The Susceptibility of Chickens to Zika Virus: A Comprehensive Study on Age-Dependent Infection Dynamics and Host Responses

Abstract

Zika virus (ZIKV) remains a public health concern, with epidemics in endemic regions and sporadic outbreaks in new areas posing significant threats. Several mosquito-borne flaviviruses that can cause human illness, including West Nile, Usutu, and St. Louis encephalitis, have associations with birds. However, the susceptibility of chickens to ZIKV and their role in viral epidemiology is not currently known. We investigated the susceptibility of chickens to experimental ZIKV infection using chickens ranging from 1-day-old chicks to 6-week-old birds. ZIKV caused no clinical signs in chickens of all age groups tested. Viral RNA was detected in the blood and tissues during the first 5 days post-inoculation in 1-day and 4-day-old chicks inoculated with a high viral dose, but ZIKV was undetectable in 6-week-old birds at all timepoints. Minimal antibody responses were observed in 6-week-old birds, and while present in younger chicks, they waned by 28 days post-infection. Innate immune responses varied significantly between age groups. Robust type I interferon and inflammasome responses were measured in older chickens, while limited innate immune activation was observed in younger chicks. Signal transducer and activator of transcription 2 (STAT2) is a major driver of host restriction to ZIKV, and chicken STAT2 is distinct from human STAT2, potentially contributing to the observed resistance to ZIKV infection. The rapid clearance of the virus in older chickens coincided with an effective innate immune response, highlighting age-dependent susceptibility. Our study indicates that chickens are not susceptible to productive ZIKV infection and are unlikely to play a role in the ZIKV epidemiology.

Keywords: STAT2; Zika virus; antibody; chicken; host restriction; innate immune response.

Figure 1

Plasma viremia of chickens inoculated with Zika virus. Chickens of varying ages (6 weeks old (w.o.), 4 days old (d.o.) and 1 (d.o.) were experimentally inoculated subcutaneously (SQ) or intravenously (IV) with varying TCID₅₀ doses of Zika virus (ZIKV) or vehicle (mock). (A–C) Experimental design of three separate infection experiments. (D–H) ZIKV viral RNA in plasma from animals euthanized at selected timepoints was quantified by real-time RT-PCR. Relative equivalent units (REUs) from a TCID₅₀ standard are shown for individual animals from Experiment (Exp) 1 (D,G), Exp2 (E,H), and Exp3 (F). Dashed lines indicate the limit of detection, horizontal lines represent mean, and vertical error bars indicate standard error of the mean.

Figure 2

Viremia and antibody response in chickens following inoculation with Zika virus. Chickens aged 6 weeks (6 w.o.), 4 days (4 d.o.), or 1 day (1 d.o.) were inoculated subcutaneously with vehicle (mock) or varying TCID₅₀ doses of Zika virus (ZIKV) and euthanized at selected timepoints. (A–C) Experimental design of three separate infection experiments. (D–F) ZIKV viral RNA in plasma, quantified by real-time PCR, are shown for individuals in Experiment (Exp) 4 (D), Exp5 (E), and Exp6 (F) as relative equivalent units (REUs) against a TCID₅₀ standard. Dashed lines indicate the limit of detection. (G–I) ZIKV-specific IgY antibody levels quantified as fold-change in optical densities at 405 nm (OD₄₀₅) over mean mock-infected values (dashed line) for Exp4 (G), Exp5 (H), and Exp6 (I) are shown for individuals. Horizontal lines represent mean, and vertical whiskers indicate standard error of the mean.

Figure 3

Longitudinal viremia and antibody response in 1-day-old (1 d.o.) chickens inoculated with Zika virus. Chickens were inoculated with 10⁷ TCID₅₀ units of Zika virus (ZIKV), and blood was collected for quantification of plasma viremia (B) and/or serum anti-ZIKV IgY antibodies at specified timepoints. (A) the experimental design of this experiment (Exp7). (B) ZIKV viral RNA in plasma quantified by real-time PCR relative equivalent units (REU) against a TCID₅₀ standard are shown for ZIKV-inoculated animals Ha22-27 individually and mock-infected animals as mean. (C) ZIKV-specific IgY antibody levels quantified as fold-change in optical densities at 405 nm (OD405) over mean mock-infected values (dashed line). Fold-change values are shown for all individuals in ZIKV- and mock-inoculated groups. Dashed line represents mean fold-change value of ZIKV-inoculated individuals.

Figure 4

Viral RNA detection in tissues of 1-day-old (1 d.o.) chickens inoculated with live or killed Zika virus. Chickens were inoculated with 10⁷ TCID₅₀ units of infectious Zika virus (ZIKV; live) or UV-inactivated ZIKV (killed) or vehicle (mock), and blood and tissues were collected following euthanasia at selected timepoints. (A) The experimental design of this experiment (Exp8). (B–D) ZIKV viral RNA quantified by real-time PCR relative equivalent units (REUs) against a TCID₅₀ standard are shown. The median of animals per timepoint is shown by height of bar graph. Symbols represent measurements from individual animals in plasma (B), spleen (C), crop (D), and brain (E). Open symbols represent individuals with undetectable viral RNA, at or below the limit of detection (LOD); the position of the symbol indicates the LOD. In B and C, LOD was not uniform across all specimens due to volume or mass variability. In D and E, LOD was equivalent across all specimens and is shown as a dashed line. All specimens tested from the killed inoculum group had undetectable viral RNA.

Figure 5

Gene expression of chickens inoculated with Zika virus. RNA of selected genes was measured against RNA from 18S by real-time PCR using tissue homogenates of 6-week-old (6 w.o.) and 1-day-old (1 d.o.) chickens inoculated with Zika virus (ZIKV) or mock-inoculated. Genes evaluated were (A) interferon alpha (IFNα), (B) interferon beta (IFNβ), (C) interleukin 1 beta (IL1β), (D) melanoma differentiation-associated protein 5 (MDA5), and (E) 2′-5′-oligoadenylate synthetase-like protein (OASL). Relative expressions of ZIKV-inoculated compared to mock-inoculated chickens are shown, with height of bar indicating mean of 2 to 4 animals per timepoint. The 6 w.o. specimens were from Exp4 (Figure 2); 1 d.o. day 16 post-inoculation specimens were from Exp6 (Figure 2), and day 1–7 specimens were from Exp8 (Figure 4).