Neutralizing antibodies to Severe Fever with Thrombocytopenia Syndrome Virus in general population, Shandong Province, China

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV) in East Asia. The research on seroprevalence of SFTSV in healthy people and risk factors had been detailed. However, the levels of neutralizing antibodies against SFTSV in general population were currently unclear. In the present study, we tested 1375 healthy persons from Penglai County, eastern China, for SFTSV neutralizing antibodies; 0.58% (8/1,375) was positive and the positive rates were not significantly different among people at different age groups, occupations and genders. Besides, a follow-up study was conducted and the titer of neutralizing antibodies decreased over time in all eight people but one, and the neutralizing antibodies of five lasted for the entire study period of seven years. Our results suggesting that subclinical infection or a relatively mild form of SFTS illness is occurring in this population, but a small percentage of sera have neutralizing capacity to SFTSV. Hence, most people are just susceptible to SFTSV infection.

ScIentIfIc REPORTS | (2018) 8:15401 | DOI: 10.1038/s41598-018-33884-z neutralization assay was used to confirm the presence of neutralizing antibodies against SFTSV in serum samples of people of different age groups, occupations and genders.
The positive rate of neutralizing antibody to SFTSV tested by neutralization assay. In our study, 0.58% (8/1,375) of the healthy persons was serum neutralizing antibody positive to SFTSV by neutralizing assay ( Table 1). The neutralizing antibody positive persons were distributed in all age groups, but few in number. After combining two neighbor groups together, no significant difference among the age groups was found (p = 0.997, fisher exact test). Besides, the difference of neutralizing antibody positive rates was not statistically significant between male and female (χ 2 = 0.461, P > 0.05), students and farmers engaged in agriculture activities (χ 2 = 0.875, P > 0.05) ( Table 1).

The titers of neutralizing antibodies in sera.
Neutralization test showed that 15.1% (8/53) of the sera with SFTSV antibodies detected by ELISA had the ability to neutralize virus, of which, the neutralization titers of four serum samples were all 1:10, and the rest were 1:40 (n = 1), 1:80 (n = 2) and 1:640 (n = 1). Subsequently we conducted a follow-up survey to the eight persons according to registered information. We collected their blood samples for 2 times in 2014 and 2017 respectively, and seven of them agreed and one refused to donate blood samples for the second time. At last, 15 specimens were obtained and tested by neutralization assay. Results showed that the titer of neutralizing antibodies decreased over time in all but one person (No. 4), who had a higher neutralizing antibody titer in 2017 than in 2011, and the neutralizing antibodies of five lasted for the entire study period of seven years ( Table 2).

Discussion
In our study, the positive rate of SFTSV antibody was found to be 3.85% in the general population. There were significant differences in positive rate among different age groups, and different occupations indicating that people at older age groups and farmers engaged in agriculture activities were susceptible to SFTSV infection. These results suggested that age and occupation were potential risk factors of SFTSV infection in general population of Penglai County. The positive rate in this study was similar to the reported percentage (3.3%) recorded in  Shandong Province 14 . However, it was much higher than that reported in Jiangsu (0.44%), lower than that in Shanxi (4.7%) 15,16 . The discrepancy may be attributed to the season of collection, population size, geographic climatic factors and different methods. But it was worth noting that ELISA method was utilized in all of these studies. Considering that the effects of non specific reactions caused by ELISA method were not excluded, we used the ELISA method as a screening experiment and the positive samples were confirmed by IFA.
Results showed that 0.58% (8/1,375) of sera were positive for SFTSV neutralizing antibodies, which was far lower than the result of ELISA (3.85%, 53/1375). This imbalance may be due to that a variety of antibodies including neutralizing antibodies were produced in virus infection but the durations of them in the body were different. These results suggested that most sera (84.9%, 45/53) positive for SFTSV had no protective effect and the majority of population in this region was still in the threat of SFTSV infection. As the neutralization assay was recognized as the gold standard for detecting virus antibodies, we believed that the positive rate of 0.58% represented the lowest positive rate of SFTSV infection in the detected population.
The result of neutralizing assay suggested that the natural SFTSV infection did exist in Penglai region but its prevalence was low. Through statistical analysis, we know that the positive rate of neutralizing antibodies is not statistically significant among different ages, occupations and genders. In the present study, eight people with neutralizing antibodies in the sera denied the experience of hospitalization for illness resembling SFTS in the past five years, indicating that SFTSV cause subclinical infections or a relatively mild form of SFTS illness in human. Consequently, the number of actual infections was more than the number of hospitalized patients but the proportion between them still needed further study.
Recent studies have shown that 100% of SFTS confirmed cases produce neutralizing antibodies to SFTSV and can last for four years with a decrease in titers 17 . We also performed a follow-up study of eight people with neutralizing antibodies in 2014 and 2017. By comparing the results of neutralizing antibodies in three surveys (2011, 2014 and 2017), we found that the titers of neutralizing antibodies in general population were low except for No. 7 (640) and none of them showed obvious clinical symptoms. That probably because mild strain of SFTSV caused low immune responses in the body or the neutralizing antibodies decreased fast over time. In additional, the neutralizing antibodies of five people lasted for seven years, while the rest of others vanished within four years. That suggested the titer of neutralizing antibody was positively correlated with duration of protection. As to be expected, the titer of neutralizing antibody decreased over time in all but No. 4; this increase may have caused by SFTSV reinfection. However, it was reported that the seroprevalence rate of SFTSV in healthy population was <1% and the chance of reinfection of a person with SFTSV was low 12 . We can not exclude that No. 4 people could have been infected with other similar phleboviruses. Few data about neutralizing antibody in healthy population are available for comparison. Consequently, more studies are needed.
In summary, our findings suggest that subclinical SFTSV infections or a relatively mild form of SFTS illness affects humans in Penglai County. We confirm that a large percentage of healthy people did not have the ability to neutralize the virus and they may be infected by SFTSV. A few of people with mild SFTS illness could produce long-lasting neutralizing antibodies to SFTSV and its titers decreased over time. We do not know the characteristics of the neutralizing antibodies against SFTSV, which need to be further investigated.

Ethics approval. The study was reviewed and approved by the Ethic Committee of China Center for Disease
Control and Prevention(China CDC). All study objects had signed an informed consent document prior to participation. A signed informed consent was required from their parents or legal guardian for minors. All data analyzed were anonymized. Human research was conducted in compliance with the Helsinki Declaration. Study site. Penglai County located in east longitude 120°34′ and 121°04′ and between north latitude 37°26′and 37°48′. It had a total area of 1, 128 square kilometers and a total population of ≈449,000, of whom 80% live in rural areas (Fig. 1).

Serological investigation.
Among the towns that had reported SFTS cases, we randomly selected 40 villages as target villages to collect serum specimens. In these selected villages, people were divided into 8 age groups  1  F  40  farmers  10  800  0  400  0  200   2  F  10  students  10  400  0  100  NT  NT   3  F  35  farmers  10  800  0  200  0  100   4  M  51  farmers  10  1600  40  3200  40  1600   5  M  22  farmers  80  3200  40  800  40  800   6  F  42  farmers  80  3200  40  1600  20  800   7  F  59  farmers  640  6400  320  6400  160  3200   8  F  38  farmers  40  1600  40  1600 20 400  Serological test. Sera were tested for antibodies to SFTSV by using a double-antigen sandwich ELISA kit (Xinlianxin Biomedical Technology Limited, Wuxi, China). Recombinant nucleoprotein (NP) of SFTSV was used as antigen for coating plates. The experiment included negative control, positive control and blank control. To each well 100 µl of a sample was added except for control wells, and then incubated at 37 °C for 30 minutes. After washing five times, each well was added 100 µl horse radish peroxidase (HRP) labeled reagent, then the plate was incubated at 37 °C for 30 minutes. After washing five times, a chromogenic agent A and B solution were added to each well to develop the color and the plate was read at 450 nm for optical density (OD). A serum sample was considered to contain SFTSV specific antibody when absorbance of the sample ≥ threshold value (cutoff). The threshold value = 0.10 + the average OD value of the negative control (if the OD value of a negative control was less than 0.04, it was considered as 0.04). In our study, undiluted samples were utilized to determine whether the samples were positive for antibodies against SFTSV. The ELISA results were confirmed by IFA. After the samples were diluted 1:10, 1:20, 1:40 and1:80 in phosphate-buffered saline (PBS)-Tween buffer, the IFA was performed and immunofluorescence was observed using an epifluorescence microscope. Goat anti-human IgG Fc-FITC (abcam) was used as second antibody. Positive and negative controls were also used. In our study, a titre of 1:40 was considered to be the most appropriate dilution.
Neutralization Assay. A microneutralization assay was performed to detect neutralizing antibodies against SFTSV as described previously 8 . Briefly, samples were diluted in 2-fold increments from 1:5 to 1:640. Each dilution of serum samples was mixed with an equal volume of 100 median tissue-culture infectious doses of SFTSV (strain HB29) and incubated at 37 °C for 1.5 hours. The mixture was then added to a 96-well plate containing Vero cells in quadruplicate. The plates were incubated at 37 °C in a 5% carbon dioxide atmosphere for 7 days. Viral infection was detected via immunofluorescence assays with mouse polyclonal antibodies against SFTSV (AbMax) and rabbit anti-mouse IgG-FITC (abcam). The end-point titer was expressed as the reciprocal of the highest dilution of serum that prevented infection.

Follow-up survey.
According to the result of neutralization assay, we conducted a follow-up study of people with neutralizing antibodies against SFTSV. Blood samples were obtained two times during a seven-year period and were tested to determine the titers of neutralizing antibodies. The ELISA and micro-neutralization assay of samples from follow-up surveys were performed in WeiFang Medical University (Weifang), in 2017.
Statistical analysis. Statistical analysis was performed using SPSS 21.0 software and P < 0.05 was considered as statistically significant difference. Chis-quare or Fisher's exact test were used for categorical variables, where appropriate.

Data Availability Statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article.