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

doi:10.21203/rs.3.rs-520795/v1

PDF

Research Article

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

https://doi.org/10.21203/rs.3.rs-520795/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted 24 May, 2021

You are reading this latest preprint version

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 fulfilled 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 significant 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.

Infectious Diseases

Severe fever with thrombocytopenia syndrome virus(SFTSV)

wild animals

Detection

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, confirmed SFTS cases have been reported in Japan and South Korea in recent years[1–7]. 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[10–13]. 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 confirm 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 fill 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.

SFTSV-RNA extraction and real-time RT-PCR.

Total RNA prepared from the serum samples from patients were extracted using an RNeasy kit (Qiagen, Germany) according to the manufacturer’s instructions. Real-time RT-PCR was performed using the QuantiTech RT-PCR kit (Qiagen, Germany). The primers were designed as previously described and used in a one-step real-time RT-PCR. The primers and MGB probe used in the real-time RT-PCR were targeted to the S segment of the viral genome [18]. Conditions for the reaction were as follows: 50°C for 30 min, 95°C for 15 min, 40 cycles at 95°C for 15 sec, and 55°C for 40 sec. Amplification and detection were performed with an Applied Biosystems 7500 Real-time PCR system (Applied Biosystems, Foster City, CA). Data were analyzed using the software supplied by the manufacturer.

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 significance 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 significant 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).

Table 1

SFTSV RNA, total antibodies detection results
Wild Animals	Total detection no.	SFTSV RNA-positive samples no. / total detection no.(%)	SFTSV total antibodies-positive samples no. / total detection no.(%)
Hedgehogs	87	5/87 (5.75)	17/87 (19.54)
Rodents	1128	10/1128 (0.89)	29/1128 (2.57)
Pheasants	140	1/140 (0.71)	9/140 (6.43)
Total	1355	16/1355 (1.18)	55/1355 (4.06)

SFTSV total antibody detection

Of the 1355 samples, all the sera samples were chosen to conduct SFTSV total antibody (IgM and IgG) detection. The detection rate of SFTSV total antibodies was 4.06% (55/1355) in all samples. The highest total antibodies detection rate was found in hedgehogs 19.54%(17/87). There was a significant difference in the detection rate of total antibodies among three species of wild animals (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 RNA-positive 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 significant difference between the detection rates of SFTSV RNA and total antibodies, and the detection consistency was very poor.

Table 2

Comparison of SFTSV RNA and total antibodies detection
Animals	SFTSV RNA	SFTSV total antibodies		P value	Kappa
		Positive N	Negative N	(McNemar test)	value
Hedgehogs	Positive	4	1	0.002	0.302
	Negative	13	69
Rodents	Positive	3	7	0.001	0.143
	Negative	26	1092
Pheasants	Positive	0	1	0.021	-0.013
	Negative	9	130
Total	Positive	7	9	0.000	0.182
	Negative	48	1291

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-specific 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 amplified hosts of SFTSV in the wild. The results is in accordance with the previous study[20].

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.

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 finalized the manuscript. All of the authors read and approved the final version of the manuscript.

Acknowledgements

We would like to thank all study participants who were involved and contributed to the data collection. We thank editors and reviewers for their warm works and valuable comments.

Ethics statement

Small wild mammals and avian were captured and handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China (Regulations for Administration of Affairs Concerning Experimental Animals, China, 1988). Handling and sampling blood collection including blood collection were approved by the Animal Ethics Committee, Jiangsu Provincial Center for Disease Control and Prevention, with the certificates No. SCXK[su] 2012–0021 and No. JSCDCLL[2012]039. The certificates also provided the permission on the protocol for sampling wild birds including Anser cygnoides.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

Not applicable.

Yu XJ, Liang MF, Zhang SY, et al, Fever with thrombocytopenia associated with a novel bunyavirus in China[J]. N Engl J Med, 2011,364(11):1523-32.
Li DX, 2013: A highly pathogenic new bunyavirus emerged in China[J]. Emerg Microb Infect, 2013,2(1):el.
Liu S, Chai C, Wang C, et al, Systematic review of severe fever with thrombocytopenia syndrome: virology, epidemiology, and clinical characteristics[J]. Rev Med Virol, 2014,24(2):90-102.
Li DX,Severe fever with thrombocytopenia syndrome: a newly discovered emerging infectious disease[J]. Clin Microbiol Infect,2015,21(7):614-620.
McMullan LK, Folk SM, Kelly AJ, et al. A new phlebovirus accociated with Severe febrile illness in Missouri[J]. N Engl J Med,2012,367(9):834-841.
Takahashi T, Maeda K,Suzuki T, et al. The first identification and restrospective study of severe fever with thrombocytopenia syndrome in Japan[J]. J Infect Dis, 2014,209(6):816-827.
Kim KH,Yi J, Kim G, et al. Severe fever with thrombocytopenia syndrome,South Korea, 2012[J]. Emerg Infec Dis, 2013, 19(11): 1892-1894.
Ding F, Zhang W, Wang L, Hu W, Soares Magalhaes RJ, et al. (2013) Epidemiologic features of severe fever with thrombocytopenia syndrome in China, 2011–2012. Clin Infect Dis 56: 1682–1683.
Liu Q, He B, Huang SY, Wei F, Zhu XQ (2014) Severe fever with thrombocytopenia syndrome, anemerging tick-borne zoonosis. Lancet Infect Dis 14: 763–772.
Wang S, Li J, Niu G, Wang X, Ding S, et al. (2015) SFTS virus in ticks in an endemic area of China. Am J Trop Med Hyg 92: 684–689.
Yun SM, Lee WG, Ryou J, Yang SC, Park SW, et al. (2014) Severe fever with thrombocytopenia syndrome virus in ticks collected from humans, South Korea, 2013. Emerg Infect Dis, 20: 1358–1361.
Ding S, Yin H, Xu X, Liu G, Jiang S, et al. (2014) A cross-sectional survey of severe fever with thrombocytopenia syndrome virus infection of domestic animals in Laizhou City, Shandong Province, China. Jpn J Infect Dis 67: 1–4.
Park SW, Song BG, Shin EH, Yun SM, Han MG, et al. (2014) Prevalence of severe fever with thrombocytopenia syndrome virus in Haemaphysalis longicornis ticks in South Korea. Ticks Tick Borne Dis 5:975–977.
Li Z#, Bao C#, Hu J, Liu W, Wang X, Zhang L, Ji Z, Feng Z, Li L, Shen A, Liu X, Zhao H, Tan W, Zhou J, Qi X, Zhu Y, Tang F, Cardona CJ, Xing Z*.Ecology of the Tick-Borne Phlebovirus Causing Severe Fever with Thrombocytopenia Syndrome in an Endemic Area of China.PLoS Negl Trop Dis. 2016,10(4):e0004574.
Bao CJ, Qi X, Wang H, 2011: A novel bunyavirus in China[J]. N Engl J Med 365:862-3.
Torres-Pérez F, Navarrete-Droguett J, Aldunate R, Yates TL, Mertz GJ, VialPA, et al. Peridomestic small mammals associated with confirmed cases ofhuman hantavirus disease on southcentral Chile. Am J Trop Med Hyg 2004;70:305–9.
Mills JN, Yates TL, Childs JE, Parmenter RR, Ksiazek TG, Rollin PE, et al. Guidelines for working with rodents potentially infected with hantavirus. J Mammal 1995;76:716–22.
Li Z, Cui L, Zhou M, Qi X, Bao C, et al. (2013) Development and application of a one-step real-time RT-PCR using a minor-groove-binding probe for the detection of a novel bunyavirus in clinical specimens. J Med Virol 85: 370–377.
Huang XX, Ding SJ, Jiang XL, et al. Detection of SFTS virus RNA and antibodies in severe fever with thrombocytopenia syndrome surveillance cases in endemic areas of China[J]. BMC Infect Dis, 2019,19(476).
Sun Y, Liu MM, Luo LM, et al. Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Virus in Hedgehog from China[J]. Vector Borne Zoonotic Dis, 2017,17(5): 347-350.

PDF

Version 1

posted 24 May, 2021

You are reading this latest preprint version

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

Status:

Version 1

Abstract

Figures

Background

Methods

Results

SFTSV RNA detection

SFTSV total antibody detection

Comparison of SFTSV RNA and total antibodies detection

Discussion

Conclusions

Declarations

References

Status:

Version 1