Investigation Of Japanese Encephalitis Virus Infection Status In The Three Gorges Reservoir Area, China

BackgroundJapanese encephalitis (JE) is a zoonose caused by Japanese encephalitis virus (JEV) which is transmitted by mosquitoes. The symptom of JE is acute infection of the nervous system, and it mainly threaten children. The fatality rate of JE is 30% to 40%,40% of survivors will be leaving sequelae of nervous system, so it brings great threat to human health. Approximately 3 billion people live in countries where JE is endemic, China is a high prevalence area of JE.From 1993 to 2009, China had built a reservoir in the Three Gorges area of Yangtze River. According to the normal water level of 175m in dam,19 counties and districts in Chongqing and Hubei province were inundated. The project involved in 15.9 million pepole and finally built a 56000 square kilometers area that called Three Gorges Reservoir Area(TGRA). Before the reservoir was impounded, JE was prevalence in this area where existing many communication medias of JE such as Armigeres subalbatus, Anopheles sinensis, Culex tritaeniorhynchus, and so on. After the reservoir was impounded, the natural characteristics of major rivers in the area were completely changed. The flow rate was slowed down, the self-purification ability was reduced, and the residual pollutants was accumulated in drawdown area, so it would cause a suitable environment for mosquitoes breeding. To understand the potential and long-term impact of JE spread caused by reservoir construction in the reservoir area, it was in need of re-evaluate JEV infection status of mosquitoes, pigs and human. ObjectiveTo understand JEV infection rate of mosquitoes, JEV infection status in pigs, and the level of JEV neutralizing antibodies in normal population in TGRA by laboratory tests of mosquitoes, piglet serum and normal human serum samples collected from this area. The data will provide background information for prevention and control of JE outbreak in TGRA.MethodsMosquitoes, pig serum and normal human serum samples were collected from TGRA in summer in 2008 and 2009. Specimens were transported to the laboratory preserving in liquid nitrogen.cDNA library was prepared after the mosquito samples were grinded by Qiagen Tissuelyser. Specific gene fragment of JEV was detected using the method of semi-nested-PCR. JEV infection rate was calculated using the software of PooledlnfRateManual. At the same time, the mosquito-grinding supernatant was inoculated into cells. The positive isolates were identified using serological and molecular biological techniques. Finally, E gene of JEV was amplificated and sequenced to analyze the nucleotide and amino acid sequence and phyletic series using software of ClustalX1.83, MegAlign, Mega4 and Genedoc3.2.IgG and IgM antibodies of piglets serum were detected using ELISA and IFA respectively.Neutralization antibody of normal human serum was detected using plaque reduction neutralization test.Results1. Mosquito samples were collected from three monitoring sites in TGRA, including Zigui county of Hubei province (HBZG), Wanzhou and Yubei districts of Chongqing city (CQWZ and CQYB) from north TGRA to south in 2008 and 2009. Totally 14183 mosquitoes were collected which belongs to 3 generas and 4 species including Culex tritaeniorhynchus, Culex quinquefasciatus, Anopheles sinensis and Armigeres subalbatus during two years. All specimens were repackaged into 286 pools. The number and species of mosquitoes collected from three monitoring sites in two years were roughly the same. But the dominant species of mosquito were different. Armigeres subalbatus was the dominate species in HBZG, and Culex tritaeniorhynchus was the dominate species in CQWZ and CQYB. Culex tritaeniorhynchus were collected from all the three monitoring sites, but the distribution taked on an increasing trend from north to south. The constituent ratio of Culex tritaeniorhynchus were 10.91%(497/4554),57.35%(2859/4985) and 76.77% (3565/4644) in HBZG, CQWZ and CQYB respectively.2. Totally 23 and 26 mosquito samples were detected positive for JEV RNA in 2008 and 2009 respectively. JEV infection of Culex tritaeniorhynchus was the highest and common one. JEV infections were exsited in all monitoring sites during two years, and it takes on the decrease trend from north to south. The mosquito infection rates were 39.23 and 31.7/1000 mosquitoes (HBZG),10.58 and 3.60/1000 mosquitoes (CQWZ),4.17 and 3.66/1000 mosquitoes (CQYB) in 2008 and 2009 respectively. Culex quinquefasciatus were collected a small amount only in CQWZ and CQYB in 2009, the infection rates were 16.09 and 0/1000 mosquitoes respectively in the two area. JEV infection of Anopheles sinensis was also higher, but JEV RNA was detected only from HBZG and CQWZ in 2009, the infection rates were 16.32 and 8.22/1000 mosquitoes. JEV infection of Armigeres subalbatus was the lowest, JEV RNA was detected only from CQWZ in 2008 and CQWZ, HBZG in 2009, the infection rates were 1.08/1000 mosquitoes,2.79 and 1.35/1000 mosquitoes respectively. Thus, there were different levels of JEV infection in four species of mosquito, the JEV infection of Culex tritaeniorhynchus was the highest, and Armigeres subalbatus was the lowest.3. Totally 22 positive isolates were obtained through the tissue culture method in TGRA in 2008 and 2009.16 isolates were identified as JEV, including one isolate co-exists JEV and Getah virus (GETV). Another six isolates were identified as GETV by serological and molecular biological methods. Phylogenetic analysis based on E gene nucleotide sequences showed that 16 JEV isolates all belonged to genotypeⅠ, the majority of strains were showed a close phylogenetic relationship with JEV strains isolated from mosquitoes in Sichuan in 2004. Compared with vaccine strain SA14-14-2, there were 14 common sites of differences in E gene segments, but these sites were not the key sites that could affect the antigenic and virulence of JEV. The length of 3'UTR gene segment of 7 GETV isolates was all 401nt, there were specific components of GETV, including three repetitive sequences element about 53nt and one conserved sequence about 19nt, there existed an lOnt missing that was similar to strains separated from other provinces in China, Mongolia and Russian.4. Totally 216 piglet serum samples were collected in TGRA on May 21 to June 14 in 2008 and 2009. Serological tests showed that the positive rates of JEV IgG and IgM antibodies were 48.76%(59/121) and 46.28%(56/121),89.47%(85/95) and 38.95%(37/95) in 2008 and 2009. The data suggested that the time of 50% sero-positivity in TGRA was about in may to June. The time taked on a gradually earlier trend from north TGRA to south in 2009, the time were June 9±5 days, May 23±5 days and May 16±5 days in CQWZ, Fuling District of Chongqing (CQFL) and CQYB respectively. The trend was not obvious in 2008, the time in CQWZ was rather earlier, probably because of the specimens were collected from pig farm, but other specimens were from individual scattered backyard, the former was higher than the later (χ2= 43.797, P<0.05).5. Totally 1222 samples of normal human serum were collected in TGRA in two years. The number of samples collected in Singshan County of Hubei province (HBXS), CQWZ and CQYB were respectively 533,496 and 193. The overall positive rate of JEV neutralizing antibody was 71.36%(872/1222). The rates were 57.04% (304/533),81.05%(402/496) and 86.01%(166/193) in HBXS, CQWZ and CQYBZ respectively. The positive rate showed an increasing trend from north TGRA to south. The differences may be related to the distribution of age. Analysis of the different age group showed that the age of samples in HBXS in 2008 was older than 30-year-old and the positive rate of antibody was 91.18%(93/102), but the age of samples in 2009 was younger than 16-years-old, and the positive rate of antibody was 48.91% (211/431); The results of CQWZ were basically the same in two years, the positive rates of antibody were 79.17%(190/240) and 82.81%(212/256) respectively. The positive rates of antibody were significantly higher than 90% of older than 10-year-old samples. The rates were 48.89%(44/90) and 53.40%(47/88) of younger than 10-year-old samples. The positive rates of antibody take on rising trend with the increasing age. The rates were 36.67%(11/30),46.67%(14/30) and 63.33%(19/30) in 2008,0%(0/22),65.63%(21/32) and 76.47%(26/34) in 2009 in 0-,1-,5-three age groups; The samples of CQYB were mainly old age group, the positive rates of antibody were 75%(72/96) and 96.91%(94/97) in two years respectivly, the results were roughly the same. Thus, the positive rate of children was low, and the children should given significant attention in TGRA.ConclusionIt was the first time to detect systematicly JEV infection in mosquitoes, intermediate host pigs and human in TGRA. The three monitoring sites were 200 km interval from north to south, which could represent the geographical scope of TGRA basicly. JEV infection in Culex tritaeniorhynchus was the highest, followed by Anopheles sinensis, which should be deserved special attention. The current epidemic strains of JEV belonged to genotype I. Compared with the vaccine strain, there were certain differences of amino acid, but these sites were not the key sites that could affect the antigenic and virulence of JEV, so the vaccine could prevent the infection of new isolates in theory. The time of JEV infection was early in local pigs, and the 50% sero-positivity in TGRA was about in May to June. The level of JEV neutralizing antibody was higher in old age group in TGRA. It may be related to the multiple silent infection. The level of JEV neutralizing antibody was lower in younger than 10-year-old children which suggested that the JEV vaccination coverage of children need to be improved. While the awareness of defending and killing mosquitos should be strengthened in order to reduce the incidence of JE.