Pathogenesis and shedding of Usutu virus in juvenile chickens

Abstract

Usutu virus (USUV; family: Flaviviridae, genus: Flavivirus), is an emerging zoonotic arbovirus that causes severe neuroinvasive disease in humans and has been implicated in the loss of breeding bird populations in Europe. USUV is maintained in an enzootic cycle between ornithophilic mosquitos and wild birds. As a member of the Japanese encephalitis serocomplex, USUV is closely related to West Nile virus (WNV) and St. Louis encephalitis virus (SLEV), both neuroinvasive arboviruses endemic in wild bird populations in the United States. An avian model for USUV is essential to understanding zoonotic transmission. Here we describe the first avian models of USUV infection with the development of viremia. Juvenile commercial ISA Brown chickens were susceptible to infection by multiple USUV strains with evidence of cardiac lesions. Juvenile chickens from two chicken lines selected for high (HAS) or low (LAS) antibody production against sheep red blood cells showed markedly different responses to USUV infection. Morbidity and mortality were observed in the LAS chickens, but not HAS chickens. LAS chickens had significantly higher viral titers in blood and other tissues, as well as oral secretions, and significantly lower development of neutralizing antibody responses compared to HAS chickens. Mathematical modelling of virus-host interactions showed that the viral clearance rate is a stronger mitigating factor for USUV viremia than neutralizing antibody response in this avian model. These chicken models provide a tool for further understanding USUV pathogenesis in birds and evaluating transmission dynamics between avian hosts and mosquito vectors.

Keywords: Usutu virus; avian model; juvenile chicken; pathogenesis; virus-host interactions.

Figure 1.

Growth kinetics of USUV isolates in DF-1 cells. The growth curve was performed three times, with one representative experiment shown. Symbols represent mean and error bars represent standard deviation of triplicate inoculated cultures. The limit of detection is indicated by dashed line. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Serum titers of two-day-old ISA Brown chickens inoculated with African and European strains of USUV. (A) Mean percentage of initial starting weight of chickens following 1500 PFU USUV inoculation. (B) Serum titers of chickens inoculated with 1500 PFU of USUV. (C) Serum titers of chickens inoculated with 10⁵ PFU of USUV. Circles represent individual samples; lines represent mean; error bars represent standard deviation. The limit of detection is indicated by dashed line. *p < 0.05.

Figure 3.

Evidence of USUV dissemination and USUV-mediated pathology in heart tissue from ISA Brown chickens inoculated with African and European strains of USUV. (A) Viral titer in heart tissue collected on dpi 3 and dpi 5. Circles represent individual samples; lines represent mean; error bars represent standard deviation. The limit of detection is indicated by dashed line. *p < 0.05. (B) Composite histopathology scores of heart tissue; lines represent median. (C) Representative image of heart tissue collected on dpi 5 from chickens inoculated PBS, with no inflammation (H&E stain). (D) Representative image of heart tissue collected on dpi 5 from chickens inoculated with European USUV strain, with foci of lymphocytic and heterophilic infiltrates (arrow) (H&E stain). (E) Representative image of heart tissue collected on dpi 5 from chickens inoculated with African USUV strain, with foci of inflammation (arrow) (H&E stain, scale bars = 200µm). (F) Viral titer in oral swabs collected on dpi 3 and dpi 5. (G) Viral titer in cloacal swabs collected on dpi 3 and dpi 5. Circles represent individual samples; lines represent mean; error bars represent standard deviation. The limit of detection is indicated by dashed line.

Figure 4.

Mortality, morbidity, and viremia of HAS and LAS chickens inoculated with USUV. (A) Kaplan-Meier survival curve of chickens inoculated with 1500 PFU USUV or PBS. (B) Mean percentage of initial starting weight of HAS chickens following USUV inoculation. (C) Mean percentage of initial starting weight of LAS chickens following USUV inoculation. *p < 0.05. (D) Viremia of HAS and LAS chickens inoculated with 1500 PFU. Circles represent individual samples; lines represent mean; error bars represent standard deviation. The limit of detection is indicated by dashed line. **p < 0.01, ****p < 0.0001.

Figure 5.

Evidence of viral dissemination, shedding, and histopathology in tissues of HAS and LAS chickens inoculated with USUV. (A) Viral titer in tissues collected on dpi 3. (B) Viral titer in tissues collected on dpi 3. (C) Viral titer in oral and cloacal swabs collected on dpi 3. (D) Viral titer in tissues collected on dpi 5. (E) Viral titer in tissues collected on dpi 5. (F) Viral titer in oral and cloacal swabs collected on dpi 5. Circles represent individual samples; lines represent mean; error bars represent standard deviation. The limit of detection is indicated by dashed line. *p < 0.05, **p < 0.01. (G) Composite score of heart tissue; line represents median. (H) Composite score of brain tissue; line represents median. (I) Composite score of kidney tissue; line represents median. (J) Representative image of heart tissue collected on dpi 5 from LAS chickens inoculated with PBS, with no inflammation (H&E stain). (K) Representative image of heart tissue collected on dpi 5 from LAS chickens inoculated with USUV, with inflammatory lesions and presence of heterophils and lymphocytes (arrow) (H&E stain). (L) Representative image of heart tissue collected on dpi 5 from HAS chickens inoculated with PBS, with no inflammation (H&E stain). (M) Representative image of heart tissue collected on dpi 5 from HAS chickens inoculated with USUV, with inflammatory lesions and presence of heterophils and lymphocytes (arrow) (H&E stain). (N) Representative image of kidney tissue collected on dpi 5 from LAS chickens inoculated with PBS, with no inflammation (H&E stain). (O) Representative image of kidney tissue collected on dpi 5 from LAS chickens inoculated with USUV, with heterophilic inflammatory foci (arrow) (H&E stain). (P) Representative image of kidney tissue collected on dpi 5 from HAS chickens inoculated with PBS, with no inflammation (H&E stain). (Q)Representative image of kidney tissue collected on dpi 5 from HAS chickens inoculated with USUV, with heterophilic inflammatory foci (arrow) (H&E stain). (R) Representative image of brain tissue collected on dpi 5 from LAS chickens inoculated with PBS, with no inflammation (H&E stain). (S) Image of brain tissue collected on dpi 5 from LAS chicken inoculated with USUV, with proliferation of glial cells in the neuropil (arrow) (H&E stain, scale bars = 200 µm).

Figure 6.

Mathematical modeling of HAS and LAS chickens inoculated with USUV. Kinetics of USUV in HAS and LAS chickens as given by model (Equation 1) versus data. A. Individual fits for HAS chickens B. Individual fits for LAS chickens. The parameters used in stimulation are given in Supplemental Table 3, $k=0.97$ per day, $p=44.7$ per infected cell per day, initial conditions are $T_0=10,000$ leukocytes/mL, $E_0=I_0=0$ leukocytes/mL, and ${\log }_{10}{V}_0=2.78$ PFU/ml. LAS chickens.