Detection of West Nile Virus Infection in Viral Encephalitis Cases,China

Abstract

This study detected West Nile virus (WNV) infection in serum samples of patients clinically diagnosed with viral encephalitis in the Japanese encephalitis virus (JEV) endemic area (seven provinces) and JEV nonendemic area (Xinjiang province) in China from 2011 to 2012. In JEV endemic areas, there were 22 positive cases of WNV immunoglobulin M (IgM) antibody in serum specimens of 65 JEV patients (JEV IgM antibody positive) in the acute phase, whereas WNV IgM antibodies were not detected in serum specimens of 63 non-JEV patients (JEV IgM antibody negative). However, the titer of JEV-neutralizing antibody was four times higher than that of WNV-neutralizing antibody in WNV-IgM-positive serum specimens. Detection was also conducted in serum specimens collected from 12 patients clinically diagnosed as viral encephalitis in Xinjiang; five serum specimens were WNV IgM antibody positive, and there were fourfold differences in WNV-neutralizing antibody titers between convalescent and acute serum. Meanwhile JEV-neutralizing antibody titer was negative or significantly lower than that of WNV-neutralizing antibody in the same specimens. WNV IgM antibodies positive were detected in acute serum specimens of patients clinically diagnosed with JEV infection in JEV-endemic areas, but no WNV neutralization antibodies were detected fourfold greater than that of the corresponding JEV antibodies. Clinical cases of WNV infection were detected in patients clinically diagnosed with viral encephalitis in Xinjiang.

Introduction

West Nile virus (WNV) is a mosquito-borne flavivirus of the family Flaviviridae. Humans can manifest transient fever following infection by WNV, and clinical manifestation is mild and mostly self-limiting. However, severe manifestation can cause encephalitis and other symptoms, or even death (Lindenbach et al. 2007). In recent years, as a newly emerging infectious disease, WNV and its infection have been expanding and increasing in terms of incidence and epidemic area (Mackenzie et al. 2004, Weaver and Reisen 2010). After WNV invaded North America in 1999, the number of WNV infections reached 46,086 in the United States during 1999–2016, including 21,574 cases of neurological symptoms and 2017 deaths (Source: ArboNET, Arboviral Diseases Branch, Centers for Disease Control and Prevention. www.cdc.gov/westnile/statsmaps/cumMapsData.html#one). WNV has been isolated from mosquito specimens collected in Xinjiang in western China in recent years, marking its first isolation in mainland China (Lu et al. 2014), and the prevalence of fever and outbreaks of viral encephalitis caused by WNV infection has been recorded at a local scale (Li et al. 2013, Cao et al. 2017), demonstrating the circulation of WNV and its prevalence in mainland China.

Japanese encephalitis virus (JEV) is also a mosquito-borne flavivirus and of the same family, Flaviviridae; its symptoms include fever, headache, nausea, and vomiting. Numerous studies have shown that, excluding Xinjiang, Tibet, and Qinghai, all provinces in mainland China are JEV-endemic areas (Zheng et al. 2012, Gao et al. 2014, Li et al. 2014). Because the JEV vaccine was introduced through the Expanded Program on Immunization (EPI) in 2008, children in China have received free JEV vaccinations. Subsequently, the incidence of Japanese encephalitis has dropped drastically. However, there are still 1000–2000 JEV cases annually (Gao et al. 2014, Li et al. 2014). WNV was isolated from mosquito specimens in the field in Xinjiang, western China, and the prevalence of fever, viral meningitis, and encephalitis caused by WNV infection in summer has been recorded (Li et al. 2013, Lu et al. 2014, Cao et al. 2017). However, it is unknown whether WNV infection or coinfection of WNV and JEV occurs in provinces other than Xinjiang, particularly in patients with viral encephalitis in summer. To determine whether there have been cases of WNV infection among patients with viral encephalitis in JEV-endemic areas of mainland China, we conducted parallel detection of infections by WNV and JEV (immunoglobulin M [IgM] antibody and neutralizing antibody) in serum specimens of patients clinically diagnosed with Japanese encephalitis or viral encephalitis in JEV-endemic areas (seven provinces).

Materials and Methods

Cells and viruses

Vero cells (African green monkey kidney cells) were preserved in our laboratory at the Department of Viral Encephalitis and Arbovirus, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (IVDC, China CDC), and maintained in Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 20,000 U/mL penicillin–streptomycin (PS) at 37°C with 5% CO₂. Cells were cultivated in PS-MEM with 2% FBS when infected with the virus. The WNV XJ11129 strain (GenBank: JX442279) used in this study was isolated in our laboratory from Culex pipiens specimens collected in the Xinjiang Uygur Autonomous Region in 2011, when WNV was first isolated in China (Lu et al. 2014). The JEV P3 strain (GenBank: JEU47032) was preserved in our laboratory (Pan et al. 2011).

Collection of serum samples and ethics statement

This study collected serum specimens from 128 acute-phase viral encephalitis patients (including JEV patients and non-JEV patients) in seven provinces with JEV-endemic areas and from 12 patients from JEV nonendemic areas (Xinjiang province) clinically diagnosed as suspected cases of acute-phase viral encephalitis. All of the samples were centrifuged to obtain serum and refrigerated for standby application after subpackaging. This study was conducted by the National Reference Laboratory of Japanese Encephalitis and the World Health Organization Japanese Encephalitis Regional Reference Laboratory, China. Serum specimens from JEV patients (JEV IgM antibody positive) and non-JEV patients (JEV IgM antibody negative) used in this study were collected and maintained during the JEV epidemiological monitoring period by these JEV-monitoring network laboratories. This research project was approved by the Ethics Committee of the Virus Disease Institute of China, Center for Disease Control and Prevention (No. 2014112509).

Detection of IgM antibodies and nucleic acid in serum samples

All of the serum specimens underwent simultaneous enzyme-linked immunosorbent assay (ELISA) detection of both JEV and WNV IgM antibodies. A JEV IgM Test Kit (JEV IgM Capture ELISA Kit; Panbio, Sinnamon Park, Queensland, Australia) was used for the JEV-IgM assay, and a WNV-IgM Test Kit (WNV IgM Capture ELISA; Panbio) was used for the WNV-IgM assay. The application and interpretation of results for both tests were conducted according to the manufacturer's instructions (Li et al. 2013, Lu et al. 2014, Cao et al. 2017). Both detection kits employed the Capture ELISA procedure (Martin et al. 2000, Cao et al. 2017). Nucleic acids in the serum specimens were extracted (QIAamp Viral RNA Mini Kit; QIAGEN, Valencia, CA) and subjected to reverse transcription one-step polymerase chain reaction (PCR) (QIAGEN OneStep RT-PCR Kits; QIAGEN) using specific primers for WNV and JEV. JEV: PrMF: 5′-CGT TCT TCA AGT TTA CAG CAT TAG C-3′ (251–275), PrMR1: 5′-CGY TTG GAA TGY CTR GTC CG-3′ (724–743), PrMR2: 5′-CCY RTG TTY CTG CCA AGC ATC CAM CC-3′; WNV: F: 5′-TTG TGT TGG CTC TCT TGG CGT TCT T-3′ (233–257), R: 5′-CAG CCG ACA GCA CTG GAC ATT CAT A-3′ (640–616) (Lanciotti et al. 2000, Wang et al. 2007, 2010). PCR products were detected by agarose gel electrophoresis.

Detection of neutralizing antibody

We used a 90% plaque reduction neutralization test (PRNT₉₀) to detect neutralizing antibodies against WNV and JEV. The serum was inactivated and diluted to 1:10 to detect WNV-neutralizing antibodies for prescreening and from 1:10 to 1:1280 to detect JEV and WNV-neutralizing antibodies in parallel. Then it was mixed with an equal volume of 200 pfu WNV and JEV suspension and incubated at 37°C for 1 h. The mixture was added to six-well Vero cells and incubated at 37°C for 1 h, followed by overlaying with 1.1% methylcellulose–MEM culture medium with 2% FBS for 4–5 days. Crystalline Violet staining was used to calculate the number of plaques of different dilution ratios and as a virus back-dropping control. The highest dilution ratio of serum specimens that could inhibit more than 90% of virus infection was the neutralizing antibody titer (Li et al. 2013, Cao et al. 2017). To further determine if the patient was infected by WNV or JEV, neutralizing antibody titers of both WNV and JEV were detected in parallel for any WNV IgM-positive specimens. Specimens were judged positive when PRNT₉₀-neutralizing antibody was ≥1:10. If the PRNT₉₀ anti-WNV antibody titer was at least fourfold greater than that of the corresponding JEV antibody titer in the same specimen, it was identified as WNV infection rather than JEV infection. Otherwise, it was identified as JEV infection (Li et al. 2013, Cao et al. 2017).

Results

Detection of WNV infection in serum specimens from viral encephalitis cases in JEV-endemic areas

Screening of WNV IgM antibodies in serum specimens from viral encephalitis cases

This study collected serum specimens from 65 acute-phase JEV patients (JEV IgM antibody positive) and 63 acute-phase non-JEV patients (JEV IgM antibody negative) from seven provinces in JEV-endemic areas, for a total of 128 serum specimens (Table 1). Parallel detection of JEV and WNV IgM antibodies was conducted for each serum specimen. The test results were positive for JEV IgM antibodies in all serum specimens from 65 JEV patients, which was consistent with the primary provincial CDC results. In addition, 33.8% (22/65) of serum specimens tested positive for WNV IgM antibodies; however, WNV and JEV IgM antibodies were not detected in serum specimens from 63 acute-phase non-JEV patients. Detailed results are presented in Table 1.

Table 1.

West Nile Virus Immunoglobulin M Antibody Positive in Viral Encephalitis Cases, China

	No. Total	Specimen of JEV-IgM (+) ^a		Specimen of JEV-IgM (−) ^a
Province	Test	No. test	No. sample of WNV IgM (+) ^b	No. test	No. sample of WNV IgM (+) ^b
Henan	19	2	2	17	0
Gansu	17	6	2	11	0
Guangdong	20	19	6	1	0
Guizhou	20	13	4	7	0
Szechwan	20	12	5	8	0
Guangxi	12	12	3	0	0
Yunnan	20	1	0	19	0
Total (%)	128	65	22/65 (33.8%)	63	0/63 (0%)

Number of JEV IgM positive (+) or negative (−) tested by IgM capture ELISA.

Number of WNV IgM positive (+).

ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; JEV, Japanese encephalitis virus; WNV, West Nile virus.

Detection of neutralizing antibodies in specimens testing positive for WNV IgM antibodies

Neutralizing WNV and JEV antibodies were further detected in parallel for each serum specimen in which WNV IgM antibodies were positively detected. The results showed that 68.2% (15/22) of serum specimens tested positive for WNV PRNT₉₀ (PRNT₉₀ ≥1:10) in WNV IgM antibody-positive specimens (distributed in five provinces: Guangdong, Guangxi, Henan, Sichuan, and Guizhou), and PRNT₉₀ was <1:10 in seven cases, 1:10 in eight cases, and 1:20 in seven cases. However, all 22 specimens tested positive for JEV PRNT₉₀, with neutralizing antibody titers ranging from 1:10 to >1:80. The results also showed that 68.2% (15/22) of specimens showed a fourfold higher titer for JEV PRNT₉₀ compared with WNV PRNT₉₀ in 22 specimens (Table 2), which suggests that these cases were JEV infections rather than WNV infections.

Table 2.

Neutralization Titers from Plaque Assay (PRNT₉₀) for Samples of West Nile Virus Immunoglobulin M Positive

					IgM-ELISA		PRNT₉₀ titer
No.	Gender	Age (years)	Province	Days postadmission	WNV	JEV	WNV	JEV
1	Female	7	Guangdong	NA	+	+	<1:10	1:10
2	Male	16	Guangdong	13	+	+	1:10	1:80
3	Male	6	Guangdong	30	+	+	1:10	1:80
4	Male	6	Guangdong	NA	+	+	1:10	1:10
5	Male	16	Guangdong	NA	+	+	<1:10	1:80
6	Male	7	Guangdong	1	+	+	1:10	>1:80
7	Female	43	Henan	NA	+	+	1:10	>1:80
8	Male	10	Henan	NA	+	+	<1:10	1:10
9	NA	NA	Guizhou	NA	+	+	1:10	1:40
10	NA	NA	Guizhou	NA	+	+	<1:10	1:10
11	NA	NA	Guizhou	NA	+	+	1:10	1:10
12	NA	NA	Guizhou	NA	+	+	<1:10	1:80
13	Male	6	Guangxi	6	+	+	1:20	>1:80
14	Male	1	Guangxi	4	+	+	1:20	1:40
15	Male	3	Guangxi	6	+	+	1:20	>1:80
16	Male	59	Szechwan	16	+	+	1:20	>1:80
17	Male	11	Szechwan	4	+	+	1:10	1:40
18	Male	9	Szechwan	5	+	+	1:20	>1:80
19	Female	40	Szechwan	2	+	+	1:20	>1:80
20	Female	70	Szechwan	2	+	+	1:20	1:40
21	Male	58	Gansu	4	+	+	<1:10	>1:80
22	Male	32	Gansu	5	+	+	<1:10	1:40

NA, not available; PRNT, plaque reduction neutralization test.

WNV infection in Xinjiang

WNV and JEV infections were detected in parallel for serum specimens of 12 Xinjiang patients clinically diagnosed with viral encephalitis (WNV was previously isolated from mosquitoes in this area). The test results showed that WNV and JEV gene tests were negative in these 12 serum specimens. All 12 specimens tested positive for WNV IgM antibodies and only 6 specimens tested positive for JEV IgM antibodies. The detection results of virus PRNT₉₀ antibody showed that, in serum specimens from five patients, the titer of WNV-neutralizing antibody (PRNT₉₀) in the recovery phase was fourfold higher than that in the acute phase, and that the titer of the WNV-neutralizing antibody was fourfold higher than that of the JEV PRNT₉₀ antibody (Table 3). Therefore, these five patients with viral encephalitis were identified as infected with WNV. Although the fourfold difference in WNV-neutralizing antibody titer was not detected between convalescent and acute serum samples in the remaining seven patients (PRNT₉₀ titer of paired acute-phase and convalescent serum samples was 1:10–20), the titer of PRNT₉₀ anti-WNV antibodies (1:20–40) was higher than that of PRNT₉₀ anti-JEV antibodies (<1:10) in serum specimens of acute-phase or recovery-phase patients (Table 3).

Table 3.

Detection for West Nile Virus Infection in Viral Encephalitis Cases, Xinjiang, China

					WNV			JEV
Year	Gender	Age (years)	Days postadmission	Symptoms	RT-PCR	IgM	PRNT₉₀	RT-PCR	IgM	PRNT₉₀
2011	Female	27	1	Hyperpyrexia 39°C, dizziness, headache, vomiting	–	+	1:20	–	–	<1:10
			83			+	1:80		–	<1:10
2011	Male	67	1	Fever, headache	–	+	1:10	–	–	<1:10
			84			+	1:80		–	<1:10
2011	Male	66	1	Hyperpyrexia 39°C, insomnia	–	+	1:10	–	+	<1:10
			63			+	1:40		+	<1:10
2012	Male	85	7	Fever, headache, vomiting, and anorexia	–	+	1:640	–	+	1:10
			17	Headache, anorexia		+	1:5120		+	1:80
2012	Female	64	8	Fever, headache, and vomiting	–	+	1:20	–	+	<1:10
			18	Headache		+	1:160		–	<1:10
2011	Female	58	1	Hyperpyrexia, coma, and headache	–	+	1:20	–	+	<1:10
			71			+	1:20		–	<1:10
2011	Male	79	1	Fever and coma		+	1:20		+	<1:10
			69			+	1:20		–	<1:10
2011	Male	64	1	Headache		+	1:10		+	<1:10
			68			+	1:20		–	<1:10
2012	Female	68	8	Fever, vomiting, and neck stiffness, hemiplegic, light coma, predying state	–	+	1:40	–	–	<1:10
			18			+	<1:10		–	<1:10
2012	Male	70	4	Fever, hemiplegic, poor consciousness	–	+	1:20	–	–	<1:10
			14			+	1:20		–	<1:10
2012	Female	62	8	Fever, vomiting, hemiplegic, and poor consciousness	–	+	1:20	–	–	<1:10
			18			+	1:20		–	<1:10
2012	Male	48	7	Fever and headache	–	+	1:40	–	–	<1:10
			Not available

RT-PCR, reverse transcription–polymerase chain reaction.

Discussion

Both JEV and WNV belong to the Japanese encephalitis serocomplex of the family Flaviviridae. Crossreactivity of antigens between the two viruses can cause interaction between IgM antibodies, IgG antibodies, and even neutralizing antibodies (Mansfield et al. 2011, Stiasny et al. 2013). The criterion for differentiating crossreactions between viruses is to detect a fourfold difference in the neutralizing antibody titer of convalescent and acute serum samples, or a fourfold difference in that of two viruses in the same specimen (Calisher et al. 1989, Pauvolid-Corrêa et al. 2014, Morales et al. 2017). A total of 22 cases simultaneously tested positive for JEV and WNV IgM antibodies (ELISA method) in serum specimens of acute JEV patients collected in JEV-endemic areas. The detection of antibodies reactive with both WNV and JEV antigens raised the possibility of coinfection with JEV and WNV (Tables 1 and 2). To further clarify whether these patients were infected with WNV, we conducted a PRNT₉₀ antibody titer for JEV and WNV in parallel. The test results showed that in 15 of these 22 specimens, the titer of JEV-neutralizing antibodies (1:10 to >1:80) was fourfold that of WNV-neutralizing antibodies (<1:10 to 1:20) (Table 2). Therefore, these cases can be considered JEV infections rather than WNV infections. Moreover, WNV IgM antibody-positive results in specimens tested using the ELISA method may have been due to crossreaction caused by JEV infection. Five specimens tested positive for both JEV and WNV IgM antibodies (Table 2), and the titers of neutralizing antibodies of both viruses were low (1:10 or <1:10) and there was no fourfold difference. Therefore, convalescent-phase samples for the above mentioned cases must be collected for further parallel detection with acute-phase serum samples, to compare these results. It was not possible to determine whether the patient's viral infection was caused by this virus alone based on only acute-phase serum specimens.

Because the serum specimens of the above patients at recovery phase were not collected, this study could not compare and analyze the neutralizing antibody titers against different viruses in serum specimens between acute phase and recovery phase. Therefore, the criteria for collection and detection of paired serum specimens must be strengthened in future studies to obtain more credible test results. In addition, the results of JEV antibodies were commonly found in the population due to the prevalence of JEV in vast areas of China and the inclusion of the JEV vaccine into the EPI. However, the neutralization test showed that a WNV IgM antibody-positive result was actually a false-positive reaction caused by crossreactivity due to JEV infection. WNV infection test results should not be based on positive WNV IgM antibody test results in acute-phase patients; therefore, this test should not be used as a laboratory diagnostic indicator when diagnosing WNV infection in China, but instead to simultaneously detect a variety of WNV and JEV antibodies in specimens, so as not to cause false-positive results.

The serum specimens used in this study were collected in summer and fall, during peak mosquito reproduction; this is also the season for epidemic JEV infection. This study detected WNV- and JEV-neutralizing antibodies in samples collected from eight provinces, and found only five cases of encephalitis patients infected with WNV in Xinjiang. However, no case of WNV infection was detected in serum specimens from viral encephalitis patients with JEV or non-JEV patients in other provinces. Previous studies have shown that there have been no cases of JEV infection in Xinjiang since 1951, and that Xinjiang is a non-JEV-endemic area (Gao et al. 2010, 2014, Zheng et al. 2012, Li et al. 2014). Our research group did not find JEV during our investigation of mosquito-borne viruses in nature in Xinjiang over a period of 10 consecutive years (Wang et al. 2009, Liu et al. 2011). Earlier studies have shown that C. pipiens and Aedes flavidorsalis are the dominant mosquito species in Xinjiang, and there are no C. tritaeniorhynchus, which is a main vector of JEV. The sizeable Uyghur population of Xinjiang traditionally does not consume pork; therefore local residents do not raise pigs, and there are no pig farms throughout Xinjiang (Lu et al. 2014). As a result, there is a lack of JEV transmission between Culex tritaeniorhynchus and pigs in Xinjiang, which may be a reason for the lack of JEV infection in Xinjiang. Samples from the other seven provinces in this study (Table 1) were collected from JEV-endemic areas in southern and central-eastern China. In these areas, peasants mainly grow rice, and C. tritaeniorhynchus is the dominant mosquito. In addition, the residents are mainly of the Han nationality, preferring to eat pork; therefore, a large number of pigs are held in these regions, providing favorable conditions for the circulation of JEV in nature. These areas are therefore JEV endemic, with frequent eruptions (Miller et al. 2012, Li et al. 2014, 2016).

Although JEV and WNV both belong to the genus Flavivirus in the family Flaviviridae and can cause viral encephalitis, the majority of cases from JEV-endemic areas are children, whereas mainly cases from WNV-endemic areas are adults (Mackenzie et al. 2004). Various age groups have been covered in our study. Similarly, our results demonstrated that cases from JEV-endemic areas are mainly children and young adults (Table 2), whereas the cases of WNV infections from Xinjiang province are mainly elderly (11 cases in 12 were over 48 years of age). We also found a low titer of WNV-neutralizing antibodies (1:20–40) in the serum specimens of seven patients with viral encephalitis in Xinjiang, although JEV-neutralizing antibodies tested negative in the same specimen (<1:10) (Table 3). There was no fourfold difference in WNV-neutralizing antibodies of paired sera in these cases; therefore, we could not determine whether this result was caused by WNV infection. Because these patients exhibited obvious fever, headache, vomiting, and even confusion and other clinical symptoms of viral encephalitis, and all of the cases occurred in summer and autumn, the local mosquito-breeding season, more extensive and in-depth research are needed to confirm whether these cases were caused by another mosquito-borne arbovirus infection.

In this study, no WNV infection was detected in serum specimens from patients with viral encephalitis collected in seven JEV-endemic areas of mainland China in 2011–2012. However, WNV has been isolated from mosquito specimens in nature in Xinjiang province in western China, and the prevalence of fever and viral encephalitis caused by WNV infection has been detected in local areas (Li et al. 2013, Lu et al. 2014, Cao et al. 2017). The WNV that presents in nature in Xinjiang can spread from western China to other parts of China along with cargo, humans, animals, and other vectors, thus expanding the geographical distribution of WNV in China and causing the prevalence of WNV outside of Xinjiang. Although WNV infection was not detected in serum specimens collected from patients with viral encephalitis in seven JEV-endemic areas of China, we could not confirm that there was a lack of WNV infection in these areas, due to the limited number of specimens. All of China, except for Xinjiang, Tibet, and Qinghai, is a JEV-epidemic area, both WNV and JEV are spread by mosquitoes, and all of the cases occur frequently in summer and autumn with similar clinical manifestations that may easily lead to misdiagnosis (Colpitts et al. 2012, Zheng et al. 2012). Therefore, it is important to strengthen the detection and monitoring of WNV infection in the vast JEV-endemic areas of China to find cases of WNV infection and natural circulation outside of Xinjiang, thereby reducing the public health hazard posed by WNV infection.

Footnotes

Acknowledgments

This work was supported by the National Key R&D Program of China (2016YFC1201904), the National Natural Science Foundation of China (81501757), the National Key Plan for Scientific Research and Development of China (2016YFD0500300), and the Development Grants of State Key Laboratory of Infectious Disease Prevention and Control (2014SKLID103, 2015SKLID505). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the article.

Author Disclosure Statement

No competing financial interests exist.

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