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. 1999 Dec;73(12):10387-98.
doi: 10.1128/JVI.73.12.10387-10398.1999.

Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome

W B Klimstra  1 , K D Ryman, K A Bernard, K B Nguyen, C A Biron, R E Johnston
Affiliations

Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome

W B Klimstra et al. J Virol. 1999 Dec.

Abstract

Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981-1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HR(sp) (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5' nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W.B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 +/- 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 +/- 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 +/- 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.

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Figures

FIG. 1
FIG. 1
Survival of neonatal mice infected with TR339 (▴), E2S1 (■), TRSB (□), nsP3 528 (◊), E172 (○), or Toto 50 (+). Mice were infected s.c. with 1,000 PFU of each virus in 50 μl of virus diluent.
FIG. 2
FIG. 2
Titers of virus in sera (A) and brains (B) of infected neonatal mice. Symbols: □, TRSB; ◊, nsP3 528; ○, E1 72; ■, E2S1; ▴, TR339; +, Toto 50; ∗, Toto 50 titer below the limit of detection of the plaque assay (3,500 PFU/ml).
FIG. 3
FIG. 3
IFN-α/β and TNF-α levels in pooled and individual sera of infected neonatal mice. Levels in mock-infected animals were below the limits of detection for these assays. (A) IFN-α/β as measured in serum pools from 5 to 10 mice. (B) TNF-α pooled samples (5 to 10 mice). Symbols: □, TRSB; ◊, nsP3 528; ○, E1 72; ■, E2S1; ▴, TR339; +, Toto 50. (C) Peak IFN-α/β levels in sera of individual mice (n = 3). (D) Peak TNF-α levels in sera of individual mice (n = 3). Levels in mock-infected animals were below the limits of detection for these assays.
FIG. 4
FIG. 4
IFN-γ and IL-6 levels in pooled sera of infected neonatal mice. Samples represent pooled sera from 5 to 10 mice that were mock infected (⧫) or infected with TRSB (□), TR339 (▴), or Toto 50 (+).
FIG. 5
FIG. 5
Corticosterone levels in pooled sera of infected neonatal mice. Samples represent pooled sera from 5 to 10 mice infected with TRSB (□), TR339 (▴), or Toto 50 (+) and mock-infected mice (⧫).
FIG. 6
FIG. 6
Histopathological and ISH analyses of virus-infected neonatal mice. (A) ISH showing virus replication in the myocardium and valves (arrow) of the heart at 24 hpi (original magnification, ×40). (B) ISH of the caudal portion of a midsagittal section through the body of a TR339-infected mouse sacrificed at 60 hpi (original magnification, ×10). Arrows indicate virus replication in the skeletal muscle (a), the diaphragm (b), and the muscularis of the gut (c). (C) ISH showing confluent virus replication in skeletal muscle of a TR339-infected mouse at 60 hpi (original magnification, ×100). (D) ISH showing focal replication of Toto 50 at 48 hpi (original magnification, ×100). Dark staining of keratinized epithelium was present in mock-infected animals and is not a consequence of virus replication. (E) H&E-stained section of skeletal muscle from a TR339-infected mouse sacrificed at 60 hpi. The arrow indicates condensed nuclei of muscle cells (original magnification, ×400). (F) ISH of a midsagittal section through the head of a TR339-infected mouse sacrificed at 60 hpi (original magnification, ×100). The arrow indicates the focus of the ISH signal over intact cells with neuronal morphology. (G) H&E-stained adjacent serial section from the area pictured in panel F. The arrow indicates the area of virus replication identified by ISH (original magnification, ×200). (H) H&E-stained section from the brain of a Toto 50-infected mouse 8 days postinfection showing perivascular cuffing and infiltration of mononuclear cells (original magnification, ×400).
FIG. 7
FIG. 7
Histopathological analysis of virus-infected and LPS-treated neonatal mice. (A) Skin of a Toto 50-infected mouse sacrificed at 48 hpi which was indistinguishable from that of mock-infected controls (the arrow indicates subcutaneous fat deposits) (original magnification, ×200). (B) Analogous section from a TR339-infected mouse sacrificed at 48 hpi (the arrow indicates apoptotic and/or necrotic cells in the dermis of the skin) (original magnification, ×200). (C to F) Arrows indicate the thymus cortex, and double arrows indicate the medulla. (C) Thymus from a Toto 50-infected mouse sacrificed at 48 hpi (original magnification, ×400). (D) Thymus from a TRSB-infected mouse sacrificed at 60 hpi (original magnification, ×400). (E) Thymus from a TR339-infected mouse sacrificed at 60 hpi (original magnification, ×400). (F) Thymus from an LPS-treated mouse sacrificed at 28 hpi (original magnification, ×400). (G) Section through the liver of a mock-infected mouse sacrificed at 60 hpi. The arrow indicates a cluster of hematopoietic cells (original magnification, ×400). (H) Analogous section from a TR339-infected mouse sacrificed at 60 hpi. The arrow indicates the condensed nucleus of a hematopoietic cell (original magnification, ×400). (I) Spleen section from a mock-infected mouse sacrificed at 48 hpi. The arrow indicates the follicle marginal zone (original magnification, ×400). (J) Analogous section from a TR339-infected mouse sacrificed at 48 hpi. The arrow indicates condensed and fragmented nuclei in the marginal zone (original magnification, ×400).
FIG. 8
FIG. 8
TNF-α, IFN-γ, IL-6, and corticosterone levels in pooled sera of LPS-treated neonatal mice. TNF-α (A), IL-6 (B), IFN-γ (C), and corticosterone (D) levels in pooled sera from 5 to 10 LPS-treated (●) and mock-treated (⧫) neonatal mice are shown.
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