Japanese Encephalitis Virus In Pigs Research Articles

Fitness adaptations of Japanese encephalitis virus in pigs following vector-free serial passaging.

Japanese encephalitis virus (JEV) is a zoonotic mosquito-transmitted Flavivirus circulating in birds and pigs. In humans, JEV can cause severe viral encephalitis with high mortality. Considering that vector-free direct virus transmission was observed in experimentally infected pigs, JEV introduction into an immunologically naïve pig population could result in a series of direct transmissions disrupting the alternating host cycling between vertebrates and mosquitoes. To assess the potential consequences of such a realistic scenario, we passaged JEV ten times in pigs. This resulted in higher in vivo viral replication, increased shedding, and stronger innate immune responses in pigs. Nevertheless, the viral tissue tropism remained similar, and frequency of direct transmission was not enhanced. Next generation sequencing showed single nucleotide deviations in 10% of the genome during passaging. In total, 25 point mutations were selected to reach a frequency of at least 35% in one of the passages. From these, six mutations resulted in amino acid changes located in the precursor of membrane, the envelope, the non-structural 3 and the non-structural 5 proteins. In a competition experiment with two lines of passaging, the mutation M374L in the envelope protein and N275D in the non-structural protein 5 showed a fitness advantage in pigs. Altogether, the interruption of the alternating host cycle of JEV caused a prominent selection of viral quasispecies as well as selection of de novo mutations associated with fitness gains in pigs, albeit without enhancing direct transmission frequency.

Survey of Japanese encephalitis reactor on pigs in north Sulawesi

Several studies have shown that pigs are considered as the main reservoir of Japanese encephalitis virus transmission in Indonesia, in addition to having isolated Japanese encephalitis virus in pigs, pigs are also the type of livestock that is most often reported to contain Japanese encephalitis antibodies with a relatively high infection rate. This study aims to determine the spread and magnitude of Japanese encephalitis virus infection in pigs by detecting the presence of Japanese encephalitis antibodies using the Competitive Enzyme Linked Immuno Assay (C-ELISA). By knowing the spread of Japanese encephalitis virus infection in pigs in North Sulawesi Province, it can indirectly be used as an indicator of the possible threat of Japanese encephalitis virus transmission to humans, especially if the location of pig farms is close to residential areas. The results of serological examination using the Competitive ELISA method on 50 samples of porcine serum in 3 districts/cities, 30% of Japanese encephalitis reactors (15 samples were positive). The JE reactor in pigs in Minahasa Regency is the highest (43.8%) compared to Tomohon City (29.4%) and South Minahasa Regency (17.6%). The proximity of pigs to humans can be seen from the location of the settlements adjacent to the location of pig farms. Pigs located in Minahasa Regency which are close to settlements are the highest risk factors for the transmission of the Japanese Encephalitis virus.

Mosquito Saliva Modulates Japanese Encephalitis Virus Infection in Domestic Pigs

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is the leading cause of pediatric viral encephalitis in Asia. Japanese encephalitis virus is transmitted byCulexspecies mosquitoes that also vector several zoonotic flaviviruses. Despite the knowledge that mosquito saliva contains molecules that may alter flavivirus pathogenesis, whether or not the deposition of viruses by infected mosquitoes has an impact on the kinetics and severity of JEV infection has not been thoroughly examined, especially in mammalian species involved in the enzootic transmission. Most JEV pathogenesis models were established using needle inoculation. Mouse models for West Nile (WNV) and dengue (DENV) viruses have shown that mosquito saliva can potentiate flavivirus infections and exacerbate disease symptoms. In this study, we determined the impact of mosquito salivary components on the pathogenesis of JEV in pigs, a species directly involved in its transmission cycle as an amplifying host. Interestingly, co-injection of JEV and salivary gland extract (SGE) collected fromCulex quinquefasciatusproduced milder febrile illness and shortened duration of nasal shedding but had no demonstrable impact on viremia and neuroinvasion. Our findings highlight that mosquito salivary components can differentially modulate the outcomes of flavivirus infections in amplifying hosts and in mouse models.

Targeting of the Nasal Mucosa by Japanese Encephalitis Virus for Non-Vector-Borne Transmission.

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.

Full-Length Genome Sequence of Japanese Encephalitis Virus Strain FC792, Isolated from Guangxi, China.

ABSTRACTWe report here the complete genomic sequence of Japanese encephalitis virus (JEV) strain FC792, isolated from aborted fetuses of sows which were unimmunized with JEV vaccines in Guangxi Province, southern China. The complete JEV genome of strain FC792 had the highest nucleotide homology (99.7%) and amino acid identity (99.4%) with the sequence of JEV strain SA14-14-2 (GenBank accession number AF315119). Phylogenetic analysis showed that strain FC792 had the closest phylogenetic relationship to the sequence of strain YUNNAN0901 (GenBank accession number JQ086762). This study will help us understand the molecular pathogenesis and genetic diversity of genotype III Japanese encephalitis virus in pigs.

Japanese encephalitis virus tropism in experimentally infected pigs.

Pigs are considered to be the main amplifying host for Japanese encephalitis virus (JEV), and their infection can correlate with human cases of disease. Despite their importance in the ecology of the virus as it relates to human cases of encephalitis, the pathogenesis of JEV in pigs remains obscure. In the present study, the localization and kinetics of virus replication were investigated in various tissues after experimental intravenous infection of pigs. The data demonstrate a rapid and broad spreading of the virus to the central nervous system (CNS) and various other organs. A particular tropism of JEV in pigs not only to the CNS but also for secondary lymphoid tissue, in particular the tonsils with the overall highest viral loads, was observed. In this organ, even 11 days post infection, the latest time point of the experiment, no apparent decrease in viral RNA loads and live virus was found despite the presence of a neutralizing antibody response. This was also well beyond the clinical and viremic phase. These results are of significance for the pathogenesis of JEV, and call for further experimental studies focusing on the cellular source and duration of virus replication in pigs.

Vector-free transmission and persistence of Japanese encephalitis virus in pigs

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.

Porcine alpha interferon inhibit Japanese encephalitis virus replication by different ISGs in vitro

Japanese encephalitis virus (JEV) causes serious zoonosis in South Asia, Southeast Asia and other areas. Pigs are an important reservoir for this virus in nature. The treatment of JEV infection in pigs is important for controlling the prevalence of JEV in humans and economic losses in pig farming. In this study, we selected a high activity porcine alpha-interferon to inhibit JEV replication in porcine kidney cell lines (PK-15). Alpha interferon exhibited high antiviral activity against JEV; the expression of three interferon-stimulated genes (ISGs), ISG15, Mx2 and OAS L, increased significantly after interferon treatment. Furthermore, we verified the anti-JEV effect of these ISGs by RNAi and overexpression. Mx2 and OAS L exhibited strong anti-JEV effects in PK-15 cells. Based on these novel results, alpha interferon (IFN-α) should be considered to be a potential drug against JEV in pigs.

Survey of Japanese encephalitis virus in pigs and wild boars on Ishigaki and Iriomote Islands in Okinawa, Japan

SUMMARY We previously revealed that Japanese encephalitis virus (JEV) seroprevalence was 4.5% in pigs on Ishigaki Island from 2005 to 2007. However, a partial E gene sequence (151 bp) of the JEV genome (JEV/sw/Ishigaki/1/2005) was detected in one pig. Phylogenetic analysis showed that JEV/sw/Ishigaki/1/2005 belonged to genotype III and to the same lineages isolated in Taiwan from 2006 to 2008. Serum samples were collected from 128 pigs on Ishigaki from 2009 to 2010, 24 wild boars on Ishigaki from 2008 to 2010, and 117 wild boars on Iriomote Island from 2008 to 2010. Four (3.1%) pigs on Ishigaki were positive for JEV antibody, but all wild boars on the island were negative. Fifty-two (44.4%) wild boars on Iriomote were positive for JEV antibody, in contrast to a seroprevalence of 3.7% in 2000 and 2004. JEV on Iriomote and/or in Taiwan might be related to transmission on Ishigaki.

Circulation of Japanese encephalitis virus in pigs and mosquito vectors within Can Tho city, Vietnam.

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing encephalitis in humans and reproductive disorder in pigs. JEV is present in large parts of Asia, where urbanization is high. Households within and outside Can Tho city, South Vietnam, were selected to monitor circulation of JEV. A nested RT-PCR was established to detect the presence of JEV in mosquitoes whereas sera from pigs belonging to households within the province were analyzed for the presence of antibodies to JEV. A total of 7885 mosquitoes were collected and divided into 352 pools whereof seven were JEV-positive, six of which were collected within the city. Fragments from four pools clustered with JEV genotype III and three with genotype I. Of the 43 pigs sampled inside the city 100% had JEV antibodies. Our study demonstrates exposure to JEV in pigs, and co-circulation of JEV genotype I and III in mosquitoes within an urban environment in South Vietnam. Thus, although JEV has mainly been considered a rural disease, the potential for transmission in urban areas cannot be ignored.

Molecular characterization of Japanese encephalitis viruses circulating in pigs and mosquitoes on pig farms in the Chinese province of Henan

Since the first Chinese case report of Japanese encephalitis, Japanese encephalitis virus (JEV) has circulated in China for at least 60years. Even though pigs play a critical role in the JEV transmission cycle information on the prevalence of JEV in pigs has not been investigated in China. As the central Chinese province of Henan has the largest human population in China, a history of serious JEV and is the largest pig producing province it was chosen for this study. We have found that currently natural infection with JEV in pigs and mosquitoes is prevalent and both genotypes 1 and 3 co-circulate in pigs and mosquitoes in central China. Phylogenetic analysis showed that all of the newly obtained pig-derived JEV isolates are more closely related to isolates from the 1950s to 1960s than to those recently isolated from humans and mosquitoes. Further analyses based on all the previous reported Chinese isolates indicates that presently genotype 3 JEV is the predominant genotype in pigs but genotype 1 JEV is emerging and spreading rapidly in recent years. Our study provides information for understanding the current epidemiology of JEV in China and suggests possible measures applicable to the further control of JEV.

Seroprevalence of Japanese Encephalitis Virus and Risk Factors Associated with Seropositivity in Pigs in Four Mountain Districts in Nepal*

Japanese encephalitis was recently reported from individuals in the mountain districts of Nepal without travel history to Japanese encephalitis virus (JEV) endemic areas. We performed a cross-sectional study to estimate the seroprevalence of JEV in pigs and subsequently conducted a survey of farmers to identify risk factors associated with seropositivity. In July and August, 2010, 454 pig serum samples were collected and tested by competitive ELISA. Data from a 35-question survey of 109 pig owners were analysed using multivariate logistic regression. Seventy-six (16.7, 95% CI 13.6-20.4) pigs tested positive for anti-JEV antibodies, none of which had been vaccinated against JEV or sourced from JEV endemic areas. Risk factors associated with JEV seropositivity were 'summer abortion', 'wells as a water source', 'urban location', 'reported presence of mosquitoes' and 'lower elevation'. Our results suggest that JEV is likely circulating in the mountain districts of Nepal, and that locally acquired JEV should be considered a risk for residents and travellers in these areas.

Survey of Japanese Encephalitis Virus in Pigs on Miyako, Ishigaki, Kume, and Yonaguni Islands in Okinawa, Japan

Serum specimens were collected from 125 pigs on Miyako Island, 112 pigs on Ishigaki Island, and 42 pigs on Kume Island from 2005 to 2007, and 54 pigs on Yonaguni Island from 2006 to 2007. Their sera were tested for Japanese encephalitis virus (JEV) antibody by hemagglutination inhibition (HI) assay. Five serum samples (4.5%) from Ishigaki Island were positive for HI antibody, and 4 of the 5 samples were positive for 2-mercaptoethanol- sensitive antibody (IgM Ab). All samples from Miyako, Kume, and Yonaguni Islands were negative for HI antibody. Our results indicate that JEV transmission activity was extremely low on Miyako, Ishigaki, Kume, and Yonaguni Islands. The JEV genome (JEV-RNA) was detected from the sera of one pig on Ishigaki Island. The partial gene of the E region (151 nt) was analyzed phylogenetically. The analysis showed that the new JEV-RNA belonged to genotype 3 and was closely related to JEV strains isolated in Taiwan from 1985 to 1996. It was suggested that JEV previously introduced from Taiwan had been maintained on Ishigaki Island.

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Biophysical characterization of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

A virus strain, showing cytopathic effect in Vero cell, was isolated from plasma of a fattening pig in Gyeonggi province, Korea in October 1999. The evaluation of physicochemical/biological properties of the isolate showed that the virus, KV1899, inoculated suckling mouse showed paralysis and died within 7 days post-inoculation, the mouse brain suspension had hemagglutinating activity with goose RBC. Pathogenicity of isolate was carried out by intracranial and intraperitoneal inoculation of 3-4 weeks mice. The mice inoculated with isolate showed 10(4.5) LD50/ 0.03 ml and 10(3.0) LD50/0.5 ml according to the inoculation route. The isolate was identified as RNA and enveloped virus using IUDR and chloroform sensitivity test. The virus particles within the infected Vero cell were measured to be 40-50 nm in size by electron microscopy. The isolate was further characterized by immuno-fluorescence assay using Japanese encephalitis virus (JEV) specific monoclonal antibodies. Reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of JE specific conserved sequences in this isolate. The artificially inoculated pigs had HI titer of 320 to 2,560 against JEV at 14 to 42 days post inoculation. We confirmed this isolate as Japanese encephalitis virus. It was the second isolation of JEV in pigs in Korea.

日本脳炎の疫学

The five years survey results of antibody rise in summer against Japanese Encephalitis Virus in pigs in Natori district of Miyagi Prefecture were analyzed with regard to (1) the time of antibody rise, (2) the steepness of the antibody positive slope to reach to the height of 100%, (3) the level of antibody titers and (4) the detection rate of 2-ME sensitive antibodies. These serological results obtained in pigs were finally correlated to the isolation rate of the Japanese Encephalitis Virus from mosquitoes collected in the same area and to the incidence of Japanese Encephalitis patients among 1, 800, 000 residents in Miyagi Prefecture.On the basis of this limited 5 years survey, one can conclude as follows:1. A hundred percent infection among pigs and a consistent isolation of the virus from mosquitoes have been the case for these 5 years. Thus the cyclic outbreak between mosquitoes and pigs existed every year, even in the absence of the spread to human.2. The time of initial and consistent isolation of virus from mosquitoes coincided completely with the time of antibody rise among pigs, as was pointed out by us 5 years ago.3. The earlier the appearance of initial antibody rise among pigs, the higher the incidence of infection among human.4. A minor pre-outbreak and a minor post-outbreak of infection among pigs were noticed before and after the main outbreak among pigs. The fact was clearly illustrated by pursuing the 2-ME sensitive antibodies.5. The height of antibody titers in pig sera was assumed to be a reliable indicator to predict the amount of virus harbored by mosquitoes. The titer in pig sera was lowest in a year of the least incidence of human infections, irrespective of the fact that 100% of pigs were infected every year.