Seoul virus in the Brown Rat (Rattus norvegicus) from Ürümqi, Xinjiang, Northwest of China

In China, hemorrhagic fever with renal syndrome (HFRS) remains a severe public health problem. There may be as many as 150,000 HFRS cases each year, with more than half occurring in China (Song et al. 1999). The case fatality rate for HFRS is as high as 12% (Vapalahti et al. 2003). Hantaan virus and Seoul virus (SEOV) have mainly caused HFRS in China (Zhang et al. 2010). Seoul virus is carried by the brown rat (Rattus norvegicus) in the urban districts of China (Lin et al. 2012). In 1982, SEOV was isolated from the brown rat in Henan Province, China, where the virus is endemic. Seoul virus is found in all areas of China endemic to HFRS, except for Qinghai Province and the Xinjiang Uygur Autonomous Region (XUAR) (Zhang et al. 2010). Interestingly, HFRS caused by SEOV occurs in areas in which HFRS has not been previously reported, suggesting that both the virus and the disease are spreading into other regions of China (Huang et al. 2012).

Xinjiang Uygur Autonomous Region is adjacent to eight countries. Ürümqi, the provincial capital of XUAR, and has a human population of more than 3.5 million. Only an indirect immunofluorescence assay was used to detect HFRS antigens in captured brown rats in Turpan city (east XUAR) during the 1980s (Xinjiang Center for Disease Control and Prevention unpubl. data). Further investigation is warranted because reports regarding Hantavirus in XUAR are limited, and the virus is emerging further into China.

To investigate the presence of hantaviruses in northern XUAR, we collected rodent samples from representative rodent habitats, which consisted of woodlands (around Shihezi City, XUAR), semideserts (surrounding the port of Alashankou, XUAR), urban areas (Ürümqi and Yining City, XUAR), and high mountain meadows (around Jinghe County, XUAR). Each habitat selection consisted of two to four trapping sites in areas in which there was a high density of the dominant species. The rodents were captured with rodent traps during 2010–14 (Table 1). The great gerbil (Rhombomys opimus) and the long-tailed ground squirrel (Spermophilus undulatus) were captured in Sherman traps (H. B. Sherman Traps, Tallahassee, Florida, USA), which were placed at the entrances of occupied burrows. Each survey site included >150 traps that were checked twice a day for a 1–2 d period. Each trap was removed before nightfall and replaced on the survey site the following day. The brown rat, gray dwarf hamster (Cricetulus migratorius), and other less-abundant rodents were captured using snap traps (Victor® Easy Set® Rat Trap, Woodstream Corporation, Lititz, Pennsylvania, USA), which were set up at approximately 5-m intervals in the evening and collected the next morning, as described by Aplin et al. (2003). In addition, 200–250 mousetraps were used in each trapping site during each survey. This study was approved by the Animal Ethics Committee of Xinjiang Medical University, Ürümqi, China (approval AECXMU2010-11).

We captured 844 rodents from the four habitats that were examined. We trapped 283 rodents (255 great gerbils [Rhombomys opimus], 9 Mongolian five-toed jerboas [Allactaga sibirica], 5 tamarisk jirds [Meriones tamariscinus], 5 midday jirds [Meriones meridianus], 7 Libyan jirds [Meriones libycus], and 2 Asian house shrews [Suncus murinus]) in the semidesert region located near Alashankou Port, 31 gray dwarf hamsters (Cricetulus migratorius) in the forest region near Shihezi City, 278 brown rats in the urban areas (72 in Ürümqi and 206 in Yining City), and 252 long-tailed ground squirrels (Citellus undulatus) in the high mountain meadow near Jinghe County.

After each captured animal was identified and euthanized, approximately 150 mg of lung tissue was collected, cut aseptically into slices <0.5 cm thick, and placed into tubes containing 5 mL RNA stabilization reagent (Ambion RNAlater®, Thermo Fisher Scientific Life Sciences, Carlsbad, California, USA). Each tube was assigned a classification number, and the samples were then placed at 5 C overnight. On the following day, the samples were transferred to −80 C for long-term cryopreservation.

Total RNA was extracted from lung tissue using the TRIzol reagent (Thermo Fisher) according to the manufacturer's instructions. The detailed cycling conditions for complementary DNA synthesis and coding DNA targeted amplification are shown in the Supplementary Material (primer nucleotide sequences; Table S1). The amplified products were analyzed on a 2% agarose gel. Positive samples were purified and ligated according to the instructions provided in the TIANgel Midi Purification Kit (TIANGEN Biotech Co., Ltd., Beijing, China) and pGEM®T Easy Vector System I (Promega, Madison, Wisconsin, USA). All of the Hantavirus-positive clones were selected for sequencing and analyzed using Clustal X 2.0 software (Larkin et al. 2007). The M gene fragment of SEOV was first amplified in 15% (11 or 72) of the brown rat samples from Ürümqi only. The captured Hantavirus-positive rodents were identified by PCR and the complete cytochrome b gene was sequenced as a phylogenetic marker (Schlegel 2012). All clones from the positive samples were classified as two representative strains, namely Ürümqi55 and Ürümqi59 (deposited in GenBank as Ürümqi55: KT188829 and Ürümqi59: KT188830 accessions). The two clones had 99.5% nucleotide identity. The silent mutations (A to G) were detected in two nucleotide sites of the M gene (2230 and 2258) in both clones.

Thirty-four Hantavirus sequences that were retrieved from GenBank are available in Table S2. The phylogenetic tree based on the M gene fragments revealed that the Ürümqi55 and Ürümqi59 branches were related to SEOV and more closely related to HuludaoRn101, HB55, L99, k24-e7, and k24-v2, which were isolated in Liaoning, China; Hubei, China; Jiangxi, China; and Zhejiang Province, China, respectively (Fig. 1). However, we found no association between the homology of the SEOV and the geographic distribution of host rodents. Our attempts to obtain the whole nucleotide sequence of L, M, S segments were unsuccessful because of nucleic acid degradation.

Many data indicate that the translocation or trafficking of domestic and wild animals has an important role in the rapid dispersal of many zoonotic pathogens (Cutler et al. 2010). More specifically, the migration of the brown rat resulted in the worldwide distribution of the Seoul virus (Lin et al. 2012). Kang et al. (2012) showed that the brown rat entered XUAR by way of the Lanzhou-Xinjiang Railway line and formed a stable population in the mid-1980s, which gave rise to the gradual spread of this species throughout XUAR, resulting in the dissemination of parasites and pathogens, although local human Hantavirus infections that result in HFRS have not been reported. Our results indicated that 15% of brown rats carry SEOV, suggesting a high risk for HFRS in Ürümqi City. Seoul virus has not yet been detected in the brown rats of Yining City. It is likely virus distribution area in XUAR is not restricted to Ürümqi City and may spread to other regions in Xinjiang and even to neighboring countries, including Kazakhstan, through transportation. Seoul virus circulates in Ürümqi City, where the brown rat may act as a natural reservoir, as is indicated by its high prevalence in that species.

To our knowledge, there have been no reports demonstrating that hantaviruses are naturally carried by the great gerbil, long-tailed ground squirrel, or gray dwarf hamster. Our negative results indicate the possibility that these species are not susceptible to Hantavirus infections or that they lack exposure the viruses. Based on our studies, long-term surveillance and examination of hantaviruses in the human and rodent populations located in XUAR, and especially in Ürümqi City, are necessary to identify novel Hantavirus strains and ultimately to reduce the rate of human exposure.

This research was supported in part by grants from the Scientific Research Fund Project of General Administration of Quality Supervision (2012IK245) and the High-Tech Develop and Research Fund Project of Xinjiang (201217153).

Supplementary material for this article is online at http://dx.doi.org/10.7589/2015-08-210.