Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3

Abstract

Coxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3) Nancy strain. Using HeLa cell plaque assays with agar overlays, we noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. We identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. We determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. Our data reinforce the idea that culture-adapted laboratory virus strains can have reduced fitnessin vivo N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.

Importance: Coxsackieviruses frequently infect humans, and although many infections are mild or asymptomatic, there can be severe outcomes, including heart inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. We used a cell culture-adapted strain of CVB3, Nancy, to examine viral replication and pathogenesis in orally inoculated mice. We found that mice shed viruses distinct from input viruses because they formed extremely large plaques in cell culture. We identified a single mutation, VP3 N63Y, that was sufficient for large-plaque formation. N63Y mutant viruses have reduced glycan binding and replication in cell culture; however, they have enhanced replication and virulence in mice. We are now using N63Y mutant CVB3 as an improved system for viral pathogenesis studies.

FIG 1

Emergence of a CVB3 large-plaque variant following oral inoculation. Four- to 6-week-old male IFNAR^−/− mice were orally inoculated with 5 × 10⁷ PFU of light-sensitive, neutral-red-labeled WT CVB3 in the dark, and fecal samples were collected at 24, 48, and 72 hpi in the dark. (A) Total viral titers determined by HeLa cell plaque assays with agar overlays. (B) Percentages of replicated (noninoculum) virus in fecal samples. Flowthrough inoculum viruses are light sensitive, whereas viruses that replicated in mice are light insensitive. Aliquots of each processed fecal sample were exposed to light or dark, and replication status was determined by dividing the number of PFU/ml of light-exposed samples by the number of PFU/ml of dark-exposed samples and multiplying by 100%. Each symbol represents one mouse. (C) Representative plaque morphologies of inoculum and 48- and 72-hpi fecal samples. (D) Plaque size quantification. Each symbol represents a plaque. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm² or larger; indicated by a dashed line). For panels A and B, n = 5 to 10.

FIG 2

The emergence of the large-plaque variant differs in various tissues. Four- to 6-week-old male IFNAR^−/− or IFNAR^+/+ mice were orally inoculated i.p. or i.m. with 5 × 10⁷ PFU of WT CVB3. Fecal samples were collected from orally inoculated mice at 72 hpi. Liver, heart, and spleen samples were harvested from i.p. inoculated mice at 48 hpi. Muscle and liver samples were harvested from i.m. inoculated mice at 48 hpi. Following processing, viruses were plated on HeLa cells by using agar overlays. The sizes of 100 randomly picked plaques were quantified with ImageJ. The total percentages of large plaques among hundreds of plaques per condition are indicated at the top. Large plaques are defined as the average inoculum plaque size multiplied by a factor of 10 (2.473 mm² or larger; indicated by a dashed line).

FIG 3

VP3 N63Y is sufficient for the large-plaque phenotype. RT-PCR products from several large plaques were sequenced and found to contain the VP3 N63Y mutation. The N63Y mutant was cloned into a new infectious clone, and virus was generated. (A) Alignment of the VP3-63 region sequences of various viruses. The highlighted residue is in position 63. (B) CVB3 structure with the location of VP3-63 in red. The inset shows one 5-fold symmetry axis, with the VP3 proteins in gray. (C) Plaque phenotypes of WT CVB3, a plaque-purified fecal isolate with the large-plaque phenotype, and N63Y mutant CVB3.

FIG 4

N63Y mutant CVB3 has a growth defect in cell culture and reduced glycan-mediated cell attachment. Single-cycle assays of viral replication in HeLa (A) and Huh7 (B) cells were performed. Infections with WT or N63Y mutant CVB3 were performed at an MOI of 0.1. Viral titers were determined by plaque assay with HeLa cells. n = 3. (C) Cell attachment of ³⁵S-labeled WT or N63Y mutant CVB3. Virus was incubated with cells at 4°C for 40 min. Cells were washed and trypsinized, and cell-associated ³⁵S was quantified. (D) ³⁵S-labeled WT or N63Y mutant CVB3 was incubated with CHO cells (CHO-K1, pgsA745, pgsD677, and pgsB761) that vary in GAG expression. Plus and minus signs indicate the relative levels of GAGs on the cell surface. (E) Effect of heparinase treatment on CVB3 cell attachment. Huh7 cells were treated with or without heparinase I for 90 min prior to quantification of ³⁵S CVB3 attachment. n = 7. (F) Heparin-agarose pulldown assay. ³⁵S-labeled WT or N63Y mutant CVB3 was incubated with heparin-agarose resin or streptavidin-agarose resin (control). Resin was washed, and bound ³⁵S-labeled CVB3 was quantified. n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

N63Y mutant CVB3 shows enhanced replication and virulence in mice. The viral fecal shedding profiles of IFNAR^−/− (A) or IFNAR^+/+ (B) mice orally inoculated with 5 × 10⁷ PFU of WT or N63Y mutant CVB3 were determined. Virus titers were determined by plaque assay. Tissue viral titers at 72 hpi were determined in IFNAR^−/− (C) or IFNAR^+/+ (D) mice orally inoculated with 5 × 10⁷ PFU of WT or N63Y mutant CVB3. (E) Survival curves of CVB3-infected mice. From left to right, IFNAR^−/− (blue lines) or IFNAR^+/+ (gray lines) mice were inoculated orally with 5 × 10⁷ PFU, i.p. inoculated with 1 × 10⁴ PFU, or i.m. inoculated with 1 × 10² PFU of WT (solid line) or N63Y mutant (dashed line) CVB3. For all panels, n = 5 to 8. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 6

N63Y mutant CVB3 shows greater dissemination to the liver and induces more liver damage than WT CVB3. IFNAR^−/− mice were orally infected with 5 × 10⁷ PFU of WT or N63Y mutant CVB3. (A) Blood was harvested at 24 and 72 hpi, and ALT levels were quantified by ELISA. n = 6. (B) IFNAR^−/− mice were inoculated i.p. with 2 × 10⁷ PFU/20,000 cpm ³⁵S labeled WT or N63Y mutant CVB3. Heart, liver, and spleen samples were harvested at 1 hpi, and tissue-associated counts per minute were quantified. (C) Binding of CVB3 to homogenized tissues. A total of 3 × 10⁶ PFU/3,000 cpm ³⁵S labeled WT or N63Y mutant CVB3 were incubated with homogenized liver tissue from IFNAR^−/− mice for 60 min; this was followed by washing and quantification of the tissue-associated counts per minute. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.