REVIEW SUMMARY. Reviews in Medical Virology

Reviews in Medical Virology REVIEW Rev. Med. Virol. 2012; 22: 301 322. Published online 8 March 2012 in Wiley Online Library (wileyonlinelibrary.com).1710 Japanese encephalitis and Japanese encephalitis virus in mainland China Yayun Zheng 1,2, Minghua Li 2, Huanyu Wang 2 and Guodong Liang 1,2 * 1 School of Public Health, Shandong University, Jinan, Shandong Province, China 2 State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China SUMMARY Japanese encephalitis (JE), caused by Japanese encephalitis virus (JEV) infection, is the most important viral encephalitis in the world. Approximately 35,000 50,000 people suffer from JE every year, with a mortality rate of 10,000 15,000 people per year. Although the safety and efficacy of JE vaccines (inactivated and attenuated) have been demonstrated, China still accounts for 50% of the reported JE cases worldwide. In this review, we provide information about the burden of JE in mainland China and the corresponding epidemiology from 1949 to 2010, including the morbidity and mortality of JE; the age, gender, and vocational distribution of JE cases; its regional and seasonal distribution; and JE immunization. In addition, we discuss the relationships among vectors, hosts, and JEV isolates from mainland China; the dominant vector species for JEV transmission; the variety of JEV genotypes and the different biological characteristics of the different JEV genotypes; and the molecular evolution of JEV. Copyright 2012 John Wiley & Sons, Ltd. Received: 1 December 2011; Revised: 16 January 2012; Accepted: 19 January 2012 INTRODUCTION Japanese encephalitis (JE), which is transmitted by mosquitoes, is globally the most important viral encephalitis. JE is induced by infection with Japanese encephalitis virus (JEV) and is prevalent in Asian and Pacific areas [1 3]. Approximately 35,000 50,000 people suffer from this disease every year, causing approximately 10,000 15,000 deaths [1 3]. Of the JE survivors, 50% have sequelae for the remainder of their lives [4 6]. Approximately three billion people live in the epidemic areas [2,3]. The morbidity rate has also increased because tourism to epidemic areas has increased [5]. Japanese encephalitis was first reported in Japan in 1924 when more than 6000 cases were detected *Corresponding author: Dr. GD Liang, State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, 155 Changbai Road, Changping District, 102206 Beijing, China. E-mail: gdliang@hotmail.com Abbreviations used: China, mainland China; IPV, inactivated purified JE vaccine; JE, Japanese encephalitis; JEV, Japanese encephalitis virus; LAV, live attenuated vaccine; TMRCA, time of the most recent common ancestor. with a 60% death rate. To differentiate JE from Von A encephalitis, it was termed Japanese B encephalitis. However, when JE was later determined to be completely different from Von A encephalitis, it became known as Japanese encephalitis and abbreviated JE [1,2]. JE is mainly prevalent in Asia [1 3], and became prevalent in Australia (in the South Pacific area) since 1995 [7 10]. Recent research reported by the World Health Organization (WHO) showed that JE is prevalent in 24 countries, including districts of Asia and the Pacific [11]. JE has experienced recent outbreaks in India in 2005 where more than 5000 cases were detected with 1300 deaths [11,12]. JE typically affects children below 15 years of age [1 4,12,13], but it can also infect adults, as seen in the 2006 outbreak of JE in Shanxi province of China. In this outbreak, more than 80 cases of JE were detected in August with more than 20 deaths. More than 85% of the cases occurred in adults older than 25 years [14]. Thus, JE has become an important public health concern that has received global attention [1 3,5]. Japanese encephalitis virus is transmitted by mosquitoes, particularly culicines; Culex tritaeniorhynchus is the most important JEV-transmitting mosquito. Mosquitoes and other insects may be Copyright 2012 John Wiley & Sons, Ltd.

302 Y. Zheng et al. infected during hemophagia, and thus JEV can replicate in their bodies and be transmitted to other animals such as birds and pigs via hemophagia. Therefore, JEV naturally cycles from mosquitoes to birds or mosquitoes to pigs [1,2,15]. In tropical and subtropical areas, mosquitoes can be found continuously throughout the year, and thus a continuous viral cycle occurs between mosquitoes and animal hosts. Infections of domestic animals and poultry by JEV during the prevailing season are mostly subclinical, but the virus can proliferate in their bodies and lead to temporary viremia. Thus, they become a temporary host for JEV and the infectious source for humans. Particularly, young pigs are highly susceptible to JEV and may be the transmitting element from pigs to mosquitoes to pigs [1,2]. Japanese encephalitis virus is a member of the Flaviviridae family and is a single-strand positive chain RNA virus. The genome is approximately 11,000 nt and contains one ORF encoding the polyprotein. The entire genome encodes three structural proteins (C, M, E) and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Noncoding regions (5 and 3 ) can be found on both sides of the ORF [1,16]. JEV structural proteins consist of the glycosylated envelope protein (E) and the membrane protein (M), which forms the icosahedron nucleocapsid embedded in the phospholipid bilayer. E protein is involved in many important biological processes, including hemagglutination, virus neutralization, and viral particle assembly. The nonstructural viral proteins provide functional regulatory enzymes for JEV replication. JEV can be divided into five genotypes according to the E gene and genomic sequences [17,18]. The first case of JE in mainland China (China) was reported in 1949. Subsequently, China has become a highly epidemic area in Asia, and over 170,000 cases were reported in 1971. Since the 1950s, more than 100 JEV strains have been isolated from different hosts. This study presents an overview of the morbidity, mortality, endemic distribution, and other epidemiologic features of JE, as well as the current immunization state in mainland China. We also discuss JE viral ecology, virology, and molecular biology, focusing on the epidemic distribution of JE strains isolated in China in the last 10 years and the relationship between viruses and vectors (hosts). JAPANESE ENCEPHALITIS IN CHINA A global study on the morbidity of JE showed that it is prevalent in 24 countries (or districts) in Asia and western Pacific areas. Approximately 67,900 cases of JE occur every year (average morbidity: 1.8/100,000); among them, almost 75% of the cases (51,000) are in children 0 14 years of age (average morbidity: 5.4/100,000). The study also showed that 50% of the cases (33,900) occurred in China (not including Taiwan) [11]. China has been one of the most prevalent countries for JE since 1951 [19 21], and JE is a legally reportable infectious disease in China [19 21]. Burden of Japanese encephalitis Japanese encephalitis cases have been legally reported every year in China since 1951. The morbidity of JE from 1960 to 1970 was higher than 10/100,000, whereas its morbidity was highest in 1971. In total, 174,932 cases were reported from the country, and the morbidity was 20.92/100,000 [19,20]. Immunization with JE vaccine became common in the late 1970s. Afterward, reported cases of JE, morbidity, and mortality rates gradually decreased [22]. Between 20,000 and 40,000 cases were reported every year in the 1990s [23]. From 2000 to 2005, the reported cases decreased from 11,779 to 5097, the morbidity rate decreased from 0.9489/100,000 to 0.3898/100,000, the death toll decreased from 375 to 214, the death rate decreased from 0.0302/100,000 to 0.0164/100,000, and the mortality rates ranged between 2.51% and 4.66% [24,25]. The decreased number of JE cases in China has been particularly obvious in the last 5 years (Figure 1, Table 1) since 7643 cases were reported in 2006 (with 463 deaths) [26,27], whereas 2541 cases were reported in 2010 (with 92 deaths) [28]. Both the number of reported cases and the number of deaths significantly decreased. The reported cases of JE in 2011 further decreased and totaled 1646 cases by the end of September 2011 [28]. Because the months prior to September comprise the prevalent period for JE in China, the number of cases in 2011 may be the lowest in history, which is only 0.94% of the highest morbidity in history (1971,1646/174,932) and 13.97% (1646/11,779) of the morbidity in 2000. Although China has established a monitoring system for JE since the 1950s and included it as a legally reportable infectious disease [19 21], the sensitivity and accuracy of these reports requires further study. A study was performed, which actively monitored acute viral encephalitis in six fixed

JE and JEV in mainland China Figure 1. Morbidity of Japanese encephalitis (JE) from 1961 to 2010 in China point hospitals in four cities distributed from north to south in central eastern China during 2006 2008 (Shijiazhuang in Hebei province, Yantai in Shandong province, Yichang in Hubei province, and Guigang in Guangxi province). This study was based on the average populations in these cities and did not focus solely on JE cases. All cases were based on laboratory examinations, and the results were used to deduce the morbidity of the general population [29]. Of 4513 cases of acute viral encephalitis (2294 samples were collected), 213 were confirmed to be JE after laboratory examination (9.2%, 213/2294). The morbidity rates of JE in local areas were estimated according to the general populations. Among them, the morbidity rate of JE in Shijiazhuang in North China was 0.08/100,000, whereas the morbidity rate in Guigang in South China was 1.58/100,000. The morbidity rates in south and north China differed by 20-fold [29]. As the WHO proposed that the morbidity rate in 303 populations below 15 years of age in epidemic areas of JE should be controlled below 0.5/100,000 by 2015 [11], the morbidity rates in the four areas previously may significantly increase if the local populations below 15 years of age are exclusively evaluated. The statistical data from the fifth population census in 2000 showed a population size of 1,242,612,226 people in China, whereas the percentage of children 0 15 years old accounted for 24.54% [30]. Furthermore, the morbidity rate of JE (deduced from laboratory examination) during the prevalent season in local areas was two to three times higher than the reported morbidity rate. In other words, the annual reported morbidity rate of JE on the basis of the monitoring network in China is two to three times lower than the actual morbidity rate. One explanation is that although JE is a national legally reportable infectious disease (every case of JE should be reported to the center for disease control and prevention (CDC)), the accuracy and timing of these reports may be affected because the diagnosis and report of JE involve many factors such as the diagnostic level of clinical physicians, laboratory detection rate for the samples from each case, reliability of laboratory detection results, and the timing of the case report. Therefore, the accuracy of reported JE cases may require improvement [21,29]. Of the 1382 JE cases reported in Guizhou province in 2006, only 1210 cases were positive by IgM and PCR for JEV infection, which accounts for 87.6% of all reported cases (1210/1382). Among the 172 negative JE samples, 38.9% (67/172) were detected with specific antibodies to enteric viruses (enteric cytopathogenic human orphan virus and coxsackie virus), herpes simplex virus, mumps virus, and Table 1. The incidence and death data of Japanese encephalitis (JE) in 2001 2010, China Year No. of JE cases Incidence (1/100 000) No. of death cases Mortality (1/100 000) Case fatality (%) 2001 9795 0.7707 246 0.0194 2.511 2002 8769 0.6548 229 0.0171 2.611 2003 7860 0.5829 366 0.0271 4.6565 2004 5422 0.4171 200 0.0154 3.6887 2005 5097 0.3898 214 0.0164 4.1985 2006 7643 0.58 463 0.035 6.1 2007 4330 0.33 227 0.017 5.2 2008 2975 0.23 142 0.011 4.8 2009 3913 0.29 172 0.013 4.4 2010 2541 0.19 92 0.0069 3.6 Total 58345 0.4428 2351 0.0179 4.0295

304 Y. Zheng et al. cytomegalovirus. This study also indicated that the legally reported cases of JE by the monitoring system in China included some other cases of viral encephalitis [31]. Therefore, improvement of JEV laboratory detection will improve the accuracy of the reported JE cases. The burden of JE can only be determined after gathering accurate data regarding the onset of JE, providing a theoretical basis for JE prevention and control [21,29,31]. Mortality due to Japanese encephalitis Japanese encephalitis causes serious viral encephalitis with a mortality rate of approximately 35 40%, or even as high as 70% [1,2]. In 1950, the mortality rate of JE in China was 35% [22,23]. The use of a JE vaccine since the 1960s significantly decreased the morbidity rate of JE in China [19,20,23], and recently, the mortality rate of JE in China was found to continue to decrease. The mortality rate decreased 10 20% in the 1960s, 10 12% in the 1970s, 5 12% in the 1980s, and 2 5% in the 1990s, and has decreased 2 3% since 2000 [19,20,22 25]. Theoretically, use of the JE vaccine should reduce the morbidity but have no effect on the mortality rate. Because significant economical differences exist between the Chinese provinces, the inoculation rate and efficacy of the JE vaccine in China is extremely unbalanced. JE vaccination in economically developed cities such as Beijing and Shanghai is complete (the full JE vaccination with inactivated JEV requires four dosages), resulting in a low JE morbidity and prevalence in these areas [32]. Whilst China has the policy of immunization in all JEV prevalent regions, failure to complete the full immunization schedule for the JE vaccine in children has been common in remote provinces or mountain areas, particularly rural areas. Thus, although many children will have received at least one dose of vaccine [33,34], the consequent low-level immunity may be incapable of preventing infection. Nevertheless, exposure to the virus might trigger an anamnestic response, which could explain why even though the JEV infection rate is relatively high in children in rural areas of China, the mortality rate is lower than that observed in India, where immunization coverage and therefore seroprevalence in children is likely to be lower and the mortality rate of JE in India in 2005 2006 was 35% [12]. Distribution of Japanese encephalitis Japanese encephalitis cases have been reported every year since establishment of the monitoring system in 1951, and the reported cases are scattered in different areas. Transformation of Japanese encephalitis distribution Japanese encephalitis onset in the 1970s and 2010 can be divided into four groups according to the morbidity rate, namely, non-prevalent areas, low-morbidity areas, moderately epidemic areas, and highly epidemic areas (Figure 2). Because the morbidity rates of the two different periods changed significantly, different criteria were used to compare the data. The most obvious feature for territory distribution of JE in China in the 1970s or 2010 is that a small number of cases were reported in west and north China, whereas a high number of cases occurred in east and southwest China. Many provinces that had high morbidity rates transformed into moderate and low epidemic areas with moderate or low morbidity rates after 2000. Five moderate JE epidemic provinces were identified in 2005 (SX, HN, HuB, HuN, and GX) along with 18 low epidemic provinces (AH, BJ, FJ, GD, GS, HaiN, HeB, HLJ, JL, JS, JX, LN, IMG, NX, SD, SH, TJ, ZJ) that were moderate epidemic areas in the 1970s. From 1970 to 2010, only Heilongjiang and Inner Mongolia were constantly low epidemic areas, and thus the major epidemic areas in China have changed from the eastern littoral areas in the 1970s to the southwestern provinces. The rapid economical development in eastern littoral areas because the reformation and opening of China allows for a relatively high JE vaccination rate in these areas, which explains the decreased JE morbidity [19,20,23,27,33,34]. Five provinces in southwest China are still highly epidemic areas for Japanese encephalitis Five provinces in China have a mean JE morbidity rate higher than 1/100,000. These provinces are located in southwest China and the middle area of China, and the highly epidemic provinces are adjacent (Figure 2). The highly epidemic provinces include Henan, Chongqing, Sichuan, Guizhou, and Yunnan. The annual number of JE cases in the five provinces accounts for more than 50% of the total cases and was as high as 74.1% in 2010, indicating that the onset of JE in China is concentrated in these highly epidemic provinces. The populations in these provinces accounts for only 26% of the total population in China. Immunization with vaccines in highly epidemic provinces is important to further decrease the morbidity rate of JE in China [21].

JE and JEV in mainland China 305 Figure 2. Comparison of geographic distribution of Japanese encephalitis (JE) case in the 1970s and 2000s, China. Symbols: AH, Anhui; BJ, Beijing; CQ, Chongqing; FJ, Fujian; GD, Guangdong; GS, Gansu; GX, Guangxi; GZ, Guizhou; HaiN, Hainan; HB, Hebei; HLJ, Heilongjiang; HN, Henan; HuB, Hubei; HuN, Hunan; IMG, Inner Mongolia; JL, Jilin; JS, Jiangsu; LN, Liaoning; NX, Ningxia; QH, Qinghai; SaX, Shaanxi; SC, Sichuan; SD, Shandong; SH, Shanghai; SX, Shanxi; TJ, Tianjin; XJ, Xinjiang; XZ, Tibet; YN, Yunnan; ZJ, Zhejiang Non-epidemic areas of Japanese encephalitis Although China is a highly epidemic country for JE and the annual morbidity accounts for 50% of the total number of estimated cases by the WHO [11], no local case of JE has been reported in Xinjiang, Tibet, or Qinghai, even during the highly epidemic 1970s and after 2000 (more details and possible explanations are provided later in the text). Therefore, these three provinces are considered to be JEfree areas [19,20]. Although JEV was isolated from mosquito samples collected in Tibet in 2009 and neutralizing JEV antibody can be detected in local populations and raised pigs [17,35], JE was not observed. Seasonal distribution of Japanese encephalitis Japanese encephalitis is typically seen from January to December, with fewer cases reported from January to May and November to December. However, the number of cases rapidly increases after June, and the morbidity in June is twice that in May. July and August have the highest morbidity. This high level of morbidity is maintained in September and then significantly decreases in October. The morbidity in August accounts for 41.14% of the total annual morbidity. As shown in Figure 3, the distribution Figure 3. Seasonal distribution of Japanese encephalitis (JE) case from 2006 to 2010 in China tendency of JE between 2006 and 2010 is very similar between different months. China has a broad territory; therefore, the number of JE cases in southern and northern China differs significantly. Normally, the morbidity in southern China increases after July and significantly decreases at the end of August, whereas the morbidity in northern China increases after August and significantly decreases in the middle of September.

306 Y. Zheng et al. Figure 4. Monthly distribution of all Japanese encephalitis (JE) cases in China. Symbols: AH, Anhui; BJ, Beijing; CQ, Chongqing; FJ, Fujian; GD, Guangdong; GS, Gansu; GX, Guangxi; GZ, Guizhou; HaiN, Hainan; HB, Hebei; HLJ, Heilongjiang; HN, Henan; HuB, Hubei; HuN, Hunan; IMG, Inner Mongolia; JL, Jilin; JS, Jiangsu; LN, Liaoning; NX, Ningxia; QH, Qinghai; SaX, Shaanxi; SC, Sichuan; SD, Shandong; SH, Shanghai; SX, Shanxi; TJ, Tianjin; XJ, Xinjiang; XZ, Tibet; YN, Yunnan; ZJ, Zhejiang Peak JE cases occur in June in some areas of HaiN, GD, and GX, south of the Tropic of Cancer. In addition, because the temperature in some local areas is relatively high all year, JE occurs year-round. The onset of JE in the nine provinces in the middle and downstream areas of the Yangtze River (ZJ, SH, JS, JX, FJ, AH, HN, HuB, and HuN) occurs in July. The onset peaks in SC, YN, CQ, GZ, GS, SD, SaX, and SX are mainly in August. The onset of JE in northern China, including IMG, NX, HB, BJ, LN, TJ, JL, and HLJ, typically occurs in September (Figure 4). Between 2006 and 2010, the morbidity and the mortality of JE significantly increased after June, peaked in July and August, and decreased after October. This suggests that morbidity and the mortality rate of JE fluctuates among different months [28,36 39]. Vocation, gender, and age Vocation The data from a vocational distribution of JE cases from 1996 to 2005 showed that scattered children (Children whose parents are working in faraway places and do not live with them lack care and may have failed to accept full immunization. They are mainly from rural areas.) account for 54.51% of the total cases in all of the vocations and 54.72% of the total deaths [19,20,24,25,27]. The vocational distributions for the cases reported in 2005 were mainly scattered children, students, and kindergarten children, which accounted for 88.5% of the total reported cases. Among them, scattered children accounted for 53.6%, students comprised 23.4%, kindergarten children represented 11.5%, and farmers accounted for 8.3%. Death cases were mainly scattered children (50.3%), followed by students (21.1%), farmers (15.8%), and kindergarten children (7.0%) [19,20,24,25,27]. Gender Male cases of JE are significantly more common than female cases, and the recent ratio (the last 5 years) is 1.3:1 [19,20,27].

JE and JEV in mainland China Japanese encephalitis occurs mainly in young children Japanese encephalitis cases are reported in different age groups from 0 to >75 years old. The constituent rates in 2, 3, and 4-year-old age groups are relatively high, which account for 10.88%, 12.88%, and 12.33% of the total cases, respectively. Children below 6 years of age account for 68.10% of the total cases. Children below 15 years of age from 2000 to 2005 account for 91.19%, 91.26%, 91.06%, 90.14%, 89.80%, and 90.6%, respectively, of the total JE cases [20,27]. Adult Japanese encephalitis As discussed previously, more than 90% of JE cases occur in children under 15 years of age in China. However, adult JE cases have also been reported in recent years [40] and showed an epidemic outbreak tendency [14]. Adult JE cases were mainly found in the provinces located north of the Yellow River in China, such as SX, HN, SD, and GS [27]. JE in adults older than 40 years (of the 45 cases of JE IgM antibody-positive patients hospitalized in Shanxi province, located in the middle area of the Yellow River) accounted for 77.8% (35/45) of the total cases, whereas only 10 cases were in children below 15 years of age. Serious and extremely serious cases of JE accounted for 60.0% (27 cases) of the total, indicating that the JE in this area is characterized by onset in higher age groups [40]. In 2006, an outbreak of JE, which was confirmed by laboratory testing, was reported in Yuncheng of Shanxi province in China [14,41]. In total, 66 cases were reported in July and August; 19 deaths occurred with a mortality rate of 28.8% (19/66). The age distribution of the cases was as follows: children below 7 years of age accounted for 13.4%, whereas adults over 30 years of age comprised 86.6%. Of the deaths, 94.74% were in age groups above 50 years [14]. The cause of adult JE remains unknown. Children in Japan and Korea are generally protected because these two countries include JE vaccination in the basic immunization program for children, whereas adults may fall ill because they are not protected by the vaccine [2]. Although the JE vaccine was developed in China in the 1960s, it was not used throughout the country until recently. Therefore, adults are not inoculated with 307 the JE vaccine, which may explain the presence of adult JE in China [23,24,42]. Japanese encephalitis vaccination program Japanese encephalitis is a preventable disease by vaccination. Because JE vaccination is included in the conventional immunization schedule in Japan and Korea, only a small number of JE cases have been reported every year since 1965, and the disease has basically been eliminated [2]. In China, following the successful development of inactivated JEV vaccine in the 1960s, an attenuated JE vaccine was developed and mass-produced in the 1990s [42,43]. JE vaccination has expanded since its successful development (Figure 1), but the generalized vaccination schedule underwent several modifications [44 46]. The cost of JE vaccination was paid by the vaccinated person from the 1960s to 2000 in China, and thus only families in good economical conditions could afford the inoculation for their children. This situation is highlighted in western provinces in China where the economy is relatively underdeveloped and inoculation with the JE vaccine is extremely unbalanced. This also explains the 10,000 JE cases in 2000, despite the two forms of JE vaccines available. Since 1978, the economic conditions in certain areas of China have improved significantly, particularly in the eastern littoral provinces. The investment in public health rapidly increased and the JE vaccine is now included in the planned immunization. Sixteen provinces in China have included JE vaccination in the local planned immunization management in 2005, and local children can be inoculated for free. Therefore, the number of JE cases in many formerly highly epidemic provinces in east China has rapidly declined. The central government of China announced in 2008 that 15 types of vaccines for infectious diseases have been included in the planned immunization, including JE. JE vaccination is now free of charge in epidemic areas in China, which explains why only 2541 cases were reported in 2010 (Table 1). For the reinforcement of the information management for JE vaccination in China, the Chinese government implemented the electronic management system for recording planned vaccination. This system requires the local CDC to establish electronic archives for every vaccinated child and connect them to the related administration in the Chinese Center for Disease Control and Prevention (China CDC). Thus,

308 Y. Zheng et al. inoculation records can be found in the local CDC, and these records will be permanently stored as historic archives for preventative inoculation of children. This allows the China CDC to control local vaccination all over the country using real-time management. This system ensures that inoculation records are not maintained by only the inoculated person [42]. In the past, because of missing inoculation records, children that hadbeeninoculatedwithonejedosagewerealways treated as complete immunization cases, leading to numerous incomplete immunizations. This explains the high number of JE cases in rural areas. Japanese encephalitis ecology Mosquitoes, birds, and pigs are major JEV propagative hosts. Mosquitoes can infect animals or humans with JEV through biting. Therefore, JE is a type of zoonosis [1,2,10,15]. In the provinces south of the Yangtze River in China, the temperature is humid all year with high amounts of precipitation, allowing for rice planting. Villages in rural areas in southern China are separated and surrounded by paddy fields. In other words, the families are surrounded by paddy fields, leading to propagation of mosquitoes. Pig is an important economic resource for farmers, and accordingly, in many regions of China, they are raised in pigpens in the farmer s yards in close proximity to the human residents. Because the mosquitoes that transmit JEV to humans also feed on pigs (Figure 5), this provides an ideal environment both for the amplification of mosquito population density and the transmission of JEV amongst humans [47,48]. In many cases, this explains why it can be so difficult to eliminate JEV in these typical rural regions. JAPANESE ENCEPHALITIS VIRUS IN CHINA China probably has the highest endemicity of JE in Asia. First reported in 1949, the disease has existed in China for more than 60 years. With its vast region, diverse climates, various environments, and abundant insect species, China is host to a particularly complex set of JEV genotypes. Therefore, it is necessary to assess the epidemic situation of encephalitis, as well as vaccine protection and disease trends in China, on the basis of the etiology and ecology of JEV. Extensive surveys of arboviruses that started in 2000 have isolated 145 JEV genotypes (Table 2) from insects (e.g. mosquitoes, midges), bats, porcine, and human samples, collected from various provinces and environments. This has provided important etiological information about JEV in China. This part analyzes JEV in China with respect to the geographical distribution, the insect vectors, and the original environment and genotype of JEVs. Figure 5. Environment for Japanese encephalitis virus transmitting in mosquito pig cycle and human infection

JE and JEV in mainland China 309 Table 2. Japanese encephalitis virus isolates in China, 1949 2009 Year Location Strain Source Genotype Accession no. 1949 Beijing A2 Human brain III AY243843 Beijing P3 Human brain III AY243844 Beijing Beijing-1 Human brain III L48916 1950s Heilongjiang 47 Human brain III AY243827 1954 Yunnan YN Human brain III AY243838 Fujian G35 Mosquito III AY243831 Fujian CBH Human brain III DQ404116 Fujian CZX Human brain III AY243828 1955 Fujian YLG Human brain III AY243837 Fujian ZSZ Human brain III AY243839 Fujian LFM Human brain III AY243833 Fujian CTS Human brain III AY243830 Fujian ZMT Human brain III AY243840 1957 Fujian LYZ Human brain III AY243834 Sichuan CH-13 Human brain III AY243835 1960 Shanxi SA14 Mosquito III AY243850 Shanxi SA14-14-2 Vaccine III AF315119 1960s Heilongjiang Ha-3 Human brain III AY243842 Beijing GSS Human brain III AY243845 1971 Liaoning TLA Human brain III AY243832 1979 Yunnan YN79-Bao83 Culex tritaeniorhynchus I DQ404128 1982 Yunnan YN82-BN8219 Culex I DQ404129 1983 Yunnan YN83-Meng83-54 Forcipomyia taiwana Shiraki I DQ404130 Yunnan YN83-83199 Culex I DQ404131 1985 Yunnan YN85-L86-99 Culex I DQ404132 1986 Yunnan YN86-B8639 C. tritaeniorhynchus I DQ404133 Yunnan YN86-86266 IU I DQ404134 Yunnan YN-XiangJE Human blood I DQ404135 1987 Shanghai SH-3 Human blood III AY243826 1989 Yunnan B58 Bat brain III FJ185036 1997 Yunnan GB30 Bat brain III FJ185037 1998 Yunnan YN98-151 Mosquito III DQ404136 2001 Shanghai SH-53 C. tritaeniorhynchus I AY555757 Shanghai SH-80 C. tritaeniorhynchus I AY243841 Shanghai SH-81 C. tritaeniorhynchus I AY555758 Shanghai SH-83 C. tritaeniorhynchus I AY555759 Shanghai SH-90 C. tritaeniorhynchus I AY243835 Shanghai SH-96 C. tritaeniorhynchus I AY555760 Shanghai SH-101 C. tritaeniorhynchus I AY555761 2002 Heilongjiang HLJ02-134 Genus culicoides III DQ404081 Heilongjiang HLJ02-136 Genus culicoides III DQ404082 Heilongjiang HLJ02-144 Aedes vexans III DQ404083 Heilongjiang HLJ02-170 A. vexans III DQ404084 Liaoning LN02-102 Culex modestus I DQ404085 Liaoning LN02-104 Culex pipiens pallens I DQ404086 Fujian 02-29 Human CSF III AY555762 (Continues)

310 Y. Zheng et al. Table 2. (Continued) Year Location Strain Source Genotype Accession no. Fujian 02-41 Human blood III AY555763 Fujian 02-43 Human blood III AY555764 Fujian 02-76 Human blood III AY555765 Fujian 02-84 Human blood III AY555766 Fujian 02-102 Human blood III AY555767 2003 Shanghai SH03-103 C. tritaeniorhynchus I DQ404096 Shanghai SH03-105 C. tritaeniorhynchus I DQ404097 Shanghai SH03-109 C. tritaeniorhynchus I DQ404098 Shanghai SH03-115 C. tritaeniorhynchus I DQ404099 Shanghai SH03-124 C. tritaeniorhynchus I DQ404100 Shanghai SH03-127 C. tritaeniorhynchus I DQ404101 Shanghai SH03-128 C. tritaeniorhynchus I DQ404102 Shanghai SH03-129 C. tritaeniorhynchus I DQ404103 Shanghai SH03-130 C. tritaeniorhynchus I DQ404104 Fujian FJ03-31 Human blood III DQ404117 Fujian FJ03-35 Human blood III DQ404118 Fujian FJ03-39 Human blood III DQ404119 Fujian FJ03-46 Human blood III DQ404120 Fujian FJ03-56 Human blood III DQ404121 Fujian FJ03-66 Human blood III DQ404122 Fujian FJ03-67 Human blood III DQ404123 Fujian FJ03-68 Human blood III DQ404124 Fujian FJ03-69 Human blood III DQ404125 Fujian FJ03-94 Human blood III DQ404126 Fujian FJ03-97 Human blood III DQ404127 2004 Henan HN04-11 Culex I DQ404087 Henan HN04-21 Culex I DQ404088 Henan HN04-40 Culex I DQ404089 Sichuan SC04-12 Culex I DQ404090 Sichuan SC04-15 C. tritaeniorhynchus I DQ404091 Sichuan SC04-16 Armigeres I DQ404092 Sichuan SC04-17 C. tritaeniorhynchus I DQ404093 Sichuan SC04-25 Culex I DQ404094 Sichuan SC04-27 Culex I DQ404095 Shanghai SH04-3 C. tritaeniorhynchus III DQ404105 Shanghai SH04-5 C. tritaeniorhynchus III DQ404106 Shanghai SH04-10 C. tritaeniorhynchus III DQ404107 Guizhou GZ04-2 Armigeres III DQ404109 Guizhou GZ04-4 Armigeres III DQ404110 Guizhou GZ04-29 Culex III DQ404111 Guizhou GZ04-36 Culex III DQ404112 Guizhou GZ04-43 Culex III DQ404113 Guizhou GZ04-71 Armigeres III DQ404114 Guizhou GZ04-89 Armigeres III DQ404115 Yunnan YNDL04-1 C. tritaeniorhynchus III DQ404137 Yunnan YNDL04-6 Culex pipiens quinquefasciatus III DQ404138 (Continues)

JE and JEV in mainland China 311 Table 2. (Continued) Year Location Strain Source Genotype Accession no. Yunnan YNDL04-29 Culex theileri III DQ404139 Yunnan YNDL04-31 C. theileri III DQ404140 Yunnan YNDL04-37 Anopheles sinensis III DQ404141 Yunnan YNDL04-39 C. tritaeniorhynchus III DQ404142 Yunnan YNDL04-42 Armigeres subalbatus III DQ404143 Yunnan YNDL04-44 C. theileri III DQ404144 Yunnan YNDL04-45 Mosquito III DQ404145 Yunnan YNJH04-18 Mosquito III DQ404146 Yunnan YNJH04-19 Unclassified Culex III DQ404147 Yunnan YNJH04-25-3 Culex fuscocephalus III DQ404148 2005 Shanghai SH05-24 C. tritaeniorhynchus I DQ404108 Guangxi GX0519 C. tritaeniorhynchus I NA Guangxi GX0523 C. tritaeniorhynchus I NA Guangxi GX0558 C. tritaeniorhynchus I NA Yunnan YN0528 C. tritaeniorhynchus I NA Yunnan YN0553 C. tritaeniorhynchus I NA Yunnan YN0555 C. tritaeniorhynchus I NA Yunnan YN0572 C. tritaeniorhynchus III NA Yunnan YN0581 C. tritaeniorhynchus I NA Yunnan YN0591 C. tritaeniorhynchus I NA Yunnan YN05124 C. tritaeniorhynchus I NA Yunnan YN05128 C. tritaeniorhynchus I NA Yunnan YN05133 C. tritaeniorhynchus I NA Yunnan YN05155 C. tritaeniorhynchus I NA Yunnan YN05194 Mosquito I NA 2006 Shanghai SH0601 Pig III EF543861 Yunnan YN0623 C. tritaeniorhynchus I NA Yunnan YN0686 C. tritaeniorhynchus I NA Guangxi BL50 Armigeres subalbatus I NA Guangxi BL53 Armigeres subalbatus I NA Guangxi BL54 Armigeres subalbatus I NA Guizhou GZ1 Human CSF I NA Guizhou GZ56 Human CSF I HM366552 Gansu GS105 Human CSF I NA Henan HN06-21 Culex I NA Henan HN06-22 Culex I NA Henan HN06-23 Culex I NA Henan HN06-26 Culex I NA Henan HN06-127 Culex I NA Henan HN06-129 Culex I NA Henan HN06-130 Culex I NA Shanxi SX06M-5 Mosquito I NA Shanxi SX06M-18 Mosquito I NA Shanxi SX06M-27 Mosquito I NA Shanxi SX06M-29 Mosquito III NA Shanxi SX06M-30 Mosquito III NA (Continues)

312 Y. Zheng et al. Table 2. (Continued) Year Location Strain Source Genotype Accession no. Shanxi SX06CSF-1 Human CSF I NA Shanxi SX06CSF-3 Human CSF III NA Shanxi SX06CSF-4 Human CSF I NA Shanxi SX06CSF-5 Human CSF III NA Shanxi SX06CSF-11 Human CSF I NA Shanxi SX06CSF-12 Human CSF I NA Gansu GS07-TS11 Culex I NA Gansu GS07-TS61 Armigeres I NA Gansu GS07-KD15 Culex I NA Gansu GS07-KD40 Culex I NA Liaoning LNDG07-02 C. tritaeniorhynchus I NA Liaoning LNDG07-16 C. tritaeniorhynchus I NA Yunnan TC07018 C. tritaeniorhynchus I NA Yunnan TC07172 C. tritaeniorhynchus I NA Zhejiang XJ69 C. pipiens pallens I EU880214 Zhejiang XJP613 C. tritaeniorhynchus I EU693899 Henan HEN0701 Pig I FJ495189 2008 Gansu GSBY0801 Culex I NA Gansu GSBY0804 Culex I NA Gansu GSBY0810 Culex I NA Gansu GSBY0816 Culex I NA Gansu GSBY0827 Culex I NA Gansu GSBY0861 Culex I NA Liaoning LN0828 C. tritaeniorhynchus I NA Shandong SD08-10 C. tritaeniorhynchus I NA Yunnan YN114 Human CSF I NA Yunnan YN135 Human CSF I NA Zhejiang JX61 Pig I GU556217 Henan CSFBY-1 Pig III GQ845081 Henan CSFBY-2 Pig III GQ845082 Henan CSFZMD-3 Pig III GQ336809 Henan CSFZMD-4 Pig III GQ845083 2009 Jiangxi JX0939 C. tritaeniorhynchus I NA Yunnan YN0911 C. tritaeniorhynchus I NA Yunnan YN0967 C. tritaeniorhynchus I NA Hubei HBZG08-09 Mosquito I NA Hubei HBZG08-55 Mosquito I NA Tibet XZ0934 C. tritaeniorhynchus V NA Tibet XZ0938 C. tritaeniorhynchus I NA IU, Information unavailable; NA, not available. History Although the first JEV strain was isolated in China in 1940, the sample was not preserved. Dr. Huang Chenhsiang, the pioneer of virology research in China, and his team isolated a virus strain from a viral encephalitis patient in August 1949 (known as Beijing-1 virus) and the P3 strain from another encephalitis patient on 2 September 1949 [2,48]. And the vaccine virus strain SA14-14-2 was derived from a wild-type JE virus SA14 isolated

JE and JEV in mainland China from pool of Culex pipiens mosquito larvae in Xi an, China [43]. Thereafter, many JEVs were isolated from various regions, but because not all isolates were preserved, only 19 JEVs were collected for molecular epidemiological study, including 2 strains isolated in 1949, 12 strains in the 1950s, 3 in the 1960s, 1 in the 1970s, and 1 in the 1980s [48,49]. China has strengthened its virus-monitoring procedures, and since 2000, 145 JEVs have been isolated from insect vectors (including mosquitoes and midges [50,51]), bats [52], porcine, and cerebrospinal fluid and serum samples of patients with viral encephalitis (Table 2). Distribution of Japanese encephalitis virus isolates Located in East Asia on the western Pacific Rim, China is the largest country in Asia, which ranges from a northern latitude of 3 51 N 53 33.5 N from south to north and across an eastern longitude of 73 33 E 135 05 E from west to east [53]. The overall geographic elevation is higher in the west, with numerous mountains such as the Pamirs and the Qinghai-Tibet Plateau, and lower in the east. Therefore, the average temperature is lower in the western 313 provinces with drought and poor vegetation, whereas the average temperature is higher in the east with a rainy climate, abundant vegetation, and diverse vector insects [53]. Owing to the high-altitude and low-average temperatures, no JEV or JE cases have been reported in three western provinces in China (Xinjiang, Qinghai, and Tibet), and these provinces are considered to be non-endemic areas [19 21]. In more than 20,000 mosquitoes collected during the arbovirus survey of different areas in Xinjiang and Qinghai provinces during 2005 2010, not only no JEV and positive-jev genome was found in specimens, but also no C. tritaeniorhynchus was found there [54 56], indicating that JEV is absent in Xinjiang and Qinghai. However, recent studies have shown that a natural foci of JEV already existed in parts of Tibet. Discovered in Tibet for the first time, abundant C. tritaeniorhynchus (the main vehicle of the JEV) were found in Motuo County in 2009. JEV was isolated from the mosquito specimens, and in addition, high levels of anti-jev antibodies were found in the serum of residents and domestic pigs [35,57]. This suggested that Tibet has JEV vectors and amplification hosts, making it a new JE-endemic Figure 6. Distribution of Japanese encephalitis virus isolates in mainland China (2000 2009). Symbols: AH, Anhui; BJ, Beijing; CQ, Chongqing; FJ, Fujian; GD, Guangdong; GS, Gansu; GX, Guangxi; GZ, Guizhou; HaiN, Hainan; HB, Hebei; HLJ, Heilongjiang; HN, Henan; HuB, Hubei; HuN, Hunan; IMG, Inner Mongolia; JL, Jilin; JS, Jiangsu; LN, Liaoning; NX, Ningxia; QH, Qinghai; SaX, Shaanxi; SC, Sichuan; SD, Shandong; SH, Shanghai; SX, Shanxi; TJ, Tianjin; XJ, Xinjiang; XZ, Tibet; YN, Yunnan; ZJ, Zhejiang

314 Y. Zheng et al. region and natural foci of JEV [35,57]. Collectively, the above studies showed that the distribution of JEVextended to 95º E in China, in which JEV was previously only found in Sichuan Province (97º 21 E) (Figure 6). The Chinese inland province, Inner Mongolia, is historically the most prominent JE-endemic area with an average annual incidence rate of 1.55 JE cases per 100,000 people. C. tritaeniorhynchus was the most locally dominant mosquito species in the 1970 1980s [58,59], and many JEVs were isolated from them. However, because of climate warming in recent years resulting in grassland desertification and reduced rainfall, the local mosquito species composition has changed greatly. Mosquito samples were collected for species identification and mosquito-borne arbovirus research during two consecutive summers (2007 and 2008) at 12 sites in four cities in Inner Mongolia, from which 10,542 specimens including three genera (Culex, Aedes, and Anopheles) and seven species were obtained. The results showed that Aedes was the locally dominant mosquito species making up 79.9% of all collected mosquitoes (8,426/10,542), followed by Culex mosquitoes (19.5%; 2,049/10,542) and Anopheles mosquitoes (0.6%; 67/10,542), whereas C. tritaeniorhynchus was not represented in the collected mosquitoes [60,61]. Six viral strains were isolated from the mosquito samples, which were identified as two strains of Tahyna virus, one strain of Banna virus, and three strains of C. pipiens pallens densovirus; however, JEV was not detected [60,61]. This suggests that Aedes is currently dominant in Inner Mongolia; however, C. tritaeniorhynchus was the locally dominant mosquito in the 1970s to the 1980s [59,62]. In summary, the current JEV distribution in China spans the latitudes of 50 in Heilongjiang to 15 in Hainan, and from the longitudes of 95º E in Tibet to 130º E in Heilongjiang (Table 2, Figure 6). Xinjiang and Qinghai remain JE-free areas. Inner Mongolia, which was the traditional JE-endemic area in northern China, has no virus distribution at present. Japanese encephalitis virus isolates and elevation Mosquitoes are metamorphic insects that require water for their various stages of development [15]. Because they are readily maintained in areas of rainfall, mosquito species and density are high at low altitudes of China, owing to high average annual temperature and heavy rainfall, whereas high altitudes are not conducive to mosquito breeding [63]. Since 2000, JEV has been identified in mosquito specimens isolated in the mountains from elevations of 2 to 3000 m in China [64,65]. A three-dimensional study examining the distribution of mosquito species and JEV for two years (2005 and 2006) was performed in the Gaoligong Mountains in the Yunnan Province (24º 00 to 29º 00 N). The sample locations ranged from 24º 00 N to 29º 00 N at a latitude of 11º with samples collected at every 30 of gradient. The altitude ranged from 900 to 3280 m, which was divided into five gradients of 900, 1500, 2000, 2500, and 3000 m. The results demonstrated that there were significant differences in the number of mosquitoes among the altitudes: the most were found at 1500 m, followed by 900 m, and the number was drastically lower above 2000 m. JEV was isolated from mosquito specimens in each gradient below 3000 m in the sample range within the latitudes of 25º 30 N to 28º 00 N, with no significant difference in strain number. JEV was not isolated above 3000 m. The maximum altitude where JEV was isolated was 2900 m, which is the highest altitude reported at present [65]. Previous studies isolated JEV at an altitude of 2000 m [63,66]. According to the data from the JEV-monitoring network, Yunnan Province has a high incidence of JEV with perennial JEV cases [34], indicating that the prevalence of JEV is consistent with its high-altitude distribution. Japanese encephalitis virus and vectors The population diversity of JEV vectors in China could be obtained from the origin of isolated virus strains in recent years (Table 2). More than 100 JEVs have been isolated in China since 2000. Of the 89 JEVs isolated from mosquito specimens of Culex, 78 were from C. tritaeniorhynchus (Table 2), indicating that C. tritaeniorhynchus is the major JEV vector in China [17,19,20,50,51,67 90]. JEVs were also isolated from C. theileri [70], C. pipiens pallens [75,81], C. modestus [81], C. pipiens quinquefasciatus, and C. fuscocephalus [70]. Ten JEVs were isolated from mosquito samples of Armigeres, and four were isolated from A. subalbatus [69,82], indicating that Armigeres is an inferior JEV vector compared with Culex. JEV was also isolated from Aedes vexans [51] and Anopheles sinensis [70]. Two strains of JEVs were isolated from blood-sucking midges in China

JE and JEV in mainland China 315 Table 3. Japanese encephalitis virus (JEV) and dominant species of mosquito in different areas of China Site JEV (strain) Dominant mosquito Species a (b/c) Reference Liaoning 2 Culex tritaeniorhynchus 100% (1500/1500) [61] Gansu 6 90.4% (6250/6917) [74] Henan 7 90.1% (5614/6231) [71] 3 80.3% (2990/3722) [66] Jiangxi 1 88.8% (2570/2893) [77] Shanghai 7 80% (8974/11218) [65] Tibet 2 75.2% (2297/3053) [17,35] Yunnan 5 56.7% (2728/4810) [68] Heilongjiang 2 Aedes vexans 87.4% (1735/1985) [81] Guangxi 3 Armigeres subalbatus 91.8% (2588/2828) [80] 3 C. tritaeniorhynchus 64.5% (771/1196) [69] Sichuan 1 Armigeres 79.5% (3710/4668) [67] 5 C. tritaeniorhynchus 20.5% (958/4668) [67] Total 47 41 strains of JEVs from C. tritaeniorhynchus a represents percentage of dominant mosquito in all the collected mosquito in the corresponding province. b represents number of dominant mosquito. c represents number of all the mosquitoes collected in the corresponding province. for the first time, from members of the genus Culicoides gathered in northeast China [51]. As mentioned previously, JEV has been isolated from many species of insect collected in various parts of China; however, the virus has been isolated from the dominant mosquito species in the various regions (Table 3). Table 3 shows that the widely distributed C. tritaeniorhynchus is the most important mosquito vector for JEV (87.23%, 41/47) in China, with JEVs isolated from C. tritaeniorhynchus specimens collected in locations from 95 E in Tibet to 130 in Liaoning, and in the northern latitude of 50 in Liaoning to 15 in Hainan Island (Figure 6). This widespread distribution may partly explain why numerous JE cases have occurred in China despite the promotion of JE vaccination since the 1960s. The abundance of mosquitoes in China carrying JEV is the principle reason for poorly controlled disease. Although C. tritaeniorhynchus is the dominant carrier of JEV, other dominant mosquito species are present in some provinces. For example, C. tritaeniorhynchus accounted for 20.5% whereas Armigeres accounted for 79.5% among mosquito samples collected in the Sichuan Province of southwest China in 2004 (Table 3), with the latter being the apparent locally dominant mosquito species. However, five JEVs were isolated from C. tritaeniorhynchus and only one JEV was isolated from Armigeres (Table 3), showing that despite the presence of a different dominant mosquito species in different local regions, the JEV-positive rate of C. tritaeniorhynchus is much higher than that for other mosquito species. In the Guangxi Province of South China, a similar situation has been observed among mosquito samples collected in different regions (Table 3). With the use of two strains of Chinese JEV isolated from brain tissue samples of bats in 1989 (B58) and 1997 (GB30), the molecular characteristics of JEV were identified, and virulence studies were performed. The results showed that the full lengths of the two viral genomes were 10,977 nucleotides, which encoded 3432 amino acids. There were high homologies in nucleotide and amino acid sequences between the two viruses. In addition, eight JEV virulence-related sites were the same between isolates from bats and local patients. Neurovirulence tests revealed that the two strains from bats could kill adult mice and that

316 Y. Zheng et al. the LD50 for suckling mice was 8.0 log. These results suggested that the virus from bats had higher virulence than the virus from mosquito samples. Through evolution analysis of the virus genome sequence, it was found that the JEVs belonging to genotype III isolated from bats were on the same branch as JEVs isolated from local patients with viral encephalitis, as well as from mosquito specimens. This close relationship between JEVs from bats and mosquitoes indicates that bats might be involved in the local natural cycle of JEV and play an important role in JEV outbreaks [52]. Genotypes of Japanese encephalitis virus isolated in China Divided into five genotypes according to the JEV E gene or the JEV genome sequence, the distribution of the five JEV genotypes has certain regional distribution rules [18]. The results from studiesin 2003 showed that the I V genotypes of JEV existed in southeast Asia such as Malaysia and Indonesia, I and II in the South Pacific regions such as Australia and New Guinea, II and III in the Philippines, II and III in Thailand, Cambodia, and Vietnam, I and III in East Asia (including China, Japan and South Korea), and III in South Asia regions such as India, Sri Lanka, and Nepal [18]. The genome-wide evolutionary analysis of JEVs isolated in China (Figure 7) revealed that genotypes I and III were isolated in China [50] and that genotype V was isolated in 2009 [17]. In genotyping studies on 19 strains of JEVs isolated in China during 1949 1987 (17 from patients with viral encephalitis and two from mosquito samples), cytopathic effect (CPE) lesions Figure 7. Phylogenetic analysis of Japanese encephalitis virus strains on the basis of the complete genome sequences. Phylogenetic analyses were performed by the neighbor-joining method using MEGA version 5.04 software package (www.megasoftware.net). Bootstrap probabilities of each node were calculated using 1000 replicates. Scale bars indicate the number of nucleotide substitutions per site