African and Asian Zika Virus Isolates Display Phenotypic Differences Both In Vitro and In Vivo

Abstract

Zika virus (ZIKV) is a mosquito-borne member of the genus Flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of Aedes aegypti mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of Ifnar1^-/- mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was > 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Figure 1.

Midpoint rooted maximum likelihood tree based on nucleotide (NT) sequence of the open reading frame. Bootstrap values of > 75% are shown at internal nodes. The scale bar represents NT substitutions per site.

Figure 2.

Plaque phenotype of African (ArD 41525) and Asian (CPC-0740 and SV0127-14) Zika virus isolates in Vero and HEK-293 cell monolayers. Plaque phenotype was assessed in Vero cell monolayers; 5 days post-infection (dpi) cells were fixed and stained with crystal violet.

Figure 3.

Replication kinetics of African (ArD 41525) and Asian (CPC-0740) Zika virus isolates in Vero-WHO, HEK-293, DEF, and RK-13 cell lines. Replication kinetics of all isolates were performed at multiplicity of infection of 0.1 plaque forming units (PFU)/cell in triplicate. Average titers ± standard deviation (SD) (error bars) are shown. P values ≤ 0.01 are indicated with *.

Figure 4.

Replication kinetics of African (ArD 41525) and Asian (CPC-0740) Zika virus isolates in C7/10, RML-12, AP-61, Anopheles gambiae, and sandfly (PP-9) cell lines. Replication kinetics of all isolates were performed at multiplicity of infection of 0.1 plaque forming units (PFU)/cell in triplicate. Average titers ± standard deviation (SD) (error bars) are shown. P values ≤ 0.03 are indicated with *.

Figure 5.

Replication kinetics of African (ArD 41525) and Asian (CPC-0740, SV0127-14) Zika virus isolates in Vero-WHO, HEK-293, C7/10, and AP-61 cell lines. Replication kinetics of all isolates were performed at an multiplicity of infection of 0.1 plaque forming units (PFUs)/cell in triplicate. Average titers ± standard deviation (SD) (error bars) are shown. P values ≤ 0.02 are indicated with *.

Figure 6.

Infection of African and Asian isolated in ifnar^−/− and IFNAR1-blocking MAb murine models. Outline of the study design in mice (A and D). African (ArD 41525) and Asian (CPC-0740 and SV0127-14) Zika virus isolates in 5-week-old C57BL/6 ifnar^−/− mice (B). Cohorts of 10 males and females were infected with 5.0 log₁₀ plaque forming units (PFUs) through intraperitoneal (IP) route. Fifty percent lethal dose (LD₅₀) studies of the African (ArD 41525) isolate in ifnar^−/− and IFNAR1-blocking MAb murine models (C and E). Cohorts of 10 males and females were infected with doses ranging from 6.0 to 2.0 log₁₀ PFU through IP route. After infection, animals were monitored for survival, weight loss, and clinical scores (C and E). P values ≤ 0.0006 are indicated with *.

Figure 7.

Infection of African (ArD 41525) (B) and Asian (CPC-0740 [C], PRVABC59 [D]) Zika virus isolates in 5-week old C57BL/6 mice treated with IFNAR1-blocking MAb. Outline of the study design in mice (A). Cohorts of 10 females were infected with 6.0–2.0 log₁₀ plaque forming units (PFUs) through intraperitoneal (IP) route. After infection, animals were monitored for survival, weight loss, and clinical scores (B–D). P values < 0.0001 are indicated with *.