Aerosol and Contact Transmission Following Intranasal Infection of Mice with Japanese Encephalitis Virus

Abstract

The Japanese encephalitis virus (JEV), a causative agent of severe viral encephalitis in humans, has a biological cycle fluctuating between transmission in mosquitoes and avian species and amplification in pigs. Contact transmission of JEV was recently shown in pigs in the absence of arthropod vectors. Here, we show JEV transmission between infected and contact mice and further demonstrate that JEV transmission occurs between animals via aerosols, as both viral RNA and infectious JEV were detected in direct contact- and aerosol-exposed contact animals. The results of this study change our understanding of JEV transmission in densely populated regions and may help to explain JEV outbreaks without the presence of arthropod vectors.

Keywords: JEV; aerosol transmission; contact transmission; intranasal infection; mice.

Figure 1

Tissue distribution of Japanese encephalitis virus (JEV) RNA in mice infected by the intranasal route. Four-week-old mice were intranasally (i.n.) inoculated with 10^6.0 plaque-forming units (pfu)/50 μL of SCYA201201 or SA14-14-2 JEV virus. The viral RNA load was measured using real-time RT-PCR in each mouse tissue collected at 3, 4, 5, 6, and 7 days post-infection (dpi) and expressed as RNA copies per gram (Mean ± SEM) (○ represent the SCYA201201 strain, and ● represent the SA14-14-2 strain). The horizontal dotted lines indicate the limit of detection (* P ˂ 0.05, ** P ˂ 0.01, *** P ˂ 0.001, and **** P ˂ 0.0001).

Figure 2

Virus replication, shedding, and seroconversion in mice intranasally inoculated with JEV. Four-week-old mice were i.n. inoculated with different doses (10², 10⁴, or 10⁶ pfu/50 μL) of the SCYA201201 or SA14-14-2 virus. The SCYA201201 virus is marked with hollow symbols (10^2.0 with □, 10^4.0 with △, and 10^6.0 with ○), while the SA14-14-2 virus is marked with solid symbols (10^2.0 with ■, 10⁴ with ▲, and 10⁶ with ●). (A) The viral RNA load in lungs at 3, 5, 7, 11, and 15 dpi is measured using real-time RT-PCR and expressed as RNA copies per gram (Mean ± SEM). (B) The viral RNA load in oronasal swabs collected at 3, 5, and 7 dpi was measured using real-time RT-PCR assay and expressed as RNA copies per mL (Mean ± SEM). (C) The sera were collected from infected or uninfected mice at 3, 5, 7, 11, and 15 dpi, and neutralizing antibodies were detected by PRNT. The horizontal dotted lines indicate the limit of detection (*P ˂ 0.05, ** P ˂ 0.01, *** P ˂ 0.001, and **** P ˂ 0.0001)).

Figure 3

Hematoxylin and eosin (H+E) analysis of mouse lung and brain sections. Four-week-old mice were i.n. inoculated with different doses (10², 10⁴, or 10⁶ pfu/50 μL) of the SCYA201201 or SA14-14-2 virus. (A) H+E staining of mouse lung sections revealed pathologic changes consistent with JEV infection. The scale indicates 50 μm. (B) The H+E staining of mouse brain sections revealed brain lesions consistent with JEV infection. The brain lesions are indicated by arrows. There are no microscopic lesions in the lung and brain of the control mice. The scale indicates 50 μm.

Figure 4

Contact transmission of JEV in mice. Mice were i.n. infected with 10^6.0 pfu/50 μL of the SCYA201201 or SA14-14-2 virus. The sentinel mice were commingled with the infected animals at 24 h post-infection. The SCYA201201 strain is marked with hollow symbols (infected with ○ and sentinel with ◇), while the SA14-14-2 strain is marked with solid symbols (infected with ● and sentinel with ◆). (A) The viral RNA load in the mice lungs harvested at 3, 5, and 7 dpi was measured using real-time RT-PCR. The horizontal dotted lines indicate the limit of detection (**** P ˂ 0.0001). (B) The virus titer in the lung samples was determined by plaque assay and was presented using plaque-forming units (pfu) per gram of lung tissue.

Figure 5

Aerosol transmission of JEV in mice. The mice were infected i.n. with 10^6.0 pfu/50 μL of the SCYA201201 or SA14-14-2 virus, respectively, and were housed in a cage with a double-walled wire partition that permitted airflow but prevented direct contact. The sentinel animals were added at 24 h post-infection. The mice were sacrificed, and their lungs were harvested at 3, 5, and 7 dpi. The SCYA201201 was marked with hollow symbols (infected with ○ and sentinel with ◇) and the SA14-14-2 was marked with solid symbols (infected with ● and sentinel with ◆). (A) The viral RNA load in the lungs was measured using real-time RT-PCR assay. The horizontal dotted lines indicate the limit of detection (**** P ˂ 0.0001). (B) The virus titer in the lung samples were determined by the plaque assay.