Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Abstract

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.

FIG. 1.

Schematic representation of the chimeric strains of EEEV. (A) The NA/SA chimera contains the 5′ UTR, the nsP1 to nsP4 genes, the subgenomic promoter (26S), and the 3′ UTR of the North American strain FL93-939 and the structural protein genes (including the capsid [C] and envelope [E2 and E1] proteins) of the South American strain BeAr436087. (B) The SA/NA chimera contains the 5′ UTR, the nsP1 to -4 genes, the subgenomic promoter (26S), and the 3′ UTR of the South American strain BeAr436087 and the structural genes (including C, E2, and E1) of the North American strain FL93-939.

FIG. 2.

Survival and in vivo replication of parental and infectious clone (I.C.)-derived EEEVs in 5- to 6-week-old NIH Swiss mice. (A) Viremia in mice after subcutaneous infection (1,000 PFU) with either the North American parental strain FL93-939 or the North American I.C.-derived virus (5 mice/cohort). (B) Survival of mice infected with either the North American parental strain FL93-939 or the North American I.C.-derived virus in which cohorts of 10 mice were infected subcutaneously with 1,000 PFU. (C) Viremia in mice after subcutaneous infection (1,000 PFU) with either the South American parental strain BeAr436087 or the South American I.C.-derived virus (5 mice/cohort). Error bars indicate the standard deviations (panels A and C).

FIG. 3.

In vitro replication of infectious clone (I.C.)-derived and chimeric strains of North American and South American EEEVs. Infectious clone (I.C.)-derived strains included the North American strain FL93-939 and the South American strain BeAr 436087, and the chimeric strains included NA/SA and SA/NA strains. Three replicate assays per virus strain were performed at MOIs of 0.1 (panels A and C) and 10 (panels B and D) PFU/cell in Vero (panels A and B) and C710 (panels C and D) cells. Error bars indicate the standard deviations.

FIG. 4.

Sensitivity of infectious clone (I.C.)-derived and chimeric strains of North American and South American EEEVs to IFNs. Vero cells were initially treated with human IFN-α, -β, or -γ, and 24 h later, the cells were infected in triplicate with either the North American strain FL93-939, the South American strain BeAr436087, the NA/SA chimera, or the SA/NA chimera at an MOI of 1.0 PFU/cell. Supernatants were collected from mock-treated and IFN-treated cultures at 24 and 48 h PI, and the virus titer was measured in the supernatants by plaque assay. The y axis represents the reduction (log₁₀-fold) of virus replication in Vero cells at 24 (panel A) and 48 (panel B) h after treatment with IFN-α, -β, or -γ (as shown on the x axis). Error bars indicate standard deviations.

FIG. 5.

Survival and viremia of 6-week-old NIH Swiss mice PI with infectious clone (I.C.)-derived and chimeric strains of North American and South American EEEVs. Mice were infected subcutaneously (1,000 PFU) with either the North American strain FL93-939, the South American strain BeAr436087, the NA/SA chimera, or the SA/NA chimera. (A) Survival (10 mice/cohort). (B) Viremia (5 mice/cohort). Error bars indicate standard deviations.

FIG. 6.

In vivo replication of infectious clone (I.C.)-derived and chimeric strains of North American and South American EEEVs in various organs of 6-week-old NIH Swiss mice. Mice were infected subcutaneously (1,000 PFU) with the North American strain FL93-939, the South American strain BeAr436087, the NA/SA chimera, or the SA/NA chimera (15 mice/cohort). At days 1, 2, 3, 5, and 7 PI, 1 to 3 mice/cohort were sacrificed, and the brain (A), heart (B), spleen (C), lung (D), liver (E), and kidney (F) were collected and titrated for virus by plaque assay. The limit of detection for the assay is 2 log₁₀ PFU/g. Error bars indicate standard deviations.