Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission.
ABSTRACT: The mosquito-borne Japanese encephalitis virus (JEV) causes severe central nervous system diseases and cycles between Culex mosquitoes and different vertebrates. For JEV and some other flaviviruses, oronasal transmission is described, but the mode of infection is unknown. Using nasal mucosal tissue explants and primary porcine nasal epithelial cells (NEC) at the air-liquid interface (ALI) and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could represent the route of entry and exit for JEV in pigs. Porcine NEC at the ALI exposed to with JEV resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines, indicating infection and replication in macrophages. Moreover, macrophages stimulated by alarmins, including interleukin-25, interleukin-33, and thymic stromal lymphopoietin, were more permissive to the JEV infection. Altogether, our data are important to understand the mechanism of non-vector-borne direct transmission of Japanese encephalitis virus in pigs.IMPORTANCE JEV, a main cause of severe viral encephalitis in humans, has a complex ecology composed of a mosquito-waterbird cycle and a cycle involving pigs, which amplifies virus transmission to mosquitoes, leading to increased human cases. JEV can be transmitted between pigs by contact in the absence of arthropod vectors. Moreover, virus or viral RNA is found in oronasal secretions and the nasal epithelium. Using nasal mucosa tissue explants and three-dimensional porcine nasal epithelial cells cultures and macrophages as ex vivo and in vitro models, we determined that the nasal epithelium could be a route of entry as well as exit for the virus. Infection of nasal epithelial cells resulted in apical and basolateral virus shedding and release of monocyte recruiting chemokines and therefore infection and replication in macrophages, which is favored by epithelial-cell-derived cytokines. The results are relevant to understand the mechanism of non-vector-borne direct transmission of JEV.
Project description:Japanese encephalitis virus (JEV), a main cause of severe viral encephalitis in humans, has a complex ecology, composed of a cycle involving primarily waterbirds and mosquitoes, as well as a cycle involving pigs as amplifying hosts. To date, JEV transmission has been exclusively described as being mosquito-mediated. Here we demonstrate that JEV can be transmitted between pigs in the absence of arthropod vectors. Pigs shed virus in oronasal secretions and are highly susceptible to oronasal infection. Clinical symptoms, virus tropism and central nervous system histological lesions are similar in pigs infected through needle, contact or oronasal inoculation. In all cases, a particularly important site of replication are the tonsils, in which JEV is found to persist for at least 25 days despite the presence of high levels of neutralizing antibodies. Our findings could have a major impact on the ecology of JEV in temperate regions with short mosquito seasons.
Project description:Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is capable of causing encephalitic diseases in children. While humans can succumb to severe disease, the transmission cycle is maintained by viremic birds and pigs in endemic regions. Although JEV is regarded as a significant threat to the United States (U.S.), the susceptibility of domestic swine to JEV infection has not been evaluated. In this study, domestic pigs from North America were intravenously challenged with JEV to characterize the pathological outcomes. Systemic infection followed by the development of neutralizing antibodies were observed in all challenged animals. While most clinical signs were limited to nonspecific symptoms, virus dissemination and neuroinvasion was observed at the acute phase of infection. Detection of infectious viruses in nasal secretions suggest infected animals are likely to promote the vector-free transmission of JEV. Viral RNA present in tonsils at 28 days post infection demonstrates the likelihood of persistent infection. In summary, our findings indicate that domestic pigs can potentially become amplification hosts in the event of an introduction of JEV into the U.S. Vector-free transmission to immunologically naïve vertebrate hosts is also likely through nasal shedding of infectious viruses.
Project description:The Japanese encephalitis virus (JEV) is responsible for zoonotic severe viral encephalitis transmitted by Culex mosquitoes. Although birds are reservoirs, pigs play a role as amplifying hosts, and are affected in particular through reproductive failure. Here, we show that a lentiviral JEV vector, expressing JEV prM and E proteins (TRIP/JEV.prME), but not JEV infection induces strong antibody-dependent enhancement (ADE) activities for infection of macrophages. Such antibodies strongly promoted infection via Fc receptors. ADE was found at both neutralizing and non-neutralizing serum dilutions. Nevertheless, in vivo JEV challenge of pigs demonstrated comparable protection induced by the TRIP/JEV.prME vaccine or heterologous JEV infection. Thus, either ADE antibodies cause no harm in the presence of neutralizing antibodies or may even have protective effects in vivo in pigs. Additionally, we found that both pre-infected and vaccinated pigs were not fully protected as low levels of viral RNA were found in lymphoid and nervous system tissue in some animals. Strikingly, the virus from the pre-infection persisted in the tonsils throughout the experiment. Finally, despite the vaccination challenge, viral RNA was detected in the oronasal swabs in all vaccinated pigs. These latter data are relevant when JEV vaccination is employed in pigs.
Project description:BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifiers of JEV. The entry mechanism of JEV into porcine cells remains largely unknown. In this study, we present a study of the internalization mechanism of JEV in porcine kidney epithelial PK15 cells. RESULTS: We demonstrated that the disruption of the lipid raft by cholesterol depletion with methyl-?-cyclodextrin (M?CD) reduced JEV infection. We also found that the knockdown of clathrin by small interfering RNA (siRNA) significantly reduced JEV-infected cells and JEV E-glycoprotein levels, suggesting that JEV utilizes clathrin-dependent endocytosis. In contrast, the knockdown of caveolin-1, a principal component of caveolae, had only a small (although statistically significant) effect on JEV infection, however, JEV entry was not affected by genistein. These results suggested that JEV entry was independent of caveolae. CONCLUSIONS: Taken together, our results demonstrate that JEV enters porcine kidney epithelial PK15 cells through cholesterol- and clathrin-mediated endocytosis.
Project description:Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes acute viral encephalitis in humans. Pigs are important amplifier hosts of JEV. Emerging evidence indicates that host microRNAs (miRNAs) play key roles in modulating viral infection and pathogenesis. However, mechanistic studies delineating the roles of miRNAs in regulating host-JEV interactions remain scarce.In this study, we demonstrated that miR-124 inhibited JEV replication in porcine kidney epithelial PK15 cells. Furthermore, using bioinformatics tools, we identified dynamin2 (DNM2), a GTPase responsible for vesicle scission, as a target of miR-124. Small interfering RNA (siRNA) depletion studies inicated that dynamin2 was required for efficient JEV replication. We also demonstrated that upregulation of miR-124 expression corresponded to decreased expression of its target, DNM2, in the JEV-infected PK15 cells.Overall, these results suggest the importance of miR-124 in modulating JEV replication and provide a scientific basis for using cellular miRNAs in anti-JEV therapies.
Project description:Japanese encephalitis virus (JEV) is a zoonotic mosquito-borne virus, persisting in pigs, Ardeid birds and Culex mosquitoes. It is endemic to China and Southeastern Asia. The case-fatality ratio (CFR) or the rate of permanent psychiatric sequelae is 30% among symptomatic patients. There were no reported local JEV human cases between 2006 and 2010 in Hong Kong, but it was followed by a resurgence of cases from 2011 to 2017. The mechanism behind this "skip-and-resurgence" patterns is unclear. This work aims to reveal the mechanism behind the "skip-and-resurgence" patterns using mathematical modelling and likelihood-based inference techniques. We found that pig-to-pig transmission increases the size of JEV epidemics but is unlikely to maintain the same level of transmission among pigs. The disappearance of JEV human cases in 2006-2010 could be explained by a sudden reduction of the population of farm pigs as a result of the implementation of the voluntary "pig-rearing licence surrendering" policy. The resurgence could be explained by of a new strain in 2011, which increased the transmissibility of the virus or the spill-over ratio from reservoir to host or both.
Project description:Japanese encephalitis (JE) is a mosquito-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). Pigs and water birds are the main amplifying and maintenance hosts of the virus. In this study, we conducted a JEV survey in mosquitoes captured in pig farms and water bird wetland habitats in Taiwan during 2005 to 2012. A total of 102,633 mosquitoes were collected. Culex tritaeniorhynchus was the most common mosquito species found in the pig farms and wetlands. Among the 26 mosquito species collected, 11 tested positive for JEV by RT-PCR, including Cx. tritaeniorhynchus, Cx. annulus, Anopheles sinensis, Armigeres subalbatus, and Cx. fuscocephala. Among those testing positive, Cx. tritaeniorhynchus was the predominant vector species for the transmission of JEV genotypes I and III in Taiwan. The JEV infection rate was significantly higher in the mosquitoes from the pig farms than those from the wetlands. A phylogenetic analysis of the JEV envelope gene sequences isolated from the captured mosquitoes demonstrated that the predominant JEV genotype has shifted from genotype III to genotype I (GI), providing evidence for transmission cycle maintenance and multiple introductions of the GI strains in Taiwan during 2008 to 2012. This study demonstrates the intense JEV transmission activity in Taiwan, highlights the importance of JE vaccination for controlling the epidemic, and provides valuable information for the assessment of the vaccine's efficacy.
Project description:Japanese encephalitis (JE) virus infection can cause severe disease in humans, resulting in death or permanent neurologic deficits among survivors. Studies indicate that the incidence of JE is high in northwestern Bangladesh. Pigs are amplifying hosts for JE virus (JEV) and a potentially important source of virus in the environment. The objectives of this study were to describe the transmission dynamics of JEV among pigs in northwestern Bangladesh and estimate the potential impact of vaccination to reduce incidence among pigs.We conducted a comprehensive census of pigs in three JE endemic districts and tested a sample of them for evidence of previous JEV infection. We built a compartmental model to describe JEV transmission dynamics in this region and to estimate the potential impact of pig vaccination. We identified 11,364 pigs in the study area. Previous JEV infection was identified in 30% of pigs with no spatial differences in the proportion of pigs that were seropositive across the study area. We estimated that JEV infects 20% of susceptible pigs each year and the basic reproductive number among pigs was 1.2. The model suggest that vaccinating 50% of pigs each year resulted in an estimated 82% reduction in annual incidence in pigs.The widespread distribution of historic JEV infection in pigs suggests they may play an important role in virus transmission in this area. Future studies are required to understand the contribution of pig infections to JE risk in humans and the potential impact of pig vaccination on human disease.
Project description:Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic flavivirus that causes encephalitis and reproductive disorders in mammalian species. However, the host factors critical for its entry, replication, and assembly are poorly understood. Here, we design a porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library containing 85,674 single guide RNAs targeting 17,743 protein-coding genes, 11,053 long ncRNAs, and 551 microRNAs. Subsequently, we use the PigGeCKO library to identify key host factors facilitating JEV infection in porcine cells. Several previously unreported genes required for JEV infection are highly enriched post-JEV selection. We conduct follow-up studies to verify the dependency of JEV on these genes, and identify functional contributions for six of the many candidate JEV-related host genes, including EMC3 and CALR. Additionally, we identify that four genes associated with heparan sulfate proteoglycans (HSPGs) metabolism, specifically those responsible for HSPGs sulfurylation, facilitate JEV entry into porcine cells. Thus, beyond our development of the largest CRISPR-based functional genomic screening platform for pig research to date, this study identifies multiple potentially vulnerable targets for the development of medical and breeding technologies to treat and prevent diseases caused by JEV.
Project description:BACKGROUND:Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS:A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE:Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.