Adaptation of Saffold virus 2 for high-titer growth in mammalian cells

Abstract

Saffold viruses (SAFV) are a recently discovered group of human Cardioviruses closely related to Theiler's murine encephalomyelitis viruses (TMEV). Unlike TMEV and encephalomyocarditis virus, each of which is monotypic, SAFV are genetically diverse and include at least eight genotypes. To date, only Saffold virus 3 (SAFV-3) has been grown efficiently in mammalian cells in vitro. Here, we report the successful adaptation of SAFV-2 for efficient growth in HeLa cells after 13 passages in the alpha/beta interferon-deficient human glial cell line U118 MG. Nine amino acid changes were found in the adapted virus, with single mutations in VP2, VP3, and 2B, while 6 mutations arose in VP1. Most capsid mutations were in surface loops. Analysis of SAFV-2 revealed virus growth and cytopathic effect only in human cell lines, with large plaques forming in HeLa cells, with minimal cell association, and without using sialic acid to enter cells. Despite the limited growth of SAFV-2 in rodent cells in vitro, BALB/c mice inoculated with SAFV-2 showed antibody titers of >1:10(6), and fluorescence-activated cell sorting (FACS) analysis revealed only minimal cross-reactivity with SFV-3. Intracerebral inoculation of 6-week-old FVB/n mice produced paralysis and acute neuropathological changes, including meningeal infiltrates, encephalitis, particularly of the limbic system, and spinal cord white matter inflammation.

Fig. 1.

Adaptation of SAFV-2 to high-titer growth in mammalian cells. (A) Mock-infected LLCMK2 rhesus monkey kidney cells showing normal morphology. (B) SAFV-2-infected LLCMK2 cells at 8 days postinfection (LLCMK2P8) showing small clusters of rounded cells slightly above the plane of the monolayer. (C) Temporal analysis of SAFV RNA replication in LLCMK2 cells at passage 6 by real-time RT-PCR showing a 2-log-unit increase in viral copy numbers. PI, postinfection. (D) Scheme of SAFV-2 adaptation in each cell line with the number of passages and development and progression of CPE over time. P1→P8, passage 1 to passage 8; P3, passage 3; cpe, cytopathic effect; d7, day 7. (E) Mock-infected U118 MG cells showing normal cell morphology. (F) SAFV-2-infected LLCMK2 P8 at 3 day p.i. showing advanced CPE at passage 7. (G) Mock-infected HeLa cells showing normal cell morphology. (H) SAFV-2 (U118 P13)-infected HeLa cells (24 h p.i.) showing advanced CPE at passage 3.

Fig. 2.

(A) Modified ribbon drawing of SAFV-2 strain BeAn VP1, VP2, and VP3 (15), with VP1 loop I shortened by four residues and VP2 puff B increased by two residues to more closely resemble the SAFV-2 VP sequence. The eight capsid mutations in adapted SAFV-2 are indicated as follows: a, L2174F; b, L3084P, c, D1080G; d, D1097 deletion, e, T1098A; f, Q1100R; g, T1101I; h, S1262Y, where according to picornavirus convention the first digit designates the capsid protein and the other three digits indicate the amino acid number. (B) SAFV-2 adaptation mutations on the surface of a Cα-carbon pentamer model generated using PyMol and the BeAn virus pdb1tme coordinates. Mutations and virus structure are shown in different colors as follows: yellow, 5 mutations clustered on VP1 loops I and II and one mutation in VP2 puff B; blue, VP1; green, VP2; and red, VP3. VP3 mutation L3084 is not visible in this orientation of the model, and VP1 S1262Y is not evident because the VP1 C-terminal 15 residues were disordered in the BeAn virus crystal structure (14).

Fig. 3.

(A) Single-step adapted SAFV-2 growth kinetics in HeLa cells at an MOI of 100, with 96% of the virus yield observed by 14 h postinfection. (B) SAFV-2 RNA replication kinetics. +Strand, plus strand.

Fig. 4.

SAFV-2 plaque morphology, cell association, and sialic acid binding. (A and B) Plaques produced in BHK-21 cells by SAFV-2 strain GDVII after 3 days and BeAn after 4 days of incubation, respectively. (C) SAFV-2 plaques in HeLa cells after 4 days of incubation. Note that the SAFV-2 lysate was not plaque purified and shows mainly large and some small plaques. (D) Cell association determined from the ratio of the total virus yield in PFU (cells and supernatant) to that in the supernatant for GDVII, BeAn, and Saffold virus 2 (SafV-2). Values are means plus standard deviations (error bars) (SD). The graph shows the results of a representative experiment of 4 experiments. (E) Binding of [S³⁵]methionine-labeled virions to cells in suspension after treatment with 1 mU/ml of Clostridium perfringens neuraminidase (Neur.) for 45 min at 37°C or with buffer alone. Values are mean ± SD. The graph shows the results of a representative experiment of 3 experiments.

Fig. 5.

Electron micrograph of SAFV-2-infected HeLa cells (MOI of 100) for 9 h showing typical picornavirus rearrangement of cellular membranes (viroplasm) with clusters of denser (electron-dense) 27-nm virions scattered throughout the viroplasm. Bar = 2 μm. (Inset) Virions are shown at higher magnification. Bar = 0.5 μm.

Fig. 6.

FACS analysis of hyperimmune ascitic fluid samples raised in SAFV-2-injected mice and incubated with SAFV-2- or SAFV-3-infected HeLa cells. HeLa cells infected at an MOI of 5 were harvested at 16 h p.i. and stained with a 1:500 dilution of hyperimmune ascitic fluid sample and 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. The small peaks of SAFV-2 and SAFV-3 overlapping the uninfected cell profile were probably cells that were not infected.

Fig. 7.

Neuropathological changes in an adult FVB/n mouse on day 6 p.i. after i.c. inoculation of 1 × 10⁶ PFU of SAFV. (A) Microglial proliferation and perivascular cuffs in the dentate gyrus of the hippocampus with loss of dentate neurons. (B) Lymphocytic meningeal infiltrates and microglial proliferation in the cerebellar molecular layer (black arrows) without involvement of Purkinje cells. (C) Mild meningitis and lymphocytic infiltration in the anterior white matter of the spinal cord (black arrow), with the anterior horn region spared. The sections were stained with hematoxylin and eosin.