| OBJECTIVEThe main endemic region of Japanese encephalitis virus (JEV) was in Asia. The first strain of JEV was isolated in Beijing of China in 1949, it was about 60 years since then, and JEV has become one of the major arboviruses in China now. The first strain of West Nile virus (WNV) was isolated in the blood of a woman with fever in Uganda, in 1937, and it distributed in the worldwidely since then. WNV can cause the West Nile fever (WNF), and it has become one of most serious arboviruse diseases in the world now. LvZhi and other researchers first isolated WNV in Culex pipiens pipiens and detected neutralizing antibodies against WNV in serum samples of patients with fever in Kashi, Xinjiang of China, in 2014, it indicated that infections with WNV might be exised in China.JEV and WNV both belong to the Flaviviridae family, Flavivirus, Japanese encephalitis serogroup genera, both of them can be spread by Culex and other vectors. Infection with JEV and WNV can cause severe neurological diseases, symptoms of infection with JEV and WNV are difficult to distinguish in clinical diagnosis. The diagnosis of Japanese encephalitis (JE) is usually based on JEV antibody test, since there are cross-reaction in antibodies of JEV and WNV, it is likely to exist WNV infection misdiagnosed. Since there is a high incidence of JE in Shandong Province, this study was focused on JE patients in Shandong Province,2013. Virus isolation, RT-PCR and serological tests were used to find if there were patients infected with WNV from JE patients of clinical diagnosis, and to find the cross-reactions of antibodies between JEV and WNV, providing scientific basis for diagnosis and prevention of JEV and WNV infection.METHODS1. Source of samples All samples were collected in Jinan Infectious Diseases Hospital in Shandong Province, from September to October in 2013.242 patients were hospitalized, clinical diagnosis of JE were according to "JE diagnostic criteria WS214-2008",214 samples were collected from the acute phase sera and cerebrospinal fluids (admitted the first time of collecting these samples to the acute phase, including 177 sera and 37 cerebrospinal fluids),58 convalescent sera, while 30 patients both had acute and convalescent sera. Patients came from Binzhou, Dezhou, Heze, Jinan, Liaocheng, Tai’an and other places, most of them came from Jinan.2. Viral isolation and identification BHK21 cells were cultured with DMEM (Containing 10% fetal bovine serum,1% penicillin-streptomycin serum) conventionaliy, when cells grew into a monolayer, the acute phase sera and cerebrospinal fluids were inoculated into them, cell cytopathic effects (CPE) were observated daily, supernatant of the CPE samples were tested with PCR of JEV and WNV RNA. Amplified positive samples was tested by nucleotide sequencing to determine whether they were infected with JEV or WNV; if it have no CPE, BHK21 cells was cultured of 3 generations, RNA was extracted from supernatant of each generation to detect WNV and JEV. If the results of nucleic acid detection are negative after 3 generations, virus isolation was negative. If JEV or WNV RNA amplificationsare confirmed by nucleotide sequencing of JEV or WNV in CPE samples, the samples were confirmed by the laboratory diagnosis of JEV or WNV infection.3. Viral RNA detection While RNA was extracted in the acute phase serum and cerebrospinal fluid, and it was detected by nested RT-PCR of JEV and WNV. If JEV or WNV RNA amplification positive samples are confirmed by sequencing of JEV or WNV, the samples were confirmed by the laboratory diagnosis of JEV or WNV infection, and the sequences will be analyzed by MEGA5.0 to determine the strain of the virus.4. Detection of antibodies against virus in sera and cerebrospinal fluids Samples were deteceted by indirect immunofluorescence (IFA) and enzyme-linked immunosorbent assay (ELISA). For single acute phase samples, JEV-IgM and WNV-IgM were deteceted. For paired samples, JEV-IgM and WNV-IgM were detected in acute phase sample, JEV-IgG and WNV-IgG were detected in convalescent phase sera. For single convalescent samples, JEV-IgG and WNV-IgG were detected. JEV-IgM, WNV-IgM of acute phase samples and JEV-IgG, WNV-IgG of convalescent sera were diluted by IFA from 1:32. JEV-IgM, WNV-IgM of acute phase sera and JEV-IgG, WNV-IgG of convalescent sera were diluted by ELISA from 1:100 (acute phase) or 1:41 (convalescent phase). The highest dilution of positive antibodies was considered as antibody titers in IFA and ELISA. Based on serological test results, the following two results were considered as potential WNV infected cases: 〣oth antibody positive samples:For antibody titers, WNV-IgM/JEV-IgM≥4 or WNV-IgG/JEV-IgG≥4.②WNV-IgM (+) and JEV-IgM (-), or WNV-IgG (+) and JEV-IgG (-) samples.RESULTS1. Viral isolation and identificationresults JEV or WNV were not isolated from 214 cases of acute phase sera and cerebrospinal fluids2. Viral RNA detection The detection of viral RNA by nested RT-PCR amplification showed that there were JEV RNA positive samples in acute phase serums, and they were confirmed as E fragments of JEV RNA by nucleotide sequencing, JEV RNA positive rate was 1.87%(4/214), we did not amplify 3’NCR of WNV RNA. E fragments of JEV RNA were detected belonging to GⅢ strain of JEV by MEGA5.0.3. Detection of antibodies against virus IFA test results showed that both the acute phase and convalescent phase samples were diluted from 1:32, with dilution increasing, fluorescence became weaker, antibody titer WNV-IgM/JEV-IgM≥4 or WNV-IgG/JEV-IgG≥4 of specimens were not detected. Results of ELISA showed that, in the acute phase samples, there were 74 samples of WNV-IgM (+),3 samples of the antibody titer WNV-IgM/JEV-IgM≥4,1 sample WNV-IgM titer was 1:800 while JEV-IgM antibody (-), it were highly suspected to the case with WNV infection. In convalescent samples, there were 18 samples of WNV-IgG and JEV-IgG (+), including 2 cases of antibody titer WNV-IgG/JEV-IgG≥4, while 9 samples JEV-IgG (-), WNV-IgG (+). Based on WNV-IgM, WNV-IgG results of ELISA, there were 15 potential WNV infection cases from JE patients.CONCLUSIONThis study was foncused on parts of patients diagnosed with JE clinically in Shandong Province,2013,4 patients were confirmed as JE infection by nucleic acid detection in laboratory, E fragments of JEV in serums were belonging to GⅢ strain of JEV. According to the results of the viral antibody detection, there might be some WNV infections of these patients, but there were no many paired acute and convalescent samples and no plaque reduction neutralization (PRNT) test results to detect WNV antibody furtherly, so the test results can not fully support the epidemic of WNV in Shandong Province. According to the presence of WNV vector in some regions of Shandong, we suggested that the government should strengthen the prevention and control of JE while increase WNV monitoring. |
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