liberian strains share 79–83% nucleotide identity across all 4 CDS. Amino acid identity was greatest in S segment-encoded proteins (95% for both NP and GPC), and lower in L segment-encoded proteins (87% for L and 84% for Z).

Arenavirus NCRs contain highly conserved 19–25 terminal nucleotides responsible for maintaining the panhandle structure of arenavirus genomes, and cis-acting promoter elements for viral RNA (vRNA) synthesis (Buchmeier et al., 2007). Aligning Josiah and NJ2015 NCRs demonstrated this conservation of the terminal nucleotide sequences. Both S and L segment NCRs varied immediately upstream of the start codon (Figure 2A). The NJ2015 L segment 5′ NCR was 67% shorter than the Josiah equivalent, but retained 32 conserved nucleotides at the terminus. Using minigenome assays based on Josiah and NJ2015 genome segments, we evaluated the relative activities of both the cis-acting (NCRs) and trans-acting elements (NP and L) required for vRNA synthesis using cognate and non-cognate combinations. Minigenome reporter gene activity values are presented as relative to activity values determined using Josiah NP and L (Figure 2B; raw values for ZsG fluorescence and gLuc luminescence in Supplementary Figure 1). The activity of both NJ2015 S and L segment-based minigenomes with cognate NP and L was comparable to activity of both Josiah S and L segment-based minigenomes with cognate proteins. When Josiah NP and NJ2015 L were used in conjunction, minigenome activity significantly increased 2- to 5-fold over activity of cognate NP and L, independently of NCR origin. Conversely, minigenome activity significantly declined when NJ2015 NP was used together with Josiah L, again independently of NCR origin. These results show that the cis-acting and trans-acting elements of these LASV strains are interchangeable, although minigenome activity changes depending on the combination of NP and L used.

A reverse genetics system to rescue infectious recombinant Josiah (rJosiah) from DNA plasmids has been described previously (Albariño et al., 2011). We created an analogous system to rescue NJ2015 (rNJ2015), cloning full-length antigenomic-sense S and L genome segments into transcription plasmids under control of a T7 polymerase promoter to rescue virus in BSR-T7/5 cells (Figure 3A). By alternating rescue plasmids, we also rescued 2 reassortant variants: one containing the Josiah S segment and NJ2015 L segment (rJos-S/NJ-L), and the other containing NJ2015 S segment and Josiah L segment (rNJ-S/Jos-L). rJosiah and rJos-S/NJ-L showed similar growth kinetics in A549 cells, reaching maximum titers at 48 hpi (Figure 3B). rNJ2015 and rNJ-S/Jos-L exhibited an attenuated phenotype compared to rJosiah and rJos-S/NJ-L, with lower titers at 24 and 48 hpi, and approximately 10-fold lower end-point titers. Successful rescue and propagation of both rJos-S/NJ-L and rNJ-S/Jos-L viruses demonstrated that reassortment between Josiah and NJ2015 genome segments can result in viable virus variants.

Reassortment altered viral protein expression profiles of S segment-encoded proteins (Figure 3C). NP levels in cells infected with rJosiah and rNJ2015 were first detectable 48 hpi and persisted until 96 hpi. Expression levels were equivalent between strains at 48 and 72 hpi, but rNJ2015 levels dropped slightly at 96 hpi. Levels of GPC and the associated cleavage product GP1 were slightly higher in rJosiah-infected cells than in rNJ2015-infected cells, with levels in the latter decreasing over time. Protein expression levels were similar in rJos-S/NJ-L-infected and rJosiah-infected cells, although the former expressed slightly more NP. The opposite was true of rNJ-S/Jos-L infected cells, with expression of both proteins appearing later and at lower levels than in NJ2015-infected cells. LASV NP and Z proteins are known to antagonize various aspects of the immune system (Martínez-Sobrido et al., 2007; Qi et al., 2010; Hastie et al., 2012; Pythoud et al., 2012; Rodrigo et al., 2012; Jiang et al., 2013; Huang et al., 2015; Xing et al., 2015). However, transcription levels of IFN-β, IFN-λ, ISG56, CCL5, and IL-6, and expression levels of STAT1, pSTAT1, and ISG15 were similar in A549 cells after infection with all 4 recombinant viruses (Supplementary Figure 2). Therefore, although viral protein expression was altered in the reassortants, these differences did not appear to result in differences in immune suppression.

To investigate how phenotypic differences observed in vitro translate in vivo, we inoculated strain 13/N guinea pigs with 1 × 10⁴ FFU of rJosiah, rNJ2015, rJos-S/NJ-L, or rNJ-S/Jos-L. Similar to previous reports, animals inoculated with rJosiah succumbed to disease 14–20 dpi (Jahrling et al., 1982; Carrion et al., 2007; Cashman et al., 2011, 2013); one animal did not display overt clinical signs and was euthanized at study completion (41 dpi) (Figure 4A). Animals inoculated with rNJ2015 exhibited weight loss and elevated temperatures, but all recovered and were euthanized at study completion (Figure 4B). The clinical course of animals inoculated with the reassortants largely mirrored that of the parental viral strain from which the S segment originated. Animals inoculated with rJos-S/NJ-L animals exhibited acute weight loss beginning on 10 dpi, and all succumbed to disease 17–24 dpi. In contrast, most rNJ-S/Jos-L-inoculated animals exhibited no observable progressive weight loss or elevated temperatures, and all survived until study completion (41 dpi). Similarly, vRNA was widely disseminated and detected at high levels in both blood and tissues of animals with terminal disease upon infection with either rJosiah or rJos-S/NJ-L (Figure 4C). Low or no vRNA levels were detected at study completion in animals infected with rNJ2015, and vRNA was detected only in the spleens of rNJ-S/Jos-L-infected animals. Clinical chemistry analyte values were comparable between rJosiah- and rJos-S/NJ-L-infected animals, and between rNJ2015- and rNJ-S/Jos-L-infected animals (Figure 5). In animals that succumbed to rJosiah or rJos-S/NJ-L infection, analyte abnormalities included hypocalcemia, hypoalbuminemia, and decreased total protein; in rNJ2015- and NJ-S/Jos-L-infected survivors, these values were within normal limits at study completion. Compared to rNJ2015- and NJ-S/Jos-L-infected survivors, mean AST increased at least 3.75- and 1.83-fold in rJosiah- and rJos-S/NJ-L-infected animals, respectively; however, mean ALT was only higher in rJosiah-infected animals, but correlated to the relative magnitude of the more elevated AST levels seen in some of the animals. Anti-LASV NP IgG was detected in all surviving animals, but not in succumbing animals (Table 3). IgM was detected in 5 of 6 rJosiah-infected guinea pigs and in 1 of 5 rJos-S/NJ-L-infected guinea pigs.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.