Sheep serve as amplifying hosts of Japanese encephalitis virus, increasing the risk of human infection

Abstract

The transmission cycle of Japanese encephalitis virus (JEV), involving pigs and birds as amplifying hosts and mosquitoes as vectors, was elucidated in the 1950s. However, factors contributing to this cycle remain unclear. Here, sheep were infected with a JEV strain isolated from sheep exhibiting neurological symptoms. The results revealed that sheep are susceptible to JEV infection and develop viremia, with levels and duration comparable to those observed in pigs, a known JEV-amplifying host. Mosquitoes fed viremic sheep blood showed an infection rate of 40.6 to 57.1%. These findings indicate that sheep can serve as amplifying hosts for JEV, potentially contributing to JEV transmission and increasing the public health risk of human infections. We propose an alternative, sheep-associated rural domestic JEV transmission cycle, which may be prevalent in specific regions where sheep are bred but pigs are not. This cycle exists along with the well-known pig-associated rural domestic and bird-associated wild cycles.

Fig. 1.. Isolation of JEV from sheep.

(A) Histopathological examination of brain tissue from sheep exhibiting neurological signs. Lymphohistiocytic perivascular cuffing is indicated by arrows. (B) Detection of pathogens associated with neurological signs using RT-qPCR or qPCR. PRV, pseudorabies virus. CAEV, caprine arthritis encephalitis virus. (C and D) BHK-21 cells were inoculated with supernatants from cytopathic effect–positive cells and incubated for 36 hours. Immunofluorescence staining was used to detect the JEV E protein (C). Western blotting was used to examine the presence of the JEV NS1 protein (D). (E) Growth kinetics of the SH2201 strain in BHK-21 cells. Data are presented as means ± SDs from three independent biological replicates. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 2.. Phylogenetic analysis of JEV strains.

Phylogenetic trees were inferred based on nucleotide sequences using the maximum likelihood method. (A) Phylogenetic analyses of the full-length genome of JEV. (B) Phylogenetic analyses of JEV E gene. The SH2201 strain is highlighted in red, and other strains from sheep are highlighted in blue. (C) Comparison of amino acid sites of protein E between JEV strains.

Fig. 3.. Virulence of the SH2201 strain in mice.

Mice (n = 8) were inoculated with the SH2201 strain at doses ranging from 10⁰ to 10⁴ PFU and then monitored for 21 days. (A) Survival curves of mice inoculated via the intracerebral route. (B) Survival curves of mice inoculated via the intraperitoneal route. (C) Histopathological examination of brain tissue from mice inoculated via the intracerebral route. (D) Histopathological examination of brain tissue from mice inoculated via the intraperitoneal route. Lymphohistiocytic perivascular cuffing and lymphohistiocytic meningitis are indicated by arrows.

Fig. 4.. Pathogenicity of the SH2201 strain in sheep.

Fifty-day-old Hu sheep (n = 5) were infected with the SH2201 strain at a dose of 10⁶ PFU and then monitored for 21 days. Clinical signs were assessed using a clinical scoring system: 0, no restriction in movement, no obvious clinical signs, and normal breathing; 1, reduced appetite, slow movement, and facial flushing; 2, slow movement, conjunctival redness, and shortness of breath; 3, moaning, restlessness, and apparent neurological symptoms (e.g., spinning and difficulty walking); 4, apparent neurological symptoms such as muscle spasms, followed by death. (A) Clinical scores of sheep inoculated via the intravenous route. (B) Clinical scores of sheep inoculated via the subcutaneous route. (C) Changes in rectal temperature. (D) Survival curves of sheep inoculated via intravenous and subcutaneous routes. (E) Histopathological examination of brain tissue from euthanized sheep. Lymphohistiocytic perivascular cuffing is indicated by arrows. (F) Viral loads in tissues collected from euthanized sheep, as measured by RT-qPCR. Data are presented as means ± SDs from three independent biological replicates.

Fig. 5.. Viremia levels in sheep infected with the SH2201 strain.

Fifty-day-old Hu sheep (n = 5) were infected with the SH2201 strain at a dose of 10⁶ PFU via subcutaneous and intravenous routes, respectively, and then monitored for 21 days. Blood samples were collected daily to detect viremia. (A) Viremia levels measured by TCID₅₀ assay. (B) RNAemia levels measured by RT-qPCR. (C to F) Ae. albopictus, Cx. pipiens pallens, and Cx. pipiens quinquefasciatus were fed viremic blood meal collected from SH2201-infected sheep. Engorged mosquitoes were randomly collected at 7 and 14 dpi to measure infection rates and viral loads. JEV NS1 gene copies in whole mosquitoes (C), midguts (D), and salivary glands (F) were measured by RT-qPCR. Viral loads in midguts and salivary glands were determined by TCID₅₀ assays (E). Two-tailed Student’s t test was performed for comparison of variables. Data are presented as means ± SDs.

Fig. 6.. Detection of virus shedding in JEV-infected sheep.

Fifty-day-old Hu sheep (n = 5) were infected with the SH2201 strain at a dose of 10⁶ PFU via subcutaneous and intravenous routes, respectively, then monitored for 21 days. Nasal and anal swabs were collected daily to detect virus shedding by RT-qPCR. (A) Viral RNA detected in nasal swabs. (B) Viral RNA detected in anal swabs. (C and D) Tissue samples were collected at the end of the experiment (21 dpi) to detect viral loads by RT-qPCR (C) and TCID₅₀ assays (D). Data are presented as means ± SDs.

Fig. 7.. Prevalence of JEV in field sheep.

A total of 3349 serum samples were collected from sheep farms located in 31 provinces in China to detect the presence of JEV and seropositivity rate by RT-qPCR and PRNT₅₀. (A) Positivity rates of JEV. (B) RNAemia levels. (C) Seropositivity rates of JEV. Data are presented as means ± SDs.