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Characterization of nonstructural protein 3 of a neurovirulent Japanese encephalitis virus strain isolated from a pig

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      Abstract

      Background

      Japanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5'-triphosphatase, and plays important roles in viral replication and pathogenesis.

      Results

      We characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum.

      Conclusions

      The NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.

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      Most cited references 21

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      Past, Present, and Future of Japanese Encephalitis

      Japanese encephalitis (JE) is a vector-borne viral disease that occurs in South Asia, Southeast Asia, East Asia, and the Pacific ( 1 ). An estimated 3 billion persons live in countries where the JE virus is endemic ( 2 ), and the annual incidence of the disease is 30,000–50,000 cases ( 1 ). The disease can cause irreversible neurologic damage. The JE virus (JEV) is mainly transmitted by the mosquito Culex tritaeniorrhynchus, which prefers to breed in irrigated rice paddies. This mosquito species and members of the Cx. gelidus complex are zoophilic. Wading ardeid water birds (e.g., herons and egrets) serve as virus reservoirs, but the virus regularly spills over into pigs, members of the family of equidae (e.g., horses and donkeys), and humans. The annual number of human deaths is 10,000–15,000, and the estimated global impact from JE in 2002 was 709,000 disability-adjusted life years (DALYs) ( 1 , 3 ). However, these statistics should be interpreted with care because the transmission of JE is highly dynamic; hence, the disease usually occurs in epidemics, and there is considerable fluctuation in estimates of its global impact. In 1999, JE caused 1,046,000 DALYs; in the 2 subsequent years, it caused 426,000, and 767,000 DALYs, respectively ( 3 ). Underlying factors that might explain these fluctuations are contextual determinants (mainly environmental factors) and spillover effects into the human population, which trigger epidemics. Reporting of JE cases depends on the quality of health information systems and the ability to clinically and serologically diagnose the disease. JE is often confused with other forms of encephalitis. Differential diagnosis should therefore include other encephalitides (e.g., conditions caused by other arboviruses and herpesviruses) and infections that involve the central nervous system (e.g., bacterial meningitis, tuberculosis, and cerebral malaria) ( 4 ). Figure 1 shows the transmission of JE and highlights contextual determinants. Because infected pigs act as amplifying hosts, domestic pig rearing is an important risk factor in the transmission to humans ( 1 ). Two distinct epidemiologic patterns of JE have been described. In temperate zones, such as the northern part of the Korean peninsula, Japan, China, Nepal, and northern India, large epidemics occur in the summer months; in tropical areas of southern Vietnam, southern Thailand, Indonesia, Malaysia, the Philippines, and Sri Lanka, cases occur more sporadically and peaks are usually observed during the rainy season ( 5 ). Thus far, the reasons for the spread of JE are not fully understood. Bird migration might play a role in dispersing JEV ( 6 ). Accidental transportation of vectors, human migration, and international travel seem to be of little importance because viremia in humans is usually low and of short duration and because humans are dead-end hosts ( 1 ). JE was likely introduced into northern Australia by wind-blown mosquitoes from Papua New Guinea ( 7 ) (Figure 1). Figure 1 Contextual determinants and transmission of Japanese encephalitis. The main pillar of JE control is the use of a live attenuated vaccine for humans, which was developed some 40 years ago ( 8 ). Currently available JE vaccines are relatively safe and effective, but a drawback is that multiple doses are required ( 1 , 9 ). Effective delivery of the vaccines to poor, rural communities therefore remains a formidable challenge, and compliance and delivery costs have to be considered ( 10 ). Two vaccine candidates are in late-stage clinical development. The first one is a second-generation, live inactivated, single-dose vaccine grown in Vero cells. It is the yellow fever virus–based chimeric vaccine and will soon enter the market ( 1 ). The second candidate is an attenuated SA 14–14–2 virus strain, adjuvanted with aluminum hydroxide and also grown in Vero cells ( 9 , 11 ). The vaccination of pigs represents another potential strategy to control JE, but it is not widely used for 2 main reasons. First, the high turnover in pig populations would require annual vaccination of newborn pigs, which would be costly. Second, the effectiveness of live attenuated vaccines is decreased in young pigs because of maternal antibodies ( 12 ). Environmental management for vector control, such as alternative wetting and drying of rice fields (also known as intermittent irrigation), can substantially reduce vector breeding while saving water, increasing rice yields, and reducing methane emission ( 13 ). However, an effective irrigation requires well-organized educational programs, sufficient water at specific times during the rice-growing cycle, and an adequate infrastructure. In addition, because vectors are largely dispersed, intermittent irrigation should be applied to all rice fields over large areas and during the entire cropping season, which is often not feasible ( 14 ). Environmental management measures are most viable if they are readily integrated into a broader approach of pest management and vector management ( 15 ). Chemical control of vector populations with insecticides such as pyrethroids, organophosphates, and carbamates plays a marginal role in JE control. In some circumstances (for example, when an outbreak of JE occurs in a densely populated area), space spraying can break the transmission cycle in the short term. However, rising levels of insecticide resistance have compromised the effectiveness of this emergency measure. Indeed, JE vectors that prefer manmade habitats, such as irrigated rice fields, are often heavily exposed to pesticide selection pressure. Although JE vectors are prone to develop insecticide resistance, usually this issue arises with insecticides that are not directly targeted to JE control, but rather are targeted to control of other pests ( 16 ). We provide a historic account of the origin of JE and disease epidemics, describe the current situation, and discuss several factors that might explain the rise of JE incidence in some countries and its decline in others. Finally, we speculate about possible future trends. Historic Account Genetic studies suggest that JEV originated from an ancestral virus in the area of the Malay Archipelago. The virus evolved, probably several thousand years ago, into different genotypes (I–IV) and spread across Asia ( 17 ). The history of the clinical recognition and recording of JE dates to the 19th century. JE appeared as recurring encephalitis outbreaks in the summer season. The first clinical case of JE was recorded in 1871 in Japan. Half a century later, also in Japan, a large JE outbreak involving >6,000 cases was documented. Subsequent outbreaks occurred in 1927, 1934, and 1935. In 1924 an agent from human brain tissue was isolated; 10 years later, it was proven to be JEV by transfection into monkey brains. The role of Cx. tritaeniorhynchus as a vector and the involvement of wading ardeids and pigs as reservoir hosts were demonstrated in 1938 ( 18 ). Table 1 shows when the first JE cases were described in countries currently considered JE-endemic. On the Korean Peninsula, the first JE cases were recorded in 1933. On the Chinese Mainland, the first JE cases were documented in 1940. In the Philippines, first reports of JE cases occurred in the early 1950s ( 19 ). Eventually, the JE epidemic reached Pakistan (1983) as the furthest extension in the West, and Papua New Guinea (1995) and northern Australia (Torres Straight) as the furthest south. In parts of southeastern Russia (Primorje Promorsij), a few JE cases have been reported occasionally (e.g., 2 cases from 1986 to 1990) ( 18 ). JE is potentially endemic to Afghanistan, Bhutan, Brunei Darussalam, and the Maldives, but to our knowledge, no cases have been reported in these countries in the past 30 years. According to the World Health Organization (WHO), JE is endemic to the Western Pacific Islands, but cases are rare ( 20 ). The enzootic cycle on those Pacific islands might not sustain viral transmission; hence epidemics occur only after introduction of virus from JE-endemic areas. Subtle changes in the spatiotemporal distribution of JEV are difficult to track; thus, the year when a first case of JE in a country is reported does not necessarily correspond with the actual first occurrence of JE in that country (Table 1) ( 21 – 35 ). Table 1 First reported case and current situation of Japanese encephalitis in the main disease-endemic countries Country First reported case Total population in rural JE-endemic areas (% of total)* Annual incidence† DALYs in 2002‡ Trend of JE incidence§ Vaccination program† National diagnostic center† References Australia 1995 NA§ 2 decades. The following key control strategies and developments might explain the successful decline of JE in these countries: 1) large-scale immunization programs for humans, 2) pig immunization and the separation of pig rearing from human settlements, 3) changes in agricultural practices (e.g., enhanced mechanization and decrease of irrigated land), and 4) improved living standards (e.g., better housing and urbanization). We speculate that JE incidence is increasing mainly in low-income countries. However, because reliable figures about JE emergence are lacking due to the absence of rigorous monitoring systems, more research is needed to support or refute this claim. In any event, lack of political will and financial resources are 2 important reasons why JE is often given low priority. These factors might explain the paucity of JE immunization programs for children in low-income countries where the disease is endemic. Nevertheless, Sri Lanka and Nepal, 2 countries with limited health budgets, and Thailand and Vietnam have managed to successfully control JE. The national situations with respect to JE in the near future could develop as follows. We hypothesize that in Cambodia, Laos, and Myanmar, severe JE outbreaks could occur in the near future, partially explained by increases in irrigated rice farming and enhanced pig rearing. The JE situation in North Korea is not well understood, but on the basis of the population’s general health status, we predict that JE will likely remain a substantial public health issue in the years to come. Bangladesh and Pakistan are among the worst affected and most populous countries in which JE is endemic, and yet effective surveillance is missing. Outbreaks are likely to occur but will remain largely undetected. Muslim countries such as Bangladesh and Pakistan have traditionally been JE free. JEV transmission ends in Pakistan, even though the JE vector is abundant further to the West. The recent rise in JE in those countries has yet to be fully investigated and shows the complexity of transmission of this disease. In Indonesia, Malaysia, the Philippines, and Singapore, JE incidence has usually been low, and transmission will remain stable at a relatively low level. Given the paucity of data in Indonesia, a monitoring system should be established to document changes over time. Occasional small JE outbreaks might also occur in Papua New Guinea with spillover to Australia. Awareness of the disease and vaccination coverage rates are high in Australia, particularly in the region of the Torres Strait; hence, it seems unlikely that larger epidemics will occur anytime soon. The overall trend of JE has been declining over the past 3 decades, and we anticipate that this trend will continue in the long term. Indeed, China and India influence JE figures on a global scale because most people living in JE-endemic areas are concentrated in these 2 countries. The incidence of JE in China has declined since 1971, coincident with economic growth and development. Meanwhile, the national JE vaccination program has been integrated into the Expanded Program on Immunization, and, at present, >110 million doses of a live, attenuated vaccine (SA14–14–2 strain) are produced annually. However, social, economic, and health policy changes in the face of privatization and a more market-based economy have led to reduced funding for immunization programs and somewhat reduced salaries for public health workers, particularly in the poorest provinces. As a consequence, these changes have contributed to increasing disparities in immunization coverage rates between the wealthy coastal and the less developed rural provinces and thus to the recently observed differences in levels of JE incidence between those regions ( 40 ). The incidence of JE in India is still increasing, and the case-fatality rate of reported cases is high, i.e., 10%–30% (Technical Appendix, supplementary reference 41). India currently has no national vaccination program, but the Ministry of Health has recently drawn up a plan in which children 1–12 years of age will be immunized. In Tamil Nadu and Uttar Pradesh, immunization programs are already running; thus, JE incidence might stabilize in those regions. However, overall trends for India are difficult to predict because JE endemicity is heterogeneous and because socioeconomic conditions for control differ substantially from 1 state to another (Technical Appendix, supplementary reference 42). Coverage of immunization programs and changes in agricultural practices will further influence JE transmission. In Taiwan, for example, the average age for the onset of confirmed JE cases shifted from children <10 years toward adulthood, explained by a high coverage of vaccinated children (Technical Appendix, supplementary reference 43). Interestingly, the peak JE transmission, which occurred in August in the 1960s, shifted to June beginning in the 1980s. Improvements in pig-feeding technologies, which resulted in shorter periods from birth to pregnancy of female pigs, has been proposed as an important reason explaining the shift in transmission (Technical Appendix, supplementary reference 44). Climate change has been implicated in the increase of transmission of several vector-borne diseases (Technical Appendix, supplementary reference 45). For example, a potential effect of climate change has been shown empirically for dengue virus, which is closely related to that of JE (Technical Appendix, supplementary reference 46). Although JE vector proliferation might be influenced in a similar way than that predicted for dengue vectors, the potential impact of climate change on JE remains to be investigated. Indeed, climate change could not only directly increase JE vector proliferation and longevity but could also indirectly increase disease because of changing patterns of agricultural practices such as irrigation (Technical Appendix, supplementary references 47,48). Areas with irrigated rice-production systems may become more arid in the future, and the impact of flooding will be more dramatic, which in turn might result in JE outbreaks. Generally, extreme rainfall after a period of drought can trigger outbreaks in situations in which vector populations rapidly proliferate and blood feeding is spilling over to humans. Climate change may also influence migration patterns of birds, which may result in JEVs being introduced into new areas. However, little is known about reservoir bird migration patterns; hence, this issue remains to be investigated ( 6 ). The culicines that transmit JE are usually highly zoophilic, and human outbreaks are therefore the result of a spillover of the virus from the animal reservoir into the human population. Studies in Sri Lanka showed that spillovers happen when there is rapid and dramatic buildup of Culex spp. populations to the extent that the number of human blood meals passes a threshold after which virus transmission begins (Technical Appendix, supplementary reference 49). Such rapid buildups are a result of extreme weather conditions or of rice fields in semi-arid areas being flooded before rice is transplanted. Information on vector population dynamics would be very useful in early warning systems and could also help improve targeting of control programs. In conclusion, JE can be controlled, with effective surveillance systems and vaccines playing key roles. Although currently available vaccines are effective, the need for 3–4 injections compromises compliance and increases delivery costs ( 10 ). The advent of second-generation, cell-culture–derived vaccines will continuously replace mouse-brain and hamster kidney cell–derived vaccines. Such developments will hopefully boost current vaccination programs and deliver safer, more efficacious, and cheaper vaccines that comply with regulatory norms. Political will and commitment, financial resources, intersectoral collaboration (between the Ministries of Health and Agriculture and other stakeholders to set up vaccination programs for young children), as well as changing agricultural practices, pig vaccination, rigorous monitoring, and surveillance will go a long way in controlling JE. Supplementary Material Technical Appendix Past, Present, and Future of Japanese Encephalitis
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        Ecology and geographical expansion of Japanese encephalitis virus.

        Japanese encephalitis virus (JEV) (Flavivirus: Flaviviridae) is a leading cause of encephalitis in eastern and southern Asia. The virus is maintained in a zoonotic cycle between ardeid wading birds and/or pigs and Culex mosquitoes. The primary mosquito vector of JEV is Culex tritaeniorhynchus, although species such as Cx. gelidus, Cx. fuscocephala, and Cx. annulirostris are important secondary or regional vectors. Control of JEV is achieved through human and/or swine vaccination, changes in animal husbandry, mosquito control, or a combination of these strategies. This review outlines the ecology of JEV and examines the recent expansion of its geographical range, before assessing its ability to emerge in new regions, using the hypothetical establishment in the United States as a case study.
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          Control of Japanese encephalitis--within our grasp?

           Tom Solomon (2006)
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            Author and article information

            Affiliations
            [1 ]Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Science, No. 518, Ziyue Road, Shanghai, 200241, PR China
            [2 ]Shanghai Entry-Exit Inspection and Quarantine Bureau, No. 1208, Min sheng Road, Shanghai, 200135, PR China
            [3 ]Forestry and Biotechnology School, Zhejiang Agriculture and Forestry University, Lin'an, Hangzhou, 311300, PR China
            Contributors
            Journal
            Virol J
            Virology Journal
            BioMed Central
            1743-422X
            2011
            9 May 2011
            : 8
            : 209
            3101164
            1743-422X-8-209
            21549011
            10.1186/1743-422X-8-209
            Copyright ©2011 Deng et al; licensee BioMed Central Ltd.

            This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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            Research

            Microbiology & Virology

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