An Investigation Of Japanese Encephalitis Virus In Commensal And Field Rodents And Establishment Of NIH Rat Infection Model Of Japanese Encephalitis Virus

Background and objectiveJapanese encephalitis (JE) caused by Japanese encephalitis virus has been reported globally, especially in Asia, western Pacific countries and Australia, etc. According to statistics of the world health organizations (WHO), there are about 67,900 JE cases in 24 countries and regions all round the world very year. Mortality of JE is 20%-30% and nearly 30%-50% individuals who survived from JE would suffer from severe neurological sequelae. Occurrence time of JE is obvious seasonally in China. JE cases are mainly reported from Jun to October and reached peak in July or August, for which is associated with the growth characteristic of the mosquito. JE cases in China are mainly found in children under 15 years old with a percentage of 90%, while adults are hardly attacked.Japanese encephalitis virus (JEV) is a member of the family Flaviviridae (Flavivirus genus) with 11 kb single strands of RNA. There is only a open reading frame (ORF) Which codes three structural proteins (C protein, PrM or M protein, E protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) in its RNA.JEV is transmitted biologically in a cycle between vertebrate hosts mainly by Culex tritaeniorhynchus. Mosquito is infected with JEV though biting the infected animals which are in phase of viremia, and then the infected mosquito spreads the virus by biting the susceptible animals or human being. Infected swine is the primary infection source of JEV. Some water birds, such as heron, egret and bats may act as important reservoir hosts. Human is considered as dead end host of JEV and cannot transmit JEV or be reservoir host, because low viral load is exhibited in the blood and duration of the viremia is rather short after infection of JEV. It is reported that some other kinds of warm-blood animals can carry JEV, such as cattle, sheep and some birds. However, the roles of these animals in the zoonotic cycle of JEV remain unclear for there is lack of large epidemiologic survey.Rodent is a member of mammals and belongs to rodents (muridae). The species and number of rodent are large, which are widely distributed in the world. Rodents can be grouped into commensal and field habitat according to their relationship with human. There are mainly three kinds of commensal rodents (Rattus norvegicus, Rattus flavipectus, Mus musculus Linnaeus) and one kind of field rodents(Rattus lossea Swinhoe).Rodents are considered as important reservoir host of viral zoonosis. Dozens of viruses have been detected or isolated form rodents, such as Hanta virus, Hepatitis E virus, Orthopoxyirus, Hepatitis C virus, Forest encephalitis virus, Borne virus, Norovirus and etc. However, the role of commensal and field rodents in JEV natural history remains unknown for lack of related reports. Several other kinds of flaviviruses have been isolated from rodents, such as tick-borne encephalitis virus (TBV), West nile virus, dengue virus, implying that rodents are commonly susceptible to flaviviruses and have potential risk of carrying JEV.Laboratory mice are often used for infection animal model of JEV. According to previous publications, lab mice are highly susceptible to flaviviruses for they are naturally lack of anti-flaviviruses gene. As mentioned above, intracerebral inoculation in three days of sucking mice is usually performed to amplify JEV. After infected with JEV, adult mice would show similar symptoms of JE with human, such as arch back, tremble, titanic spasm, paralysis and etc. Previous study exhibited that lab mice would appear viremia after infecting with JEV by intraperitoneal injection; suggesting rodents have potential risk of carrying or transmission of JEV. However, there is still lack of related reports about whether commensal and field rodents could retain JEV. Thus, we collected rodent samples in Guangzhou and Xiamen to detect JEV and established NIH mice infection model of JEV to evaluate its possibility of retaining JEV.Methods1. Collection and sample of commensal and field rodentsCommensal and field rodents were captured in parks, hospitals, markets, colleges in Guangzhou and Xiamen during the period between 2013 and 2015. The catch sites include drain, garbage can, grass, paddy field, corner of wall. Blood samples were collected by extraction from their hearts under anesthesia with diethyl ether. Serum samples were separated after one hour stand and with centrifugation in speed of 3500g/min for 15 minutes. Brain samples were collected and preserved in tubes in presence with RNAlater. All the samples were stored at-80℃ until use.2. Detection of JEV in brain samples of commensal and field rodentsExtraction of viral RNA from brain specimens was conducted using the Roche High Pure Viral RNA kit. cDNA was synthesized using the Transcriptor First-Strand cDNA Synthesis Kit (Roche, America). Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of the JEV genome.3. Detection of JEV antibodies in serum samples of commensal and field rodentsDetection of serum anti-JEV IgG antibodies in rodents was based on a commercially available indirect enzyme-linked immunosorbent assay (ELISA) kit. The serum microneutralization test was conducted to evaluate the titers of neutralizing antibodies in the positive samples of IgG antibodies. The positive samples of IgG antibodies were also detected by indirect ELISA for anti-dengue IgG antibodies to exclude infection of dengue virus.4. Establishment of NIH mice infection model of JEV4.1 Virus strains:Two strains of JEV which belongs to GⅢ genotype of JEV were used in this study. The NAK strain of JEV was originally isolated from a Japanese JE patient in 1935. The GD strain of JEV was isolated from a Myotis ricketti bat in Guangdong province in 2009. These viral strains were propagated by intracerebral inoculation in three days of sucking mice and BHK-21 cells line.50% tissue culture infective dose assay (TCID50) of the virus solution was estimated.4.2 Evaluation of median lethal dose of the two JEV stains:There were 3 groups: NAK, GD and control group. In NAK and GD groups, there were six dose groups (10～105 TCID50/100μL), respectively. Every dose group has 6 NIH mice (3 were male,3 were female,5-6 weeks old). There were 6 NIH mice in control group. The original virus solution was diluted into 6 concentration gradients (100～105 TCID50/100μL). Mice were infected with JEV by intraperitoneal injection and with 100μL per mice according to the different dose groups. Mice in control group were injected with 100μL PBS. Then these mice were observed with a period of 21 days after infection of JEV. The dead mice were dissected under sterile and brains were sampled to detect JEV. Mice that survived after 21days were took the blood by removing eyeball and serum was separated to detect neutralizing antibodies against JEV.4.3 NIH mice infected with JEV:Three groups were included:NAK, GD and control group. There were 30 NIH mice (female,5-6 weeks old) in NAK, GD group, respectively and 15 NIH mice in control group.(1) Primary infection:According to various strains of JEV, every mouse was injected intraperitoneally with 103 TCIDso/100μL, 100μL of JEV. Mice in control group were injected with 100μL PBS. Blood and tissues samples were harvested from one to three anesthetized and sacrificed mice at days 4,7,8,10,11,12,13,14,17 after infection.(2) Secondary infection:The survived mice were infected with JEV again in NAK and GD groups as mentioned above. Every mouse was injected intraperitoneally with 103 TCID50/100μL, 100μL of NAK strain JEV in control group. Blood samples were harvested from all mice at days 7,14,21 after infection.5. Quality control(1) All lab materials used in experiment, such as pipette tip, EP tube, frozen pipes and gloves were one-time use products.(2) Extraction of RNA and reverse transcription should be particularly careful to prevent contamination of RNase. And the EP tube, pipette tip and grinding devices should be treated by DEPC before experiment.(3) Cell culture was carried out in the Biosafety Class Ⅱ laboratory to prevent pollution.(4) Wearing hats, masks and gloves during the test and the anatomy of flying foxes was operated on the biosafety cabinet in a biological clean room.(5) Positive, negative and blank controls were included in the process of experiments.(6) The collection and preservation of rodent serum should be protected from bacterial contamination.Results1. Collection and sample of commensal and field rodentsThe study included 198 rodents belonging to 4 species, including Rattus norvegicus (159), Rattus flavipectus (10), Rattus lossea Swinhoe (27), and Mus musculus Linnaeus (2). Sixty-six rodents were collected from Xiamen City and 132 from Guangzhou City. One hundred and six brain samples and 98 serum samples were collected in this study.2. Detection of JEV in brain samples of commensal and field rodentsWe observed no detectable JEV genomic sequences among the 186 brain specimens examined with real-time RT-PCR.3. Detection of JEV antibodies in serum samples of commensal and field rodentsThe overall seroprevalence of antibodies against JEV was 45.83%(30.0 to 52.17%) for the 96 serum samples from the four rodent species. The detection rates of serum antibodies in rodents from Guangzhou and Xiamen were 44.1%(15/34) and 46.0%(29/62), respectively. Among the 44 serum samples positive for anti-JEV IgG antibodies,36 had sufficient amounts of sera for measuring the levels of neutralizing antibodies. Neutralizing antibodies against JEV were detected in 24 (61.54%) serum samples, with titers ranging from 1:10 to 1:56. Among the 44 serum samples positive for anti-JEV IgG antibodies, none was positive for anti-dengue IgG antibodies.4. Establishment of NIH mice infection model of JEV4.1 Symptoms of NIH mice after infecting with JEV:Mice began to show JE symptoms, such as arch back, tremble, titanic spasm, paralysis and etc. in 8 days of infection in NAK group. No mice has onset of JE or died after 14 days of infection. In GD group, mice show began to show JE symptoms in 7 days of infection. There were no mice dead in control group.4.2 Evaluation of median lethal dose of the two JEV stains:Among NAK group, the number of deaths in mice in the six dose groups were 3,1,4,1,3,5, which among GD group were 0,2,4,5,1,5. The median lethal dose of the two JEV stains could not be estimated for the irregular deaths of mice in the six dose groups. However, the average titers of mice neutralizing antibodies rose with the rise of TCID50 of virus. Thus we used 103TCID50/100μL in experiment of Establishment of NIH mice infection model of JEV.4.3 Primary infection of JEV:(1) Detection of JEV in serum samples:In NAK group, serum samples were positive of JEV in days of 8,10,11 after infection and viral load in blood were 3.8x10’～1.6×104 copies/mL. In GD group, serum samples were positive of JEV in days of 4,7,10,11,14 after infection and viral load in blood were 0.7～9.8*102 copies/mL.(2) Detection of JEV in brain samples:In NAK group, serum samples were positive of JEV in days of 4,7,8,10,11,14 after infection and viral load in blood were 7.25×105-1.70×109 copies/mL. In GD group, serum samples were positive of JEV in days of 4,7,8,10,11 after infection and viral load in blood were 6.7-7.4×108 copies/mL.(3) Detection of JEV in spleen samples:In NAK group, serum samples were positive of JEV in days of 4,11 after infection and viral load in blood were 2.2～13.5 copies/mL. In GD group, serum samples were positive of JEV in days of 4,8,10 after infection and viral load in blood were 0.3-84.9 copies/mL.(4) Detection of JEV in liver samples:Liver samples were all negative of JEV in NAK, GD or control group.(4) Detection of neutralizing antibodies against JEV in serum samples:The average titers of mice neutralizing antibodies rose with infection time in both of NAK and GD group. The average titers of mice neutralizing antibodies were large than 1:20 after 10 days of infection in NAK group and 11 days in GD group.4.4 Secondary infection of JEV:(1) Deaths of mice:In control group, mice began to die in days of 8 after infection and three mice survived in the final day of observation. None of mice died in NAK or GD group.(2) Detection of neutralizing antibodies against JEV in serum samples in the secondary infection:Neutralizing antibodies were large than 1:300 after 7 days of secondary infection in NAK and GD group, while neutralizing antibodies decreased with infection time. The average titers of mice neutralizing antibodies were large than 1:20 after 14 days of infection in control group.Conclusions1. All brain samples of commensal and field rodents in Guangzhou and Xiamen City collected in this study were negative of JEV, while high rate of anti-JEV antibodies were found in their blood, which suggested these rodents were exposed to JEV previously. This study suggests the role of rodents in transmission of JEV was worth further work.2. NIH mice were susceptible to both of NAK and GD strain of JEV and show similar symptom of JE patients. NIH mice showed viremia after infecting with NAK and GD strain of JEV, which were accompanied with neutralizing antibodies in a period of 3-6 days, implying the potential possibility of transmitting JEV in mice. High titers neutralizing antibodies against JEV were protective for mice while the efficiency of low titers neutralizing antibodies remains unclear.