A Preliminary Study On Experimental Infection Of Human And Bat-derived Japanese Encephalitis Virus Strains To Flying Foxes

Background and objectiveJapanese encephalitis virus (JEV) is an enveloped virus with a positive single stranded RNA genome approximately 11 kb in length and belongs to the genus Flavivirus of family Flaviviridae. And it (the Nakayama strain, NAK) was first isolated from a patient brain in 1935 in Tokyo, Japan. JEV only contains a single open reading frame (ORF) (NTRs), the nucleotide sequence encodes three structural proteins (capsid protein C, precursor membrane protein prM and envelope protein E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Five genotypes (GI-V) of JEV have been described based on the nucleotide sequence of the envelope (E) gene. sucking mice 2-3 days old are commonly used to be propagated and harvested JEV. The onset of the lesions is usually 3-4 days after injecting JEV into mouse brain, and the infection mouse died within a week. Inoculation of virus by sucking mice is a reliable method to isolate JEV and prepare JEV antigen. In addition, baby hamster syrian kidney cells (BHK-21), a mosquito cells (C6/36), and vero cells are aslo sensitive cells to isolate JEV. Morphological changes would be observed in the cells (cells turned round, falling off and intercellular spaces dilatation).JEV is a mosquito-borne flavivirus in tropical and subtropical regions, which causes Japanese encephalitis (JE) that destroys the central nervous system (CNS) of humans and some species of animals. JE is a major infectious disease which threatens the health of human, especially the health of children. The incubation period of JE is usually 10-14 days, and the symptomatic JEV infection manifests with high fever, severe headache, nausea and vomiting, disturbance of consciousness and so on, the severe cases may be have the symptom of tetanic spasm, convulsions, meningeal irritation, respiratory failure and even death. JEV infection in human is most often asymptomatic, only a minority of cases produces clinical symptoms. The morbidity rate ranges from 2/100000 to 10/100000 in humans, and fatality is seen in 20 to 30% of the cases. And 30 to 50% of survivors have persistent neurological sequelae. Thus, JE has brought huge burden of disease to society and the family, and it has become an inportant global public health concern.JE is first found in 1871 in Japan. Then, JE extend its range to Indian, Pakistan, China, Singapore and Australia and so on, but it mainly limited in Southeast Asia. China has a high incidence of JE, and JE cases have been reported in almost all regions of China (excluding Qinghai). In 1957,1966 and 1977, there have been three JE epidemics in China. The number of JE cases of the latter two epidemics is more than 150000 and 200000, respectively. And the incidence of JE is up to 20/100000. Since the 1980s, the incidence of JE in China has declined significantly as JE vaccine is applied in children. Although the incidence of JE in China remain at a relatively low level, the annual number of JE cases still fluctuating at around 10,000 cases, the local outbreak or epidemic happens sometimes.The reservoir hosts of JEV are mainly pigs and aquatic birds, and the primary mosquito vector of which is Culex tritaeriorhynchus. Human is dead end host because of low level and short-lived viremia. Besides, some of animals are proved to be naturally infected with JEV, such as bats. More than 80 kinds of viruses have been detected or isolated from bats so far, such as Ebola virus, Hendra virus, SARS-coronavirus-like viruses, Nipah virus and JEV. So bat has been known as an important reservoir host of a large number of zoonoses viruses. Several studies have shown that JEV or serum antibodies against JEV may exist in bats in China and Japan During 2008 to 2009, our laboratory also isolated four strains of JEV virus (GD1,HN2,YY87 and YY158) from bats in Guangdong, Hainan and Hunan province of China. The full-length nucleotide sequences of GD1 and HN2 JEV isolates were determined, and they all belonged to GⅢ. Although our study showed bat-derived JEV isolates (GD1 and HN2) and human-derived JEV strains (NAK) shared high identities in nucleotide and amino acid sequences, the differences of virulence and immune response in these JEV strains are not clear.Flying foxes feeding on fresh fruits daily, and are bigger than other species of bats. They are easy to breed and closely related to humans and mosquitoes. Previous study has shown that flying foxes could be infected with JEV by inoculation in the experiment. Thus, we selected flying foxes as the experimental animals, and explore the differences of infectivity, virulence and immune response in them after flying foxes infecting artificially with bat-derived JEV strains and human-derived JEV strain.Methods1. Catching and handing of batBats were captured from palms and caves in Guangdong and Hainan provinces between June 2013 and June 2014. Pregnant bats and breast-feeding bats were excluded from the study and released. Then the remaining flying foxes were took back to the laboratory and kept in the animal room. They were fed with fresh fruits (guava, apple, banana, pear, mango, longan and litchi) daily.2. Virus strainThe strain NAK and two isolates of JEV, HN2 and GD1 were used in the study. The NAK strain of JEV was originally isolated from a Japanese JE patient in 1935. The HN2 strain of JEV was isolated from a Miniopterus schreibersii bat in Hainan province in 2008 and the GD1 strain of JEV was isolated from a Myotis ricketti bat in Guangdong province in 2009, respectively. These viral strains were propagated in the BHK-21 cells line prior to be inoculated into bats, and virus levels in the supernatant of infected BHK-21 cells was estimated by 50% tissue culture infective dose assay (TCID50).3. Bat infection with JEVTwo experiments were included in the study. In the experiment one, eight flying foxes were divided randomly into three groups (named NAK1, HN2-1 and GD1-1 group, respectively), in which flying foxes of group NAK1 (three flying foxes, named bat1, bat 2 and bat 3, respectively), flying foxes of group HN2-1 (three flying foxes, named bat4, bat5 and bat6, respectively), and flying foxes of group GD1-1 (two flying foxes, named bat7 and bat8, respectively) were hypodermically inoculated with 0.1 ml of the strain NAK (10423 BHK-21 TCID50/0.1ml), HN2 (10524 BHK-21 TCIDso/0.1ml), and GDI (105.86 BHK-21 TCID50/0.1ml) of JEV in the upper thigh area, respectively.Eleven flying foxes in experiment two were also divided randomly into three groups (named NAK2, GD1-2 and NEG group, respectively), in which flying foxes of group NAK2 (four flying foxes, named bat9, bat10, bat11 and bat12, respectively) and flying foxes of group GD1-2 (five flying foxes, named bat13, batl4, batl5, bat16 and bat17, respectively) were hypodermically inoculated with 0.1 ml of the NAK (10350 BHK-21 TCIDso/0.1ml) and GD1 (103.00 BHK-21 TCID50/0.1 ml) of the virus. The NEG group (two flying foxes, named group 18 and group 19, respectively) was used as negative control group in the study and these two flying foxes were hypodermically inoculated with 0.1 ml of the cell supernatant without virus in the upper thigh area.All experimental flying foxes were observed daily for clinical symptoms of encephalitis before and after infection with JEV. We also obtained serum samples from each bat on days 0,4,14,21 after inoculating JEV. All flying foxes were executed by cardiac exsanguination on day 21 and observed for the skins and internal organs by sterile dissection. Brain tissue samples of flying foxes were collected and preserved in the tubes with the presence of RNA later, then stored at -80℃ for further use.4. Detection of JEV in batsBHK-21 cells were inoculated with all the serum samples of flying foxes. The cytopathic effect (CPE) was observed for ≤7 days. Viral RNA was extracted from the cell or brain tissue supernatant using the High Pure viral RNA kit and reverse transcription using the Transcriptor First Strand cDNA Synthesis Kit before JEV being tested using the TaqMan real time RT-PCR.5. Detection of JEV antibodies in batsTo determine if immune reactions to JEV had produced in flying foxes after JEV exposure, an enzyme linked immunosorbent assay (ELISA) was developed using Recombinant Protein A/G, Peroxidase Conjugated (Thermo, America) to detect antibodies against JEV in them. The ELISA kit was brought from Wuhankeqian Animal Biological Products Co., Ltd, and it was a commercially available manufacturer’s recommendation. After detection by ELISA, all ELISA-positive samples were detected by virus neutralization (VN) test. The operating steps of VN test mainly refer to the health of the People’s Republic of China industry standard (WS214-2008).6. Histology and immunohistochemistryParts of the flying foxes brain tissue was taken, and then making pathological section to examine the histological change under light microscopy.7. Quality control1) 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) 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 bats serum should be protected from bacterial contamination.Results1. Clinical observation of batsAll flying foxes studied were healthy before and after infection with JEV. They displayed neither any clinical symptoms of encephalitis nor bruise on the bodies. Moreover, the internal organs of flying foxes looked normal, apart from a little ischemia.2. ViremiaBHK-21 cells were inoculated with all the serum samples of flying foxes. In the experiment one, CPE was occurred in the JEV-infected BHK-21 cells. The main expressions of CPE consisted of cells turned round, intercellular spaces dilatation and cells falling off. JEV was found in most of serum samples (7/13) of infected flying foxes by using TaqMan real time RT-PCR. There were no differences between three groups in the experiment one. However, none of the flying foxes in the experiment two developed a detectable viremia.3. Detection of JEV in bats brainIn the experiment one, all flying foxes brain tissue specimens (except for bat5 of HN2-1 group) were positive for JEV by using TaqMan Real-time RT-PCR, while were negative for JEV in the experiment two.4. Detection of JEV serum antibodies by ELISAFlying foxes exposed to the NAK. strain and HN2, GDI isolates of JEV were serum antibody-positive, indicating a seroconversion to different sources of JEV strains. All flying foxes in the experiment one were serum antibody-negative on day 4 after infection with JEV. And in both of experiments, most flying foxes produced the peak of JEV IgG antibodies on days 14-21 post-infection.5. The result of neutralizing antibodies against JEV in bats serumOf the 15 serum samples of the JEV-infected bats on the 21th day,14 serum samples had neutralizing antibodies against JEV (93.33%). Only a serum sample was negative for neutralizing antibodies (bat14, GD1-2 group). The neutralizing antibody titers of serum samples in the experiment one were much higher than that in the experiment two (1:23.41-1:56.30 vs.1:5.00-1:15.87, t=-5.733, P<0.001)6. Pathological changes in bats brainIn the experiment two, the flying foxes brain tissues showed mild inflammatory reaction. The alterations included vascular congestion, edema and lymphocyte infiltration. But the structure of flying foxes brain tissues was normal. The pathological examination for bats in the experiment one was absent.Conclusions1. Flying foxes were susceptible to different origins of JEV, but they all were asymptomatic infected with JEV, indicating that bats maybe have adapted to JEV.2. Human-derived JEV strain (NAK) and bat-derived JEV isolates (HN2 and GDI) had the similar virulence and immune response to flying foxes. There were no differences of the infection with JEV in flying foxes between NAK strain and HN2, GDI JEV isolates.3. Flying foxes could produce immune response after they infection with JEV, even at low concentrations of the virus. However, a dose-response relationship existed between viral concentrations and titers of neutralizaing andibodies.4. Viral nucleic acids of JEV were detected in serum and brain tissue samples of bats expoused to JEV in the experiment one. However, there was no detectable JEV in bats in the experiment two, indicating that a dose-response relationship existed between viral concentrations and infectivity of JEV in bats.