Japanese Encephalitis and Japanese Encephalitis Virus in Mainland China

Rev. Med. Virol. 2012; 22: 301–322. Published online 8 March 2012 in Wiley Online Library (wileyonlinelibrary.com) Reviews in Medical Virology DOI: 10.1002/rmv.1710 REVIEW Japanese encephalitis and Japanese encephalitis virus in mainland China Yayun Zheng1,2, Minghua Li2, Huanyu Wang2 and Guodong Liang1,2* 1School of Public Health, Shandong University, Jinan, Shandong Province, China 2State 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 with a 60% death rate. To differentiate JE from Japanese encephalitis (JE), which is transmitted by Von A encephalitis, it was termed Japanese B mosquitoes, is globally the most important viral encephalitis. However, when JE was later deter- encephalitis. JE is induced by infection with Japa- mined to be completely different from Von A nese encephalitis virus (JEV) and is prevalent in encephalitis, it became known as Japanese enceph- Asian and Pacificareas[1–3]. Approximately alitis and abbreviated JE [1,2]. JE is mainly prev- 35,000–50,000 people suffer from this disease alent in Asia [1–3], and became prevalent in every year, causing approximately 10,000–15,000 Australia (in the South Pacific area) since 1995 deaths [1–3]. Of the JE survivors, 50% have [7–10]. Recent research reported by the World sequelae for the remainder of their lives [4–6]. Health Organization (WHO) showed that JE is Approximately three billion people live in the prevalent in 24 countries, including districts of Asia epidemic areas [2,3]. The morbidity rate has also and the Pacific [11]. JE has experienced recent out- increased because tourism to epidemic areas has breaks in India in 2005 where more than 5000 cases increased [5]. were detected with 1300 deaths [11,12]. JE typically Japanese encephalitis was first reported in Japan affects children below 15 years of age [1–4,12,13], in 1924 when more than 6000 cases were detected 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 *Corresponding author: Dr. GD Liang, State Key Laboratory for Infec- tious Disease Prevention and Control, Institute for Viral Disease Control 85% of the cases occurred in adults older than and Prevention, Chinese Center for Disease Control and Prevention, 155 25 years [14]. Thus, JE has become an important Changbai Road, Changping District, 102206 Beijing, China. public health concern that has received global E-mail: [email protected] attention [1–3,5]. Japanese encephalitis virus is transmitted by Abbreviations used: mosquitoes, particularly culicines; Culex tritaenior- China, mainland China; IPV, inactivated purified JE vaccine; JE, Japanese encephalitis; JEV, Japanese encephalitis virus; LAV, live hynchus is the most important JEV-transmitting attenuated vaccine; TMRCA, time of the most recent common ancestor. 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 rep- JAPANESE ENCEPHALITIS IN CHINA licate in their bodies and be transmitted to other A global study on the morbidity of JE showed that it is animals such as birds and pigs via hemophagia. prevalent in 24 countries (or districts) in Asia and Therefore, JEV naturally cycles from mosquitoes western Pacific areas. Approximately 67,900 cases of to birds or mosquitoes to pigs [1,2,15]. In tropical JE occur every year (average morbidity: 1.8/100,000); and subtropical areas, mosquitoes can be found among them, almost 75% of the cases (51,000) are continuously throughout the year, and thus a con- in children 0–14 years of age (average morbidity: tinuous viral cycle occurs between mosquitoes 5.4/100,000). The study also showed that 50% of and animal hosts. Infections of domestic animals the cases (33,900) occurred in China (not including and poultry by JEV during the prevailing season Taiwan) [11]. China has been one of the most preva- are mostly subclinical, but the virus can proliferate lent countries for JE since 1951 [19–21], and JE is a in their bodies and lead to temporary viremia. legally reportable infectious disease in China [19–21]. Thus, they become a temporary host for JEV and the infectious source for humans. Particularly, Burden of Japanese encephalitis young pigs are highly susceptible to JEV and may Japanese encephalitis cases have been legally be the transmitting element from pigs to mosqui- reported every year in China since 1951. The toes to pigs [1,2]. morbidity of JE from 1960 to 1970 was higher than Japanese encephalitis virus is a member of the 10/100,000, whereas its morbidity was highest in Flaviviridae family and is a single-strand positive 1971. In total, 174,932 cases were reported from the chain RNA virus. The genome is approximately country, and the morbidity was 20.92/100,000 [19,20]. 11,000 nt and contains one ORF encoding the poly- Immunization with JE vaccine became common protein. The entire genome encodes three structural in the late 1970s. Afterward, reported cases of JE, proteins (C, M, E) and seven nonstructural proteins morbidity, and mortality rates gradually decreased (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). Non- [22]. Between 20,000 and 40,000 cases were reported coding regions (5’ and 3’) can be found on both every year in the 1990s [23]. From 2000 to 2005, the sides of the ORF [1,16]. JEV structural proteins reported cases decreased from 11,779 to 5097, the consist of the glycosylated envelope protein (E) morbidity rate decreased from 0.9489/100,000 to and the membrane protein (M), which forms 0.3898/100,000, the death toll decreased from 375 to the icosahedron nucleocapsid embedded in the 214, the death rate decreased from 0.0302/100,000 phospholipid bilayer. E protein is involved in to 0.0164/100,000, and the mortality rates ranged many important biological processes, including between 2.51% and 4.66% [24,25]. The decreased hemagglutination, virus neutralization, and viral number of JE cases in China has been particularly particle assembly. The nonstructural viral pro- obvious in the last 5 years (Figure 1, Table 1) since teins provide functional regulatory enzymes for 7643 cases were reported in 2006 (with 463 deaths) JEV replication. JEV can be divided into five gen- [26,27], whereas 2541 cases were reported in 2010 otypes according to the E gene and genomic (with 92 deaths) [28]. Both the number of reported sequences [17,18]. cases and the number of deaths significantly The first case of JE in mainland China (China) decreased. The reported cases of JE in 2011 further was reported in 1949. Subsequently, China has decreased and totaled 1646 cases by the end of become a highly epidemic area in Asia, and over September 2011 [28]. Because the months prior to 170,000 cases were reported in 1971. Since the September comprise the prevalent period for JE in 1950s, more than 100 JEV strains have been isolated China, the number of cases in 2011 may be the low- from different hosts. This study presents an over- est in history, which is only 0.94% of the highest view of the morbidity, mortality, endemic distribu- morbidity in history (1971,1646/174,932) and tion, and other epidemiologic features of JE, as well 13.97% (1646/11,779) of the morbidity in 2000. as the current immunization state in mainland Although China has established a monitoring China. We also discuss JE viral ecology, virology, system for JE since the 1950s and included it as a and molecular biology, focusing on the epidemic legally reportable infectious disease [19–21], the distribution of JE strains isolated in China in the sensitivity and accuracy of these reports requires last 10 years and the relationship between viruses further study. A study was performed, which ac- and vectors (hosts). tively monitored acute viral encephalitis in six fixed

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, Figure 1. Morbidity of Japanese encephalitis (JE) from 1961 to 2010 in China the annual reported morbidity rate of JE on the basis of the monitoring network in China is two point hospitals in four cities distributed from north to three times lower than the actual morbidity rate. to south in central–eastern China during 2006–2008 One explanation is that although JE is a national (Shijiazhuang in Hebei province, Yantai in legally reportable infectious disease (every case of Shandong province, Yichang in Hubei province, JE should be reported to the center for disease control and Guigang in Guangxi province). This study was and prevention (CDC)), the accuracy and timing of based on the average populations in these cities these reports may be affected because the diagnosis and did not focus solely on JE cases. All cases were and report of JE involve many factors such as the based on laboratory examinations, and the results diagnostic level of clinical physicians, laboratory were used to deduce the morbidity of the general detection rate for the samples from each case, reli- population [29]. Of 4513 cases of acute viral enceph- ability of laboratory detection results, and the timing alitis (2294 samples were collected), 213 were con- of the case report. Therefore, the accuracy of reported firmed to be JE after laboratory examination (9.2%, JE cases may require improvement [21,29]. 213/2294). The morbidity rates of JE in local areas Of the 1382 JE cases reported in Guizhou province were estimated according to the general popula- in 2006, only 1210 cases were positive by IgM and tions. Among them, the morbidity rate of JE in PCR for JEV infection, which accounts for 87.6% Shijiazhuang in North China was 0.08/100,000, of all reported cases (1210/1382). Among the 172 whereas the morbidity rate in Guigang in South negative JE samples, 38.9% (67/172) were detected China was 1.58/100,000. The morbidity rates in with specific antibodies to enteric viruses (enteric south and north China differed by 20-fold [29]. As cytopathogenic human orphan virus and coxsackie the WHO proposed that the morbidity rate in 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

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv 304 Y. Zheng et al. cytomegalovirus. This study also indicated that the le- system in 1951, and the reported cases are scattered gally reported cases of JE by the monitoring system in in different areas. China included some other cases of viral encephalitis [31]. Therefore, improvement of JEV laboratory Transformation of Japanese encephalitis distribution detection will improve the accuracy of the reported Japanese encephalitis onset in the 1970s and 2010 can JE cases. The burden of JE can only be determined be divided into four groups according to the morbid- after gathering accurate data regarding the onset ity rate, namely, non-prevalent areas, low-morbidity of JE, providing a theoretical basis for JE prevention areas, moderately epidemic areas, and highly epi- and control [21,29,31]. demic areas (Figure 2). Because the morbidity rates of the two different periods changed significantly, Mortality due to Japanese encephalitis different criteria were used to compare the data. Japanese encephalitis causes serious viral encephali- The most obvious feature for territory distribution tis with a mortality rate of approximately 35–40%, or of JE in China in the 1970s or 2010 is that a small even as high as 70% [1,2]. In 1950, the mortality rate number of cases were reported in west and north of JE in China was 35% [22,23]. The use of a JE China, whereas a high number of cases occurred in vaccine since the 1960s significantly decreased the east and southwest China. Many provinces that morbidity rate of JE in China [19,20,23], and recently, had high morbidity rates transformed into moderate the mortality rate of JE in China was found to and low epidemic areas with moderate or low mor- continue to decrease. The mortality rate decreased bidity rates after 2000. Five moderate JE epidemic 10–20% in the 1960s, 10–12% in the 1970s, 5–12% provinces were identified in 2005 (SX, HN, HuB, in the 1980s, and 2–5% in the 1990s, and has de- HuN, and GX) along with 18 low epidemic pro- creased 2–3% since 2000 [19,20,22–25]. Theoretically, vinces (AH, BJ, FJ, GD, GS, HaiN, HeB, HLJ, JL, JS, use of the JE vaccine should reduce the morbidity JX, LN, IMG, NX, SD, SH, TJ, ZJ) that were moder- but have no effect on the mortality rate. Because ate epidemic areas in the 1970s. From 1970 to 2010, significant economical differences exist between the only Heilongjiang and Inner Mongolia were con- Chinese provinces, the inoculation rate and efficacy stantly low epidemic areas, and thus the major of the JE vaccine in China is extremely unbalanced. epidemic areas in China have changed from the JE vaccination in economically developed cities such eastern littoral areas in the 1970s to the southwest- as Beijing and Shanghai is complete (the full JE vacci- ern provinces. The rapid economical development nation with inactivated JEV requires four dosages), in eastern littoral areas because the reformation resulting in a low JE morbidity and prevalence in and opening of China allows for a relatively high these areas [32]. Whilst China has the policy of JE vaccination rate in these areas, which explains immunization in all JEV prevalent regions, failure the decreased JE morbidity [19,20,23,27,33,34]. to complete the full immunization schedule for the JE vaccine in children has been common in remote Five provinces in southwest China are still highly provinces or mountain areas, particularly rural areas. epidemic areas for Japanese encephalitis Thus, although many children will have received at Five provinces in China have a mean JE morbidity least one dose of vaccine [33,34], the consequent rate higher than 1/100,000. These provinces are low-level immunity may be incapable of preventing located in southwest China and the middle area of infection. Nevertheless, exposure to the virus might China, and the highly epidemic provinces are trigger an anamnestic response, which could explain adjacent (Figure 2). The highly epidemic provinces why even though the JEV infection rate is relatively include Henan, Chongqing, Sichuan, Guizhou, and high in children in rural areas of China, the mortality Yunnan. The annual number of JE cases in the five rate is lower than that observed in India, where im- provinces accounts for more than 50% of the total munization coverage and therefore seroprevalence cases and was as high as 74.1% in 2010, indicating in children is likely to be lower and the mortality rate that the onset of JE in China is concentrated in these of JE in India in 2005–2006 was 35% [12]. highly epidemic provinces. The populations in these provinces accounts for only 26% of the total popula- Distribution of Japanese encephalitis tion in China. Immunization with vaccines in highly Japanese encephalitis cases have been reported epidemic provinces is important to further decrease every year since establishment of the monitoring the morbidity rate of JE in China [21].

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). There- fore, these three provinces are considered to be JE- free areas [19,20]. Although JEV was isolated from mosquito samples collected in Tibet in 2009 and neu- tralizing JEV antibody can be detected in local populations and raised pigs [17,35], JE was not observed. Figure 3. Seasonal distribution of Japanese encephalitis (JE) case Seasonal distribution of Japanese encephalitis from 2006 to 2010 in China Japanese encephalitis is typically seen from January to December, with fewer cases reported from January tendency of JE between 2006 and 2010 is very similar to May and November to December. However, the between different months. number of cases rapidly increases after June, and China has a broad territory; therefore, the number the morbidity in June is twice that in May. July and of JE cases in southern and northern China differs August have the highest morbidity. This high level significantly. Normally, the morbidity in southern of morbidity is maintained in September and then China increases after July and significantly decreases significantly decreases in October. The morbidity in at the end of August, whereas the morbidity in August accounts for 41.14% of the total annual northern China increases after August and signifi- morbidity. As shown in Figure 3, the distribution cantly decreases in the middle of September.

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, (Children whose parents are working in faraway GD, and GX, south of the Tropic of Cancer. In addi- places and do not live with them lack care and may tion, because the temperature in some local areas is have failed to accept full immunization. They are relatively high all year, JE occurs year-round. The mainly from rural areas.) account for 54.51% of the to- onset of JE in the nine provinces in the middle and tal cases in all of the vocations and 54.72% of the total downstream areas of the Yangtze River (ZJ, SH, JS, deaths [19,20,24,25,27]. The vocational distributions JX, FJ, AH, HN, HuB, and HuN) occurs in July. The for the cases reported in 2005 were mainly scattered onset peaks in SC, YN, CQ, GZ, GS, SD, SaX, and children, students, and kindergarten children, which SX are mainly in August. The onset of JE in northern accounted for 88.5% of the total reported cases. China, including IMG, NX, HB, BJ, LN, TJ, JL, and Among them, scattered children accounted for 53.6%, HLJ, typically occurs in September (Figure 4). students comprised 23.4%, kindergarten children Between 2006 and 2010, the morbidity and the mor- represented 11.5%, and farmers accounted for 8.3%. tality of JE signiﬁcantly increased after June, peaked Death cases were mainly scattered children (50.3%), in July and August, and decreased after October. followed by students (21.1%), farmers (15.8%), and This suggests that morbidity and the mortality rate kindergarten children (7.0%) [19,20,24,25,27]. of JE ﬂuctuates among different months [28,36–39].

Vocation, gender, and age Gender Vocation Male cases of JE are signiﬁcantly more common than The data from a vocational distribution of JE cases female cases, and the recent ratio (the last 5 years) from 1996 to 2005 showed that scattered children is 1.3:1 [19,20,27].

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

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv 308 Y. Zheng et al. inoculation records can be found in the local CDC, and proximity to the human residents. Because the mos- these records will be permanently stored as historic quitoes that transmit JEV to humans also feed on pigs archives for preventative inoculation of children. This (Figure 5), this provides an ideal environment both for allows the China CDC to control local vaccination all the amplification of mosquito population density and over the country using real-time management. This the transmission of JEV amongst humans [47,48]. In system ensures that inoculation records are not main- many cases, this explains why it can be so difficult tained by only the inoculated person [42]. In the past, to eliminate JEV in these typical rural regions. because of missing inoculation records, children that hadbeeninoculatedwithoneJEdosagewerealways JAPANESE ENCEPHALITIS VIRUS IN CHINA treated as complete immunization cases, leading to China probably has the highest endemicity of JE in numerous incomplete immunizations. This explains Asia. First reported in 1949, the disease has existed the high number of JE cases in rural areas. in China for more than 60 years. With its vast region, diverse climates, various environments, Japanese encephalitis ecology and abundant insect species, China is host to a par- Mosquitoes, birds, and pigs are major JEV propaga- ticularly complex set of JEV genotypes. Therefore, tive hosts. Mosquitoes can infect animals or humans it is necessary to assess the epidemic situation of with JEV through biting. Therefore, JE is a type of encephalitis, as well as vaccine protection and zoonosis [1,2,10,15]. disease trends in China, on the basis of the etiology In the provinces south of the Yangtze River in and ecology of JEV. Extensive surveys of arbo- China, the temperature is humid all year with high viruses that started in 2000 have isolated 145 JEV amounts of precipitation, allowing for rice planting. genotypes (Table 2) from insects (e.g. mosquitoes, Villages in rural areas in southern China are sepa- midges), bats, porcine, and human samples, col- rated and surrounded by paddy fields. In other lected from various provinces and environments. words, the families are surrounded by paddy fields, This has provided important etiological informa- leading to propagation of mosquitoes. Pig is an tion about JEV in China. This part analyzes JEV in important economic resource for farmers, and China with respect to the geographical distribution, accordingly, in many regions of China, they are the insect vectors, and the original environment raised in pigpens in the farmer’s yards in close and genotype of JEVs.

Figure 5. Environment for Japanese encephalitis virus transmitting in mosquito–pig cycle and human infection

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)

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)

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 INA Guangxi GX0523 C. tritaeniorhynchus INA Guangxi GX0558 C. tritaeniorhynchus INA Yunnan YN0528 C. tritaeniorhynchus INA Yunnan YN0553 C. tritaeniorhynchus INA Yunnan YN0555 C. tritaeniorhynchus INA Yunnan YN0572 C. tritaeniorhynchus III NA Yunnan YN0581 C. tritaeniorhynchus INA Yunnan YN0591 C. tritaeniorhynchus INA Yunnan YN05124 C. tritaeniorhynchus INA Yunnan YN05128 C. tritaeniorhynchus INA Yunnan YN05133 C. tritaeniorhynchus INA Yunnan YN05155 C. tritaeniorhynchus INA Yunnan YN05194 Mosquito I NA 2006 Shanghai SH0601 Pig III EF543861 Yunnan YN0623 C. tritaeniorhynchus INA Yunnan YN0686 C. tritaeniorhynchus INA Guangxi BL50 Armigeres subalbatus INA Guangxi BL53 Armigeres subalbatus INA Guangxi BL54 Armigeres subalbatus INA Guizhou GZ1 Human CSF I NA Guizhou GZ56 Human CSF I HM366552 Gansu GS105 Human CSF I NA Henan HN06-21 Culex INA Henan HN06-22 Culex INA Henan HN06-23 Culex INA Henan HN06-26 Culex INA Henan HN06-127 Culex INA Henan HN06-129 Culex INA Henan HN06-130 Culex INA 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)

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 INA Gansu GS07-TS61 Armigeres I NA Gansu GS07-KD15 Culex INA Gansu GS07-KD40 Culex INA Liaoning LNDG07-02 C. tritaeniorhynchus INA Liaoning LNDG07-16 C. tritaeniorhynchus INA Yunnan TC07018 C. tritaeniorhynchus INA Yunnan TC07172 C. tritaeniorhynchus INA Zhejiang XJ69 C. pipiens pallens I EU880214 Zhejiang XJP613 C. tritaeniorhynchus I EU693899 Henan HEN0701 Pig I FJ495189 2008 Gansu GSBY0801 Culex INA Gansu GSBY0804 Culex INA Gansu GSBY0810 Culex INA Gansu GSBY0816 Culex INA Gansu GSBY0827 Culex INA Gansu GSBY0861 Culex INA Liaoning LN0828 C. tritaeniorhynchus INA Shandong SD08-10 C. tritaeniorhynchus INA 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 INA Yunnan YN0911 C. tritaeniorhynchus INA Yunnan YN0967 C. tritaeniorhynchus INA Hubei HBZG08-09 Mosquito I NA Hubei HBZG08-55 Mosquito I NA Tibet XZ0934 C. tritaeniorhynchus VNA Tibet XZ0938 C. tritaeniorhynchus INA

IU, Information unavailable; NA, not available.

History viral encephalitis patient in August 1949 (known Although the ﬁrst JEV strain was isolated in China as Beijing-1 virus) and the P3 strain from another in 1940, the sample was not preserved. Dr. Huang encephalitis patient on 2 September 1949 [2,48]. Chenhsiang, the pioneer of virology research in And the vaccine virus strain SA14-14-2 was China, and his team isolated a virus strain from a derived from a wild-type JE virus SA14 isolated

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv JE and JEV in mainland China 313 from pool of Culex pipiens mosquito larvae in Xi’an, provinces with drought and poor vegetation, whereas China [43]. Thereafter, many JEVs were isolated the average temperature is higher in the east with from various regions, but because not all isolates a rainy climate, abundant vegetation, and diverse were preserved, only 19 JEVs were collected for vector insects [53]. molecular epidemiological study, including 2 Owing to the high-altitude and low-average strains isolated in 1949, 12 strains in the 1950s, 3 in temperatures, no JEV or JE cases have been the 1960s, 1 in the 1970s, and 1 in the 1980s [48,49]. reported in three western provinces in China China has strengthened its virus-monitoring proce- (Xinjiang, Qinghai, and Tibet), and these provinces dures, and since 2000, 145 JEVs have been isolated are considered to be non-endemic areas [19–21]. In from insect vectors (including mosquitoes and more than 20,000 mosquitoes collected during the midges [50,51]), bats [52], porcine, and cerebrospinal arbovirus survey of different areas in Xinjiang fluid and serum samples of patients with viral and Qinghai provinces during 2005–2010, not only encephalitis (Table 2). no JEV and positive-JEV genome was found in specimens, but also no C. tritaeniorhynchus was found Distribution of Japanese encephalitis there [54–56], indicating that JEV is absent in virus isolates Xinjiang and Qinghai. However, recent studies have Located in East Asia on the western Pacific Rim, shown that a natural foci of JEV already existed in China is the largest country in Asia, which ranges parts of Tibet. Discovered in Tibet for the first time, from a northern latitude of 351′N–5333.5′N from abundant C. tritaeniorhynchus (the main vehicle of south to north and across an eastern longitude of the JEV) were found in Motuo County in 2009. JEV 7333′E–13505′E from west to east [53]. The over- was isolated from the mosquito specimens, and in all geographic elevation is higher in the west, with addition, high levels of anti-JEV antibodies were numerous mountains such as the Pamirs and the found in the serum of residents and domestic pigs Qinghai-Tibet Plateau, and lower in the east. There- [35,57]. This suggested that Tibet has JEV vectors fore, the average temperature is lower in the western 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

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv 314 Y. Zheng et al. region and natural foci of JEV [35,57]. Collectively, low altitudes of China, owing to high average the above studies showed that the distribution of annual temperature and heavy rainfall, whereas JEVextended to 95º E in China, in which JEV was pre- high altitudes are not conducive to mosquito breed- viously only found in Sichuan Province (97º 21′ E) ing [63]. Since 2000, JEV has been identified in (Figure 6). mosquito specimens isolated in the mountains The Chinese inland province, Inner Mongolia, is from elevations of 2 to 3000 m in China [64,65]. historically the most prominent JE-endemic area A three-dimensional study examining the distri- with an average annual incidence rate of 1.55 JE bution of mosquito species and JEV for two years cases per 100,000 people. C. tritaeniorhynchus was (2005 and 2006) was performed in the Gaoligong the most locally dominant mosquito species in the Mountains in the Yunnan Province (24º 00′ to 29º 1970–1980s [58,59], and many JEVs were isolated 00′ N). The sample locations ranged from 24º 00′ from them. However, because of climate warming N to 29º 00′ N at a latitude of 11º with samples col- in recent years resulting in grassland desertification lected at every 30′ of gradient. The altitude ranged and reduced rainfall, the local mosquito species from 900 to 3280 m, which was divided into five composition has changed greatly. Mosquito gradients of 900, 1500, 2000, 2500, and 3000 m. samples were collected for species identification The results demonstrated that there were signifi- and mosquito-borne arbovirus research during cant differences in the number of mosquitoes two consecutive summers (2007 and 2008) at 12 among the altitudes: the most were found at sites in four cities in Inner Mongolia, from which 1500 m, followed by 900 m, and the number was 10,542 specimens including three genera (Culex, drastically lower above 2000 m. JEV was isolated Aedes, and Anopheles) and seven species were from mosquito specimens in each gradient below obtained. The results showed that Aedes was the lo- 3000 m in the sample range within the latitudes of cally dominant mosquito species making up 79.9% 25º 30′ N to 28º 00′ N, with no significant difference of all collected mosquitoes (8,426/10,542), followed in strain number. JEV was not isolated above by Culex mosquitoes (19.5%; 2,049/10,542) and 3000 m. The maximum altitude where JEV was iso- Anopheles mosquitoes (0.6%; 67/10,542), whereas lated was 2900 m, which is the highest altitude C. tritaeniorhynchus was not represented in the reported at present [65]. Previous studies isolated collected mosquitoes [60,61]. Six viral strains were JEV at an altitude of 2000 m [63,66]. According to isolated from the mosquito samples, which were the data from the JEV-monitoring network, Yunnan identified as two strains of Tahyna virus, one strain Province has a high incidence of JEV with perennial of Banna virus, and three strains of C. pipiens pallens JEV cases [34], indicating that the prevalence of JEV densovirus; however, JEV was not detected [60,61]. is consistent with its high-altitude distribution. This suggests that Aedes is currently dominant in Inner Mongolia; however, C. tritaeniorhynchus was Japanese encephalitis virus and vectors the locally dominant mosquito in the 1970s to the The population diversity of JEV vectors in China 1980s [59,62]. could be obtained from the origin of isolated virus In summary, the current JEV distribution in China strains in recent years (Table 2). More than 100 JEVs spans the latitudes of 50 in Heilongjiang to 15 in have been isolated in China since 2000. Of the 89 Hainan, and from the longitudes of 95º E in Tibet to JEVs isolated from mosquito specimens of Culex, 130º E in Heilongjiang (Table 2, Figure 6). Xinjiang 78 were from C. tritaeniorhynchus (Table 2), indicat- and Qinghai remain JE-free areas. Inner Mongolia, ing that C. tritaeniorhynchus is the major JEV vector which was the traditional JE-endemic area in in China [17,19,20,50,51,67–90]. JEVs were also iso- northern China, has no virus distribution at present. lated from C. theileri [70], C. pipiens pallens [75,81], C. modestus [81], C. pipiens quinquefasciatus, and C. fuscocephalus [70]. Ten JEVs were isolated from Japanese encephalitis virus isolates and mosquito samples of Armigeres, and four were iso- elevation lated from A. subalbatus [69,82], indicating that Mosquitoes are metamorphic insects that require Armigeres is an inferior JEV vector compared with water for their various stages of development [15]. Culex. JEV was also isolated from Aedes vexans Because they are readily maintained in areas of [51] and Anopheles sinensis [70]. Two strains of JEVs rainfall, mosquito species and density are high at were isolated from blood-sucking midges in China

Table 3. Japanese encephalitis virus (JEV) and dominant species of mosquito in different areas of China Dominant mosquito JEV Site (strain) 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 southwest China in 2004 (Table 3), with the latter Culicoides gathered in northeast China [51]. being the apparent locally dominant mosquito As mentioned previously, JEV has been isolated species. However, five JEVs were isolated from C. from many species of insect collected in various tritaeniorhynchus and only one JEV was isolated parts of China; however, the virus has been isolated from Armigeres (Table 3), showing that despite the from the dominant mosquito species in the various presence of a different dominant mosquito species regions (Table 3). Table 3 shows that the widely in different local regions, the JEV-positive rate of distributed C. tritaeniorhynchus is the most impor- C. tritaeniorhynchus is much higher than that for tant mosquito vector for JEV (87.23%, 41/47) in other mosquito species. In the Guangxi Province China, with JEVs isolated from C. tritaeniorhynchus of South China, a similar situation has been specimens collected in locations from 95 E in Tibet observed among mosquito samples collected in to 130 in Liaoning, and in the northern latitude of different regions (Table 3). 50 in Liaoning to 15 in Hainan Island (Figure 6). With the use of two strains of Chinese JEV iso- This widespread distribution may partly explain lated from brain tissue samples of bats in 1989 why numerous JE cases have occurred in China (B58) and 1997 (GB30), the molecular characteris- despite the promotion of JE vaccination since the tics of JEV were identified, and virulence studies 1960s. The abundance of mosquitoes in China were performed. The results showed that the full carrying JEV is the principle reason for poorly lengths of the two viral genomes were 10,977 controlled disease. nucleotides, which encoded 3432 amino acids. Although C. tritaeniorhynchus is the dominant There were high homologies in nucleotide and carrier of JEV, other dominant mosquito species amino acid sequences between the two viruses. In are present in some provinces. For example, C. addition, eight JEV virulence-related sites were tritaeniorhynchus accounted for 20.5% whereas the same between isolates from bats and local Armigeres accounted for 79.5% among mosquito patients. Neurovirulence tests revealed that the samples collected in the Sichuan Province of two strains from bats could kill adult mice and that

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv 316 Y. Zheng et al. the LD50 for suckling mice was 8.0 log. These of the five JEV genotypes has certain regional results suggested that the virus from bats had distribution rules [18]. The results from studiesin higher virulence than the virus from mosquito 2003 showed that the I–VgenotypesofJEV samples. Through evolution analysis of the virus existed in southeast Asia such as Malaysia genome sequence, it was found that the JEVs and Indonesia, I and II in the South Pacific belonging to genotype III isolated from bats were regions such as Australia and New Guinea, II on the same branch as JEVs isolated from local and III in the Philippines, II and III in Thailand, patients with viral encephalitis, as well as from Cambodia, and Vietnam, I and III in East Asia mosquito specimens. This close relationship be- (including China, Japan and South Korea), tween JEVs from bats and mosquitoes indicates and III in South Asia regions such as India, that bats might be involved in the local natural Sri Lanka, and Nepal [18]. The genome-wide cycle of JEV and play an important role in JEV evolutionary analysis of JEVs isolated in China outbreaks [52]. (Figure 7) revealed that genotypes I and III were isolated in China [50] and that genotype V was isolated in 2009 [17]. Genotypes of Japanese encephalitis virus In genotyping studies on 19 strains of JEVs isolated in China isolated in China during 1949–1987 (17 from Divided into five genotypes according to the JEV E patients with viral encephalitis and two from mos- gene or the JEV genome sequence, the distribution quito 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

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv JE and JEV in mainland China 317 on JEV-infected baby hamster kidney cells were virulence. Future research is required to determine observed with all strains. At 56 h post-infection, the virulence of the re-emergent type V, and about 50% cell loss was observed (CPE +++); whether the current JE vaccine is protective. 3-day-old suckling mice died within 72 h after Recent studies have demonstrated that changes inoculation with the virus. The results of poly- in the regional distribution of different JEV geno- merase chain reaction amplification and nucleo- types have emerged. For example, type V has been tide sequence analysis of the prM-C and E gene isolated in areas of China and Korea in which only I regions suggested that all strains were genotype and III were originally present [18,50,67], and type I III [49]. has been isolated from viral encephalitis cases According to an analysis of more than 80 JEVs in China and India where III originally existed from specimens derived from patients, mosqui- [97,98]. The emergence of new genotypes, prompted toes, and midges in JE-endemic areas during by the genetic variation of ancient JEVs, indicates 1949–2005, there are two genotypes (I and III) of that studying and monitoring JEV genotype distri- JEV in China. The first III strain was isolated in bution is important for understanding genetic 1949, whereas genotype I was first isolated in variation among JEVs as well as JE prevention 1979. The results of a geographical study that and control. ended in 2005 demonstrated that the strains isolated from Heilongjiang, Beijing, Shanxi, Guizhou, and Fujian were III; from Henan was I; and that Phenotype of genotypes I and III of Japanese both strains were isolated from Liaoning, Sichuan, encephalitis viruses isolated in China Shanghai, and Yunnan. JEVs isolated before 1979 The current JEV vaccines, the attenuated JE vaccine were III, whereas I emerged after 1979 (YN79- (SA14-14-2), or the inactivated vaccine (P3), are Bao83) in Yunnan [50]. derived from genotype III isolates, which were Analyses of the PrM gene nucleotide sequence obtained separately in 1953 and 1949 [42,43]. With from 19 JEVs collected in Yunnan during 1977–2006 many genotype I JEVs isolated recently in China, showed that 17 JEVs were genotype III [91], it is worth investigating whether the vaccines are whereas two were genotype I that were collected protective. Analyses of the biological characteristics separately in 1982 and 1977(M28). Genotype I JEV of the 17 JEVs of genotypes I and III, isolated in in 1977 (M28) or 1979(YN79-Bao83) was first iso- different years (five were isolated in the 1950s, latedinYunnaninsouthwestChinaandtheniso- whereas the remaining 14 were isolated after lated in other provinces. Further monitoring is 2000), have shown that plaques on baby hamster needed to confirm whether JEVs in China were kidney cells have the same diameter after infection introduced from other countries or regions in with the different strains. In addition, there is no South-East Asia [92]. significant difference in virulence among the In recent years, several JEVs of genotypes I and strains in mouse brain, indicating that, regardless III were isolated in China; one strain of JEV of the genotype, no significant differences were (XZ0934) isolated from C. tritaeniorhynchus speci- detected among the JEVs isolated in the different mens collected in Tibet in 2009 belonged to geno- years in terms of virus [99]. To evaluate the protec- type V, as identified by whole genome sequence tion efficacy of the JEV vaccine against the newly analysis [17]. This genotype was first isolated from isolated genotype I JEVs, a virus attack trial was con- tissue samples of viral encephalitis case in Malaysia ducted. It was demonstrated that both live attenu- in 1952 [93–95] but was never again isolated until ated vaccine (LAV, ≥234 plaque forming units, pfu) 2009, indicating that the virus has begun to re- and the inactivated purified JE vaccine (IPV, 1:5 emerge in areas not limited to Malaysia, having dilution) vaccines effectively conferred protection spread to China [17] and South Korea [96]. This against all 16 isolates tested following ip challenge illuminates the fact that variation in and the spread in mice. However, when vaccinated mice were chal- of JEV have undergone recent and rapid change, lenged via ic injection, ≥60% of the LAV-vaccinated highlighting new issues for JE prevention and animals were protected with most JEV isolates, but control. Furthermore, the first strain of genotype ≤40% protection was observed following ic vaccina- V (Muar) JEV was isolated from the viral encepha- tion with IPV. These results indicate that the JE litis case, suggesting that the virus has strong vaccines used in China confer effective protection

Copyright © 2012 John Wiley & Sons, Ltd. Rev. Med. Virol. 2012; 22: 301–322. DOI: 10.1002/rmv 318 Y. Zheng et al. against both JEV genotypes now prevalent in China There are three measures that could be used to con- and that the LAV formulation conferred higher trol JE incidence. Firstly, vaccination is the most levels of protection than IPV did [100]. effective way. As we have known, JE is a preventable disease by vaccination, and China has achieved great control of JE incidence by wide Emergence of Japanese encephalitis virus range of the JE vaccination. Since 2008, children in genotype I as the dominant genotype in Asia highly epidemic areas could be inoculated for free, Nakayama, the ﬁrst JEV isolated in the world in thus the incidence of JE in these areas will continue 1935, is a member of genotype III. In the decades that to reduce greatly. Secondly, changing the raising followed, various genotypes of JEV were isolated. mode of pigs is also important because pigpens The continued emergence of JEV, with different built in the yards make the propagation cycle genotypes in new areas, highlights the importance “mosquito–pig–mosquito” occurs very close to of conducting research on JEV molecular evolution, human environments. In addition, improving the molecular variation, and population dynamics with living condition for rural population could lessen respect to JEV genotypes. mosquito breeding environment so that it can cut Phylogenetic analyses of the complete genomic off the infection link of JEV to people. Obviously, sequences of JEVs from patients with encephali- vaccination is most realizable in three measures tis, mammals (pigs and bats), and various insect mentioned previously, whereas the others would vectors (more than 20 species of mosquitoes and be directly related to national economic conditions midges) have shown that the JEV genome is and different life style. As a good example, Japan divided into four genotypes. The time of the and Korea had JE that highly endemic areas in the most recent common ancestor (TMRCA) of the past, but there are very few JE cases that exist at JEV genome is 1695 years ago. JEV underwent present, as the two countries have developed their four population differentiations and derived the economics greatly. We believe JE morbidity in individual strains in the following order: IV, III, China will further reduce following the rapid II, and I. The TMRCA of type I is 193 years, the development of national economy and great latest in the independent division and the youn- improvement of rural natural environment. gest of the four genotypes. Moreover, type I JEV Japanese encephalitis is mainly epidemic in Asia has expanded rapidly in endemic areas over in the past but has spread to Australia in Oceania almost 30 years and has gradually replaced the and became vector-borne disease epidemic in two original genotype (genotype III) to become the continents, which attracted international attention. new most common species [101–104]. Strengthening the surveillance on JE and JEV in According to the JEV evolutionary rate and the other areas and regions, especially in the European calculated optimal population model of genotypes countries, which connected with Asia, is particu- I and III, the population of genotype III JEV pla- larly important. As JEV has been isolated from teaued from 1925 to 1975 and began to decline after the mosquitoes in former Soviet Union, it worth 1975, whereas the population of I has been in a sta- further research in whether JEV exists in other ble growth period since 1954. Therefore, since the European countries. 1950s, type I has been dominant, whereas type III has declined [101]. CONFLICT OF INTEREST The WHO has proposed the objective that by the The authors have declared that no competing year of 2015, JE morbidity of children aged interests exist. 0 ~ 14 years decrease to 0.5/100,000 in the JE- endemic countries. The average JE morbidity of China is far below the number by now, and inci- ACKNOWLEDGEMENTS dence in most low-morbidity areas and moderately This work was supported by grants from The epidemic areas such as eastern littoral areas is also Ministry of Science and Technology, China lower than the index. But the average incidence in (2011CB504702), Development Grant of State Key highly endemic areas including Yunnan, Guizhou, Laboratory for Infectious Disease Prevention and Guangxi province are relatively high, and China Control (2008SKLID105), and the China CDC-US still has many work to do to achieve the index. CDC Cooperative Agreement (U19-GH000004).

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