West Nile Virus Infection in Xinjiang,China

Introduction

A n outbreak of fever and meningitis/encephalitis occurred in Jiashi County, Xinjiang, China, from August 5 to September 3, 2004. Cases were reported from 10 of 12 villages in this county. A total of 80 patients were hospitalized and 10 died. Patient ages ranged from 2 from 83 years; 63% (50/80) of patients were aged 50–60 years, and 5 patients were>60 years old (Li et al. 2007). In preliminary diagnostic testing, several cerebrospinal fluid (CSF) and serum samples tested at Jiashi County Center for Disease Control (CDC) showed positive immunoglobulin M (IgM) antibody to Japanese encephalitis virus (JEV) with JE IgM Capture enzyme-linked immunosorbent assay (ELISA), suggesting the possibility of an outbreak of JEV or a similar flavivirus (Li et al. 2007). Here, we report further analysis of samples collected from several of the patients from this outbreak that were JE IgM positive in preliminary testing in an effort to determine if JEV was circulating in 2004 in an area of Xinjiang province where JEV was not thought to occur (Gao et al. 2010).

Materials and Methods

Of the patients with preliminary diagnostic results suggesting infection with JEV, CSF and multiple serum samples were available for 3 of the cases and single serum samples were available for 3 additional patients (Table 1). CSF and serum samples were collected between 3 and 32 days following admission to the hospital. Samples were stored at −80°C and transported to the laboratory on dry ice. All specimens were tested at the Institute for Viral Disease Control and Prevention, China CDC for the presence of IgM against West Nile virus (WNV), JEV, Dengue virus (DENV), yellow fever virus (YFV), eastern equine encephalitis virus, (EEEV), western equine encephalitis virus (WEEV), and Lacrosse virus (LACV) by immunofluorescence assay (IFA) using commercially available test kits (Antigen Slide for Arbovirus Screen-USA; Antigen Slide for Arbovirus Screen-Non-USA (PANBIO, Ltd., Windsor, Australia). Subsequently, the serum specimens from these patients were tested at the U.S. CDC Division of Vector-Borne Diseases for the presence of IgM against WNV, St. Louis encephalitis virus (SLEV) (Johnson et al. 2005) and JEV (Martin et al. 2000) and for neutralizing antibody against WNV (virus strain: EG-101), JEV (SA 14-14-2), tick-borne encephalitis (TBEV) (chimeric virus comprised of the TBEV Far Eastern subtype [Sofjin strain] prM and E antigens inserted into a dengue virus type 4 backbone) and Dengue-2 (ChimeriVaxTM-DEN2 [strain PUO218] expressing DEN2 PrM and E antigens on a yellow fever 17D vaccine strain backbone) using a 90% end point plaque reduction neutralization assay (PRNT) (Beaty et al. 1995). CSF volumes were insufficient for testing at U.S. Centers for Disease Control (CDC).

Results

CSF from cases 1 and 3 were WNV IgM positive and JEV IgM negative by IFA and the WNV PRNT titer on both the first and second serum samples from these cases was 4-fold greater than the JEV PRNT titer. These results are indicative of a recent WNV infection likely associated with the current illness (Table 1). Cases 4 and 6, from which only single serum samples were available, demonstrated differences in PRNT titers between WNV and JEV that suggest evidence of WNV infection. However, without additional samples, WNV cannot be confirmed as the etiology of the current illness in these cases. PRNT results from cases 2 and 5 cannot differentiate between WNV and JEV and suggest only infection with a flavivirus. All samples were negative for DENV and TBEV neutralizing antibodies and none were IFA-positive IgM against the other viruses (DENV, YFV, EEEV, WEEV, and LACV) (data not shown). Although relatively few samples were available for testing, the results indicate that several patients were hospitalized with fever and meningitis/encephalitis associated with WNV infection and suggest that WNV contributed to the outbreak that occurred in Jiashi County, Xinjiang in 2004.

Discussion

Although serological results showing WNV infection in dogs and cats in Shanghai (Lan et al. 2011) and birds in Yunnan province (Yang et al. 1988) suggest enzootic transmission of WNV in China, and results of HI test suggest antibodies against numerous flaviviruses, including WNV, in humans in Xinjiang province (Chen et al. 1983), this is the first report of human infection and disease resulting from WNV in China with PRNT-supported serological results. This is not unexpected because WNV infection causing neuroinvasive disease has been reported from countries adjacent to China in the former Soviet Union (Mackenzie et al. 2004) and India (Khan et al. 2011), and several mosquito species that are widely distributed in China are known to be competent WNV vectors (Jiang et al. 2010). A subsequent study of serum samples taken from fever cases in Xinjiang province detected evidence of IgM against several flaviviruses including JEV, DENV, and WNV in 25 of 641 samples, but the exact etiology could not be determined due to extensive cross-reaction among these viruses (Lu et al. 2011a). Mosquitoes collected in Xinjiang province have been tested for the presence of arboviruses using virus isolation in C6/36 and BHK-21 cells have yielded Tahyna virus and Liaoning virus, but no WNV (Lu et al, 2009, 2011b). Additional studies are in progress to attempt isolating WNV from mosquitoes in the area where the 2004 cases occurred.

Because Japanese encephalitis is prevalent in mainland China (Gao et al. 2010), its clinical symptoms and seasonality are similar to those of WNV infection (Yang et al. 1988, Gao et al. 2010) and diagnostic differentiation of JEV from WNV infection is problematic. It is essential to improve the accuracy of laboratory procedures and diagnosis of viral encephalitis that may be caused by JEV and WNV in regions of China, in areas where JEV is found. It is also important to implement monitoring programs to evaluate the potential contribution of WNV to seasonal encephalitis cases in areas where JEV does not occur.