Detection of SFTS Virus RNA and Total Antibodies in Wild Animals, Jiangsu, China, 2014-2019

Background Severe fever with thrombocytopenia syndrome (SFTS) is a novel infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV). Currently, SFTS is endemic to some areas in China, and wild animals are considered to play important roles in the circulation of SFTSV in the environment. Wild animals monitoring for SFTSV has been fullled since 2014 in Jiangsu Province. We studied the results of the detection to provide basic data for better diagnosis of wild animals. Methods This research was conducted in Jiangsu Province from 2014 to 2019. Sera of wild animals (Rodents, pheasants and hedgehogs) were collected to detect SFTSV both RNA and total antibodies by qRT-PCR and enzyme-linked immunosorbent assay. Statistical analysis was performed by using SPSS 25.0 (Chicago, IL, USA). Results A total of 95.8% (1298/1355) of the specimens had the same SFTSV RNA and total antibodies detection results. However, there was a signicant difference between the detection rates of SFTSV RNA and total antibodies, and the detection consistency was very poor. The detection rate of SFTSV total antibodies was highest in hedgehogs(19.54%). Conclusions SFTSV total antibodies testing was preferred method during wild animals surveillance. Also, hedgehog could be a competent reservoir for SFTSV.


Abstract
Background Severe fever with thrombocytopenia syndrome (SFTS) is a novel infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV). Currently, SFTS is endemic to some areas in China, and wild animals are considered to play important roles in the circulation of SFTSV in the environment. Wild animals monitoring for SFTSV has been ful lled since 2014 in Jiangsu Province. We studied the results of the detection to provide basic data for better diagnosis of wild animals.
Methods This research was conducted in Jiangsu Province from 2014 to 2019. Sera of wild animals (Rodents, pheasants and hedgehogs) were collected to detect SFTSV both RNA and total antibodies by qRT-PCR and enzyme-linked immunosorbent assay. Statistical analysis was performed by using SPSS 25.0 (Chicago, IL, USA).
Results A total of 95.8% (1298/1355) of the specimens had the same SFTSV RNA and total antibodies detection results. However, there was a signi cant difference between the detection rates of SFTSV RNA and total antibodies, and the detection consistency was very poor. The detection rate of SFTSV total antibodies was highest in hedgehogs(19.54%).
Conclusions SFTSV total antibodies testing was preferred method during wild animals surveillance. Also, hedgehog could be a competent reservoir for SFTSV.

Background
Sporadic cases with clinical manifestations of acute onset of fever, low white blood cell and platelet counts, high levels of alanine and aspartate transaminases, and proteinuria, have been observed in China since 2005. A new bunyavirus, which was then named severe fever with thrombocytopenia syndrome virus (SFTSV) in 2010, was found to be related to this disorder. SFTSV is endemic in the central and eastern China. In addition, con rmed SFTS cases have been reported in Japan and South Korea in recent years [1][2][3][4][5][6][7]. SFTSV is thought to be a tick-borne zoonotic virus [1,8,9], and has been detected in or isolated from several species of ticks including Haemaphysalis longicornis, Amblyommatestudinarium, and Ixodes nipponensis in China and Korea [10][11][12][13]. Spotted doves, one of the most abundant bird species in China, could be a competent amplifying host for SFTSV and play an important role in its ecology [14]. SFTS could be transmitted not only by means of tick bites, but also via human to human transmission caused by direct contact with blood [15]. Severe fever with thrombocytopenia syndrome (SFTS), as an emerging infectious disease with mortality up to more than 10%, was listed as one of the most severe infectious disease by WHO in 2007.
Virus RNA detection by real-time RT-PCR and antibody detection by enzyme-linked immunosorbent assay (ELISA) are common methods to indentify virus infection. The former is usually used to con rm SFTSV infection. The latter is often used to know recent and past infection status. However, the detection results of SFTSV RNA or antibodies in routine SFTS monitoring were not very clear in wild animals.To ll this gap, we performed SFTSV RNA and antibody detection and analysis on the wild animals collected in Jiangsu Province in 2014-2019. The aim was to understand the surveillance of the virus distribution and the detection results of SFTSV RNA and antibodies, so as to explore appropriate conventional laboratory pathogenic detection strategies to provide a pathogenic and serological basis for better diagnosis of the wild animals.

Data collection
This study was conducted in three cities (Lianyungang, Zhenjiang and Yancheng) and four counties (Yixing, Lishui, Dongtai and Xuyi ), Jiangsu province, Eastern China, the main SFTS epidemic area where human SFTS cases had been reported before (Fig. 1). We randomly selected 10 villages as study site. The wild animals were selected in every study site, including small wild mammals (rodents and hedgehogs) and avian (pheasants).
A total of 1355 serum of wild animals were collected from 2014 to 2019. Blood samples from wild animals were collected directly in serum tubes. The samples were centrifuged at 2560 × g for two minutes and the serum was transferred to small vials, which were kept at − 18 • C until the time of analysis. Rodents, pheasants and hedgehogs were collected with live traps in accordance with standard protocols, as previously described [16][17]. Trapping grids were set up at sites adjacent to case households and in locations chosen to provide geographic diversity. We abided by established safety guidelines for rodent capture and processing. All trapped animals were anesthetized using ketamine, blood was drawn from the retro-orbital sinus. ELISA for SFTSV antibody detection.
Serum samples from patients were tested for SFTSV IgG and IgM antibodies with commercial ELISA kits from DAAN GENE(Zhongshan, China). For initial screening, an 1:40 diluted serum sample was used to determine whether the sample was positive for viral antibodies. Positive serum samples were further diluted in a 2-fold serial dilution starting at 1:80 for the assay to obtain endpoint titers determined by the cutoff values set by positive and negative controls as provided with the ELISA kit.
Statistical analysis SPSS 25.0 (Chicago, IL, USA) was used for all statistical analysis and statistical signi cance level was set at 0.05. Pearson chi-square test was conducted to compare the detection rates of SFTSV RNA and total antibodies among samples. The McNemar test was applied to compare SFTSV RNA and total antibodies detection rates in the paired specimens, and the kappa value was calculated to compare the detection consistency of the two methods.

SFTSV RNA detection
Among the 1355 samples, 16 (1.18%) were positive for SFTSV RNA. There was a signi cant difference in the detection rate of RNA among hedgehogs, rodents and pheasants. The highest positive rate of SFTSV RNA in hedgehogs was 5.75% (5/87). The lowest positive rate of SFTSV RNA in pheasants was 0.71% (Table 1).

Comparison of SFTSV RNA and total antibodies detection
The detection rates of SFTSV total antibodies were 46.7% (7/16) and 3.6% (48/1339) in SFTSV RNApositive and SFTSV RNA-negative samples, respectively (Table 2). A total of 95.8% (1298/1355) of the samples had the same SFTSV RNA and total antibodies detection results. However, there was a signi cant difference between the detection rates of SFTSV RNA and total antibodies, and the detection consistency was very poor.

Discussion
Pathogen detection mostly aims at RNA and antibodies. Real-time RT-PCR is an ordinary method for virus RNA detection, which directly targets the RNA of pathogens. However, ELISA is an indirect method to determine whether there is virus infection by detecting virus antibody (IgG and IgM). Real-time RT-PCR requires higher testing environment and skilled personnel, which has a higher detection sensitivity in the acute phase. Whereas ELISA needs a simpler operating environment and is much simpler to operate by professionals in county level. These two methods are both applied in the early stage after disease onset. Thus, we used these two method to detect pathogen in this study.
Jiangsu Province is a highly endemic area for SFTS. We compared the detection rates of SFTSV RNA and SFTSV-speci c antibodies among wild animals in seven study sites of our province. We found that approximately 4.06% of surveillance wild animals had SFTSV total antibodies and 1.18% had SFTSV RNA. This result showed that the detection rates between SFTSV RNA and total antibodies were different, and the detection consistence were all very poor in hedgehog, rodents and pheasants. In addition, 3.6% of SFTSV RNA negative specimens were positive for SFTSV total antibodies. Therefore, SFTSV RNA negative specimens could not be completely ruled out as SFTS when only relying on RNA detection. It was suggested that the monitoring of wild animals was different from that of human beings [19]. SFTSV antibodies detection was superior to RNA detection for wild animals.
Besides, we found that the detection rate of SFTSV total antibodies was highest in hedgehogs(19.54%).
The positive rate of SFTSV RNA was also highest in hedgehogs 5.75%. It implied that hedgehogs might be the ampli ed hosts of SFTSV in the wild. The results is in accordance with the previous study [20].

Conclusions
Our study suggested that SFTSV antibody detection conducted with ELISA was preferred method for wild animals surveillance. Also, We found that hedgehog could be a competent reservoir for SFTSV and play an important role in its ecology.

Declarations
Availability of data and materials The datasets generated and/or analysed during the current study are not publicly available due to data transcripts including personal participant information not suitable for sharing, but are available from the corresponding author on reasonable request.
Authors' contributions CJB and JLH conceived and designed the study. SYL and ZFL collected the data. SYL, ZFL, NZ, XCW implemented the study. SYL prepared the manuscript. CJB and JLH revised and nalized the manuscript. All of the authors read and approved the nal version of the manuscript. Location of the three cities (blue) and four counties (red ) in Jiangsu Province. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.