Epidemiological Survey Of Japanese Encephalitis Virus Infection In Domestic Swine In Several Areas Of South China

Background and ObjectiveJapanese encephalitis (JE), also known as epidemic encephalitis, is a zoonotic disease. JE damages the central nervous system of human and leads to reproductive dysfunction of swine, making harm to human health and development of animal husbandry. JE is a severe zoonotic disease caused by Japanese Encephalitis Virus (JEV). JEV belongs to the genus Flavivirus of the family Flaviviridae. The virus genome is a positive single-stand RNA approximately 11kb in length. It contains a single open reading frame (ORF) flanked by 5’and 3’nontranslated regions (NTRs), encoding three structural proteins [capsid (C) protein, precursor membrane/membrane (prM/M) protein, envelope (E) protein] and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). On the basis of the nucleotide sequence of the envelope (E) gene, five genotypes (GI-V) of JEV have been identified. Before the year 2000, GⅢ was the predominant type in China; Between 2001 and 2005, G I and GⅢ coexisted; G I has been the mainly popular type since 2005.JE is epidemic in many countries all around the world, of which, China belongs to the highly endemic areas. Except for Qinghai, JE cases have been reported in almost all regions of China, outbreak or epidemic of JE irregularly happens in local region.JEV is transmitted through the natural cycle of JEV consists of pig-mosquito-pig or bird-mosquito-bird cycles. Culex tritaeniorhynchus is the major vector of JEV. JE has obvious seasonal prevalence relying on the spread of mosquitoes, occurs more common in summer and autumn. Human is the dead end host because of low level and short-lived viremia. Pigs are important natural breeding animals of JEV, which is the main source of human infection. Pig breeding has large quantity and fast update rate in China, with a high average infection rate of JEV. The incidence peak of JE among human usually appear 3-4 weeks after fifty percents of antibodies against JEV seroconvert in swine sera. To a certain extent, epidemiological survey of porcine JEV infection can be used to predict the occurrence of JE in human.Prevention of JE depends on reducing susceptibility of the population, eradicating of the media and managing the source of infection. Knowing more about the JE virus infection in pigs is necessary and conducive to get hold of the current epidemic factors, and then scientifically complete the prevention and control of JE.In this study, we detect the prevalence of JEV and anti-JEV IgG to understand current situation of porcine JEV infection in parts of South China (Guangdong, Hainan and Jiangxi). We intend to isolate and identify the suspected JEV strains from healthy swine sera for further homology analysis.Currently, several bands of commercial swine anti-JEV IgG antibody ELISA kit are available with varying quality, which makes it difficult to select. By evaluating their reliability and stability, we try to screen out a reliable and stable commercial swine anti-JEV IgG antibody ELISA kit for detection of all the swine sera by diagnostic evaluation.Methods1. Collection of swine serum samplesApparently healthy, JEV vaccine un-immune swine blood was sterilely collected from slaughter houses in Guangdong, Hainan and Jiangxi provinces during January to August 2013. All the plasma turned to sera samples after centrifuged at 3000r/min for 15 minutes. The supernatant liquor was mixed and packed into three equal parts. Samples were transported back to the laboratory by foam boxes with ice packs in 12 hours and stored at -80℃.2. Evaluation of commercial ELISA kits for detection of JEV IgG antibodyNinety swine serum samples were tested simultaneously by three kinds of commercial anti-JEV IgG antibody ELISA kits (Keqian, Lvshiyuan and Tianchen). Neutralization test was used as the’gold standard’test. Diagnostic test methods were used to evaluate the validity and reliability of the kits.3. Infection of JE in domestic swine3.1 Detection of JEV in swine sera3.1.1 Initial screening of JEV by ELISACommercial swine JEV-Ag ELISA kit was bought for initial screening of JEV in all the swine sera, steps were in strict accordance with the instructions.3.1.2 BHK-21 cell cultureThe supernatants of controlled MEM and positive sera samples detected by ELISA were inoculated into BHK-21 cell. Morphological changes were observed for a week, then the culture fluid were collected and stored at -80℃ for further detection. Sample was initially judged as positive if there was a suspected cytopathic phenomenon, such as death or rounded, gathering and other morphological changes.3.1.3 Quasi-neutralization testTen-fold diluted suspicious positive samples were equally mixed with different concentrations of porcine JEV IgG antibody. Mixed liquor was cultured at 37℃ for 1hour before inoculating into BHK-21 cell. JEV Nakayama strain was positive control and MEM was blank control. BHK-21 cells were cultured in cell culture box with 37℃,5% CO2 setting after inoculation for a week.3.1.4 Suspicious virus reproductionFive suspicious swine sera (JH12、JH33、HK121、HK151、HK212) were inoculated into BHK-21 cell. Virus was blind passed three generations for reproduction.The supernatants of controlled MEM, suspicious cytopathic samples and positive JEV Nakayama strain were inoculated into NIH sucking mice aged 3 days, and observed several times each day until typical symptoms appeared. Dying mice were sterilely dissected, brain tissue was kept in RNA later at -80℃ or used for production of pathological section. Blind passage three generations was necessary if there was no suspicious positive symptom, if still symptoms were not observed, it was decided as negative.3.1.5 Detection of JEV in suspicious samples by TaqMan real time PCRWe extracted RNA of suspicious sample by using Roche High Pure viral RNA kit, and then reverse transcribed into cDNA by using Roche Transcriptor First Strand cDNA Synthesis Kit. The cDNA was used as template during detection of JEV by TaqMan Real-time PCR.3.1.6 Detection of JEV in all the sera samples by TaqMan real time PCRWhen above procedures failed to isolate JEV, all the sera samples were detected by TaqMan Real-time PCR to avoid possible errors.3.2 Detection of anti-JEV IgG antibody in swine seraKeqian anti-JEV IgG antibody ELISA kits were used to detect the incidence of anti-JEV IgG antibodies in all the 465 swine sera.4. Quality controlWe had taken quality control seriously and observe laboratory safety code to ensure experimental correct, safety and well-off.Results1. Swine seraA total of 465 swine sera were sterilely collected from slaughter houses in Guangdong, Hainan and Jiangxi provinces during January to August 2013.2. Evaluation of commercial ELISA kits for detection of JEV IgGAll the three kinds of commercial ELISA kits had higher positive detection rate than neutralization test (34.4%,31/90). The rates of anti-JEV IgG antibodies in Keqian, Lvshiyuan and Tianchen were 50.0%(45/90),47.8%(43/90) and 38.9% (35/90), respectively. Sensitivity and specificity of Tianchen was 83.87% and 79.66%, respectively, Keqian had the highest sensitivity (90.32%) and a lower specificity (71.19%), while Lvshiyuan had the lowest sensitivity and specificity, which was 41.94% and 16.95%, respectively. No significant difference of detected rates were found between Tianchen and the’gold standard’serum neutralization test with the highest goodness of fit (κ=0.63). Additionally, internal consistency analysis suggested that all the ELISA kits had good stability.3. Infection of JEV in domestic swine3.1 Results of detection of JEV in swine seraForty-three swine sera were detected positive by commercial swine JEV-Ag ELISA kit, of which, nineteen samples were cytopathic, and eleven samples kept cytopathic ability after blind passed three generations. After quasi-neutralization test, the cytopathic effect of five samples (JH12、JH33、HK121、HK151、HK212) could be protected by porcine anti-JEV IgG antibody. N1H sucking mice aged 3 days died in 20-30 hours after inoculating supernatants of these five suspicious cytopathic samples without any special reaction. The suspicious samples were JEV negative detected by TaqMan Real-time PCR. Finally, all the sera samples were JEV negative detected by TaqMan Real-time PCR.3.2 Results of detection of anti-JEV IgG antibody in swine seraThe prevalence of anti-JEV IgG antibodies in all the 465 swine sera was 95.7%. There was significant difference of detected rates between different lots of Keqian ELISA kits.Conclusions1. Currently commercially available ELISA kits for detection of anti-JEV IgG antibody in swine sera were quite various. A high false positive rate was still found in these kits compared with neutralization test. Our results suggest the necessity of improving the validity reliability of domestic commercial anti-JEV IgG antibody ELISA kits.2. The rate for JEV specific antigen was 9.25% in healthy swine in three areas. However, no JEV was detected and isolated from all the swine sera. Of 465 swine sera, the prevalence of anti-JEV IgG antibodies was 95.7%. Our results suggest that there is a high infection of JEV in swine in Guangdong, Hainan and Jiangxi provinces. We should pay more attention to preventing and controlling JEV infection of swine.