Japanese encephalitis virus (JEV) is a zoonotic pathogen that is maintained by mosquito vectors and vertebrate hosts including birds in a natural transmission cycle. Domestic ducklings are sensitive to JEV infection, but the clinical responses of domestic ducklings to natural JEV infection are unknown. In this study, we simulated the natural JEV infection of domestic ducklings via JEV-infected mosquito bites to evaluate the pathogenicity of JEV in domestic ducklings. Specific pathogen-free domestic ducklings were infected at day 2 post-hatching with JEV-infected Culex pipiens mosquito bites and monitored for clinical responses. Among 20 ducklings exposed to JEV-infected mosquitoes, six showed mild and non-characteristic clinical signs starting at two days post-infection, then died suddenly with neurological signs of opisthotonos (a condition of spasm of the back muscles causing the head and limbs to bend backward and the trunk to arch forward) between two and three days post-infection. The mortality of the affected ducklings was 30% (6/20). Multifocal lymphohistiocytic perivascular cuffs and lymphohistiocytic meningitis were macroscopically observed in the affected duckling brains. JEV was detected in the cytoplasm of neuronal cells in the affected duckling brains by immunohistochemical assays and was recovered from the affected duckling brains by viral isolation. These observations indicated that JEV infection via mosquito bites causes mortality associated with viral encephalitis in newly hatched domestic ducklings, thus demonstrating the potential pathogenicity of JEV in domestic ducklings under natural conditions.

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Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission. IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection: mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.

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18Japanese encephalitis virus (JEV) is a viral zoonosis which can cause viral encephalitis, death 19 and disability. Culex is the main vector of JEV, but little is known about JEV transmission by 20 this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of 21 JEV on target cells via both direct and indirect pathways. Mosquito defensin bound the ED III 22 domain of viral E protein and directly mediated efficient virus adsorption on the target cell 23 surface, Lipoprotein receptor-related protein 2 expressed on the cell surface is the receptor 24 affecting defensin dependent adsorption. Mosquito defensin also indirectly down-regulated 25 the expression of an antiviral protein, HSC70B. As a result, mosquitos defensin enhances JEV 26 infection in salivary gland while increasing the possibility of viral transmission by mosquito. 27 These findings demonstrate that the novel effects of mosquito defensin in JEV infection and 28 the mechanisms through which the virus exploits mosquito defensin for infection and 29 transmission. 30 31 32 3 33 4 49 animals, the virus is transmitted to the skin through the saliva. The mosquito vector also 50 induces an immune response to JEV [14-16]. For example, C-type lectin and a series of 51 proteins increase rapidly after infection [17, 18]. C-type lectin plays an important role in 52 infection by JEV and other Flaviviridae viruses in mosquitoes, but the role of defensin has 53 not yet been clearly characterized. 54 Defensins are antimicrobial peptides consisting of 25-60 amino acids that are produced 55 by innate immune system [15, 19]. Defensin is one of the crucial immune effectors in insects 56 [20]. The antiviral effects of defensins have been well described in mammalian cells. Human 57 defensins have been reported to inhibit herpes simplex virus type 2 (retrocyclin-1, 58 retrocyclin-2) [21], human immunodeficiency virus (human beta defensin-1, human beta 59 defensin-2, Human beta defensin-3) [22, 23] and other viruses. However, human beta 60 defensin-6, expressed by adenovirus vectors, enhances parainfluenza virus type 3 replication 61 [24]. Normally, mammalian defensins can directly destroy the virus particles by binding to 62 the surface of envelope protein. They can also interact to the cell surface receptor and 63 influence cell signal transduction [19, 25]. Although there are many differences between the 64 mammalian and mosquito immune systems, defensins are considered important effectors in 5 65 the mosquito immune response. Therefore, the role of mosquito defensins during the process 66 of JEV infection requires further study. 67 In this study, we observed complex roles of mosquito defensin in JEV infection: a weak 68 antiviral effect and a strong effect enhancing binding. In the latter, defensin directly binds the 69 ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by 70 interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. 71 Mosquito defensin also down-...