Serological Prevalence Against Japanese Encephalitis Virus-Serocomplex Flaviviruses in Commensal and Field Rodents in South China

Abstract

Background:

Japanese encephalitis caused by Japanese encephalitis virus (JEV) is an endemic zoonotic disease of high public health importance in the Asian Pacific region. The aim of this study was to investigate the presence of JEV infection in commensal and field rodents in South China.

Materials and Methods:

RNA copies of JEV were detected in brain samples of rodents using real-time RT-PCR. Detection of serum against JEV-reactive antibodies was performed using indirect enzyme-linked immunosorbent assay and microneutralization test.

Results:

In total, 198 rodents were collected from Guangzhou City and Xiamen City between November 2013 and May 2014. JEV RNA was not detected in 188 brain samples. Forty-four in 96 serum samples (45.8%) were positive for JEV-reactive IgG antibodies. The prevalence of neutralizing antibodies to against JEV-reactive in these serum samples was 61.5% (24/39), with titers ranging from 1:10 to 1:56.

Conclusion:

Rodents are not known to play a role in transmission of JEV in Asia, nor is there an evidence to support a role for rodents in transmission of other related flaviviruses in China. However, in the current study, we detected evidence of JEV-reactive antibodies in large numbers of Rattus norvegicus and Rattus losea Swinhoe. Further studies of rodents as potential hosts of JEV or other related flaviviruses are warranted.

Introduction

Japanese encephalitis (JE) is the leading cause of viral encephalitis in a number of countries in Asia (Hills et al. 2014). Japanese encephalitis virus (JEV) is a member of the family Flaviviridae (Flavivirus genus) and maintained in a cycle among susceptible avian and swine species through bites of competent mosquito species (Van den Hurk et al. 2009, Lindahl et al. 2013). Swine and some species of birds such as ardeid birds serve as the major amplifying host of the virus (Liu et al. 2013). JEV also infects various incidental hosts, including horses, humans, and bats (Liu et al. 2013).

China is one of the high-incidence areas of JE in Asia. Morbidity of JE was 1–1.5‰ during 1960–1979, while it sharply decreased after immunization with the live-attenuated JEV vaccine (Wang and Liang 2015). However, there were still 2541 JE cases in 2010 according to statistics. Humans are end dead hosts of JEV, and JEV is mainly enzootic transmission among animal reservoirs (Van den Hurk et al. 2009). It is necessary to evaluate the epidemic situation of JE in China, based on the etiology and ecology of JEV (Wang et al. 2009). According to surveys in various provinces in China from 1949 to 2009, 145 JEV genotypes isolated from insects, bats, swine, and human specimens were reported (Zheng et al. 2012). To our knowledge, no evidence has been documented showing that JEV infects rodents in China.

Rodentia are natural reservoirs of some zoonotic viruses with significant impacts on public health, including Hantavirus pulmonary syndrome, hemorrhagic fever with renal syndrome, and Lassa fever (Schmaljohn and Hjelle 1997, Charrel and de Lamballerie 2010). Several other kinds of Flavivirus species, such as West Nile virus and tick-borne encephalitis virus, have been isolated from rodents (Mansfield et al. 2009, Jeffrey Root 2013), suggesting that rodents might be naturally susceptible to Flavivirus. In the laboratory, mice are highly susceptible to JEV, although the routes of the viral infection are quite different from in the nature. Experimental viremia of JEV in mice can persist for as long as 13 days after intraperitoneal injection of the virus (Saxena et al. 2008, Wang and Deubel 2011) indicating that rodents pose a potential risk of transmitting JEV.

Some species of rodents are referred to as “commensal rodents” because of their intimate relationships with humans, such as Rattus norvegicus, Rattus flavipectus, and Mus musculus Linnaeus (Garba et al. 2014). Some species of rodents that are very common in the wild, but are usually not too far from the human environment, are referred to as “filed rodents,” such as Rattus losea Swinhoe (Garba et al. 2014). In the current investigation, we sampled commensal and field rodents from two cities of South China and molecular screening for JEV in brain tissues and detection of anti-JEV-reactive antibodies by enzyme-linked immunosorbent assay (ELISA) serology to explore whether rodents play a role in the enzootic cycle of JEV.

Materials and Methods

Samples

Rodents were trapped in Guangzhou city and Xiamen city between November 2013 and May 2014. We collected rodent samples with the help of local Centers for Disease Control and Prevention (CDCs) that perform routine monitoring of rodent density monthly. The sample capture sites were located in the sites for rodent density surveillance in urban community residences of the selected cities, which were routine monitoring sites for rodent density by the CDCs. The capture sites were places of people's daily life, work, and study. Sampling was conducted three times in Guangzhou city and twice in Xiamen city. The night trap method was used. After conveying the rodents to biological safety level-2 laboratory, their species were identified on the basis of their morphology. Serum and brain samples of rodents were collected under anesthesia with diethyl ether and stored at −80°C until use.

Detection of the JEV genome

Extraction of viral RNA from brain specimens was performed using the Roche High Pure Viral RNA kit (Roche) according to the manufacturer's instructions. Real-time RT-PCR was used for detection of the JEV genome. cDNA was synthesized using the Transcriptor First-Strand cDNA Synthesis Kit (Roche). Real-time PCR was performed using Roche LightCycler 480 II. Specific primers and TaqMan probes (Takara, Japan) targeting the NS3 gene of JEV were designed to quantitatively amplify an 88 bp fragment, based on earlier studies (Toriniwa and Komiya 2006, Santhosh et al. 2007). Amplicons were quantified using the comparative Ct value method, as described previously (Livak and Schmittgen 2001). Specimens were considered JEV positive at Ct values <35.0.

Detection of serum anti-JEV IgG antibodies

Detection of serum anti-JEV IgG antibodies in rodents was based on a commercially available indirect ELISA kit (Wuhan-Keqian Animal Biological Products Co., Ltd., China) with some modifications, briefly described as follows. In the kit, antigen precoated in plates is prM-E protein of JEV. Each rodent serum sample (diluted 1:40 in buffer) was added in duplicate wells and incubated at 37°C for 30 min. The horseradish peroxidase-conjugated recombinant protein A/G (Thermo), instead of a specific secondary antibody provided by the kit, was used as a secondary antibody to bind to the JEV IgG antibody. After washing, the horseradish peroxidase-conjugated recombinant protein A/G diluted 1:20,000 in buffer was added to each well. The plate was incubated at 37°C for 30 min and rewashed. Colorimetric reactions were performed by the addition of SureBlue. Following incubation at room temperature for 10 min, the reaction was terminated by the addition of 0.2 N H₂SO₄. Within 10 min, optical density (OD) value of each mixture at 650 nm was determined on a microplate reader. Serum specimens were considered positive at S/P (sample OD/positive control OD) values equal to or greater than 0.21.

Serum microneutralization test

Neutralizing antibodies to JEV were detected by using the microneutralization test, as described previously (Cui et al. 2008). In brief, serum samples positive for anti-JEV IgG antibodies were inactivated by heating at 56°C for 30 min. Sera were serially diluted twofold (1:10–1:1280) with Dulbecco's modified Eagle's medium (Hyclone). Diluted serum samples were mixed with 100 TCID₅₀ JEV strain (Nakayama) (Lewis et al. 1947) and incubated at 37°C for 1 h. The mixtures were added to 96-well microtiter plates in the presence of monolayer BHK-21 cells (4 wells per dilution, 50 μL per well). An equal volume of culture medium containing 2% fetal bovine serum (Gibco) was added to each well after a 1-h incubation at 37°C. Plates were subsequently incubated in a humidity chamber at 5% CO₂ and 37°C for 7–10 days, and the cytopathic effect (CPE) on monolayer BHK-21 cells examined. Neutralizing antibody titers were measured as the reciprocal of the highest serum dilution, through which 50% of the wells did not show CPE (Cui et al. 2008). The geometric mean titer (GMT) was calculated by averaging the logarithms of test values.

Detection of serum anti-dengue virus IgG antibodies

As there is potential cross-reaction between flaviviruses and South China is epidemic area of dengue virus, but not of other kinds of flaviviruses, serum samples with anti-JEV IgG antibodies were also screened for dengue virus-specific antibodies by a commercially available indirect ELISA kit (Panbio, Australia) to exclude cross-reaction between flaviviruses. The procedure was as mentioned above.

Statistical analyses

This is a cross-sectional study on serological and viral prevalence of JEV-serocomplex flaviviruses. Data on proportions were obtained from categorical variables. Difference in serological prevalence for the two collection sites was analyzed using a chi-squared test. All statistical analyses in this study were carried out by using SPSS 13.0 software (SPSS, Inc., Chicago, IL). A p-value <0.05 was set to characterize statistically significant results.

Ethics

The protocol was performed in accordance with the ethics principles of the Committee of Southern Medical University on Animal Experiments.

Results

The study included 198 rodents belonging to 4 species, including R. norvegicus (159), R. flavipectus (10), R. lossea Swinhoe (27), and M. musculus Linnaeus (2). Sixty-six rodents were collected from Xiamen City and 132 from Guangzhou City. One hundred eighty-eight brain samples and 96 serum samples were obtained. All rodents did not show apparent signs of diseases. The results of serology and JEV detection are presented in Table 1.

Table 1.

Detection of Japanese Encephalitis Virus and Anti-Japanese Encephalitis Virus-Serocomplex Flaviviruses Antibodies in Rodent Samples Collected From Two Cities in South China

	IgG antibodies against JEV in serum specimens			Neutralizing antibodies in serum specimens			JEV nucleic acids in brain specimens
Species	N	Positive	%	N	Positive	%	N	Positive
Rattus norvegicus (n = 159)	61	28	45.90	25	19	76.00	150	0
Rattus flavipectus (n = 10)	10	3	30.00	2	0	0	9	0
Rattus losea Swinhoe (n = 27)	23	12	52.17	11	5	45.45	27	0
Mus musculus Linnaeus (n = 2)	2	1	1/2	1	0	0	2	0
Total (n = 198)	96	44	45.83	39	24	61.54	188	0

JEV, Japanese encephalitis virus.

No detectable JEV genomic materials were observed among the 188 brain specimens examined with real-time RT-PCR.

All of 96 serum samples were tested for antibodies reacting with JEV or other antigenically related flaviviruses and 44 were positive. The overall seroprevalence of antibodies reacting with JEV or other antigenically related flaviviruses was 45.8% (44/96). The detection rates in rodents from Guangzhou and Xiamen city were 44.1% (15/34) and 46.0% (29/62), respectively. Difference in Guangzhou and Xiamen city was not statistically significant (p > 0.05).

Among the 44 serum samples positive for antibodies against JEV or other antigenically related flaviviruses, 39 had sufficient amounts of sera for measuring the levels of neutralizing antibodies. Twenty-four of 39 (61.5%) serum samples were positive for neutralizing antibodies to JEV, with titers ranging from 1:10 to 1:56. For R. norvegicus, 19 of 25 (76.0%) serum samples were positive for neutralizing antibodies with GMT of 21.6. For R. losea Swinhoe, 5 of 11 (45.5%) samples were positive with GMT of 16.5. R. flavipectus and M. musculus Linnaeus were negative for neutralizing antibodies.

The 44 serum samples positive for antibodies reacting with JEV or other antigenically related flaviviruses were further screened for anti-dengue virus IgG antibodies, but none of them was positive.

Discussion

Antibodies reacting with JEV or other antigenically related flaviviruses were detected in 45.8% (44/96) of serum samples of rodents using ELISA. Considering that the epidemic area of dengue virus in South China and the over cross-reactivity between dengue virus and JEV exist but to a limited extent, we further detected anti-dengue virus IgG antibodies in these 44 serum samples. There was no evidence that showed prior dengue virus exposure in these serum samples. To confirm these findings and assess the neutralizing capacity of the antibodies reacting with JEV or other antigenically related flaviviruses, we determined the titers of neutralizing antibodies in samples with the microneutralization test. Neutralizing antibodies to JEV were detectable in 61.5% (24/39) of serum samples. The positive neutralizing antibodies to JEV suggested that commensal rodents and some species of field rodents might be exposed to JEV or other antigenically related flaviviruses and subsequently underwent seroconversion under natural conditions.

Kinetics of neutralizing antibodies in rodents naturally exposed to JEV or other antigenically related flaviviruses and their protective effects against reinfection with the virus are currently unclear. In the current investigation, titers of neutralizing antibodies to JEV or other antigenically related flaviviruses in rodents were low (GMTs of R. norvegicus and R. losea Swinhoe were 21.6 and 16.5, respectively), implying that rodents naturally exposed to JEV might be induced low immune response or JEV did not replicate in rodents so that no strong antibody responses were mounted against it. As the serological techniques are time-dependent and the limitation of cross-sectional nature of the sampling in the current study, protective effects of low neutralizing antibody titers in rodents remain to be proved.

The issue of whether rodents play a reservoir host role for JEV under natural conditions is under debate. In this study, we failed to detect JEV nucleic acid in brain samples of rodents using real-time RT-PCR. One possible explanation for this result is that all the rodents collected were out of the viremia period, since viremia is short. Considering that relatively limited volumes of serum samples were obtained, we have not detected JEV nucleic acids in serum samples using real-time RT-PCR. Viremia of JEV in rodents remains unclear. Previous studies have reported a high prevalence of antibodies against JEV in wild birds and pigs that are known principal reservoirs of JEV (Pant 2006, Yang et al. 2011), while detection of JEV in these host animals is usually not feasible, owing to the short period of viremia (Nemeth et al. 2012). The finding that commensal and field rodents naturally acquired high prevalence of IgG antibodies reacting with JEV or other antigenically related flaviviruses, but not detectable JEV nucleic acids in brain samples, implies that these rodent species could not be excluded as reservoir hosts.

As a preliminary study on JEV ecology of rodent, several limitations should be discussed. First, the sample procedure in the current study should be improved. The rodent sample size was limited and the nature of cross-sectional studies can only provide a snapshot on the incidence of JEV and the prevalence of antibodies in rodents. Second, only nearly 50% serum samples of rodents could be obtained and a part of them were under sufficient volume, which might affect the detection rate of antibodies. Two aspects of future research should be considered. One is that viremia should be verified and the other is that protective effects of low neutralizing antibody titers in rodents remain to be proved.

To our knowledge, this is the first report documenting serologic evidence of JEV exposure in commensal and field rodents in China. Rodents are not known to play a role in transmission of JEV in Asia, nor is there an evidence to support a role for rodents in transmission of other related flaviviruses in China. However, in the current study, we detected evidence of JEV-reactive antibodies in large numbers of R. norvegicus and R. losea Swinhoe. Further studies of rodents as potential hosts of JEV or other related flaviviruses are warranted.

Footnotes

Acknowledgment

This work was supported by the National Natural Science Foundation of China (grant no. 81373051).

Author Disclosure Statement

The authors declare no conflict of interest.

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