www.nature.com/scientificreports OPEN Pathogenic characteristics of hand, foot and mouth disease in Shaanxi Province, China, 2010–2016 Yi Xu1,2, Yuan Zheng2, Wei Shi2, Luyuan Guan2, Pengbo Yu2, Jing Xu2, Lei Zhang2, Ping Ma2 & Jiru Xu1* Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by enteroviruses. We analyzed the pathogenic characteristics of HFMD in Shaanxi province, China, during 2010–2016. Clinical samples were collected from HFMD cases. Real-time PCR and RT-PCR were used to identify the enterovirus(EVs) serotypes. Viral RNA sequences were amplifed using RT-PCR and compared by phylogenetic analysis. Descriptive epidemiological methods were used to analyze. A total of 16,832 HFMD positive cases were confrmed in the laboratory. EV-A71 and CV-A16 were the main pathogens in 2010. EV-A71 was the dominant pathogen in the periods of 2011 to 2012 and 2014, 2016. In 2013 and 2015, other EVs increased greatly, in which CV-A6 was the predominant pathogen. EV-A71 was more frequently detected in deaths and severe cases. Phylogenetic analysis revealed that EV-A71 belonged to the C4a evolution branch of C4 sub-genotype and CV-A16 belonged to the B1a or B1b evolution branch of B1 sub-genotype, whereas CV-A6 strains were assigned to D2 or D3 sub-genotype. The pathogen spectrum of HFMD has changed in 7 years, and the major serotypes EV-A71, CV- A16 and CV- A6 alternated or co-circulated. Long-term surveillance and research of EVs should be strengthened for the prevention and control of HFMD. Hand foot and mouth disease (HFMD) is a common infectious disease in children caused by human enterovi- rus (EV)1–3. Te main clinical manifestations of HFMD are fever, rash on the palms, soles, mouth and buttocks. Most HFMD patients have a good prognosis. A few patients may have complications such as aseptic meningitis, encephalitis, acute faccid paralysis, neurogenic pulmonary edema and myocarditis. Individual severe children may die due to exacerbations3–5. Since the outbreak of HFMD in Anhui Province in March 20086, there has been a widespread epidemic in mainland of China7–9. It has become one of the main public health problems which seriously endanger the health of infants and young children. EVs belong to the family of Picornaviridae, enterovirus genus10. A variety of EVs can cause HFMD. According to its gene and antigen characteristics, EVs can be divided into four species: EV-A, EV-B, EV-C, and EV-D. Human enterovirus 71 (EV-A71) and Coxsackievirus A16 (CV-A16) in EV-A species are generally considered to be the common and major pathogens causing HFMD11–13. In recent years, however, more and more studies have shown that other EVs also play an important role in the outbreak or epidemic of HFMD14-18. In particular, CV-A6 has replaced EV-A71 and CV-A16 as the main pathogen for the outbreak or epidemic of HFMD in mainland of China since 201219–21. Te change of pathogen spectrum put forward a new challenge to the prevention and control of HFMD. In this study, we analyzed the pathogenic characteristics of HFMD in Shaanxi province during 2010–2016, and clarifed the epidemiological characteristics and pathogen spectrum changes of HFMD. It could provide scientifc basis for prevention and control of HFMD in Shaanxi Province. Results Pathogenic surveillance in shaanxi province during 2010–2016. A total of 392,400 HFMD cases were reported in Shaanxi Province during 2010–2016, including 5469 severe cases and 137 deaths. Te average annual incidence rate was 148.5319 per 100,000. Te average annual mortality rate and fatality rate were 0.04806 per 100,000 and 2.32%, respectively. A total of 16,832 HFMD positive cases were confrmed in the laboratory, of 1Department of Microbiology and Immunology, School of Medicine, Xi’an Jiaotong University, Xi’an, China. 2Department of Viral Disease Control and Prevention, Shaanxi Center for Disease Control and Prevention, Xi’an, China. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:989 | https://doi.org/10.1038/s41598-020-57807-z 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Pathogen proportion of HFMD confrmed cases in Shaanxi Province, China, 2010–2016. Te histogram showed the proportion of EVA71, CVA16 and other EVs detected in the confrmed HFMD cases. Te proportion of pathogens detected in 7 years has diference. Figure 2. Monthly distribution of pathogen proportion of HFMD confrmed cases in Shaanxi Province, China, 2010–2016. Te histogram showed the distribution of EVA71, CVA16 and other EVs detected in each month, the number of confrmed HFMD cases appeared seasonal distribution with the peaks in April to July. which 7042 (44.84%) were positive for EV-A71 infection, 3473 (20.63%) were positive for CV-A16 infection and 6317 (37.53%) were other EVs. Te proportion of pathogens detected in 7 years was diferenced (Fig. 1). In 2010, EV-A71 and CV-A16 were the main pathogens causing HFMD, and the positive rates of EV-A71 and CV-A16 were 44.62% and 37.01%, respectively. In 2011 and 2012, EV-A71 was the dominant pathogen which the positive rates were 64.42% and 57.17%, respectively. In 2013, the pathogen spectrum of HFMD changed greatly, and the proportion of other EVs increased signifcantly as high as 61.17%. In 2014, the dominant position of EV-A71 was recovered to 50.91%. In 2015, HFMD was still mainly caused by other EVs, accounting for 57.38%, and in 2016, EV-A71 became the dominant pathogen again, accounting for 46.68%. During 2010–2016, HFMD cases were reported monthly in Shaanxi Province, China, the incidence peak appeared from April to July. Te peak of positive pathogen was also detected in April to July, accounting for 69.05% of the total number of confrmed samples. Monthly distribution of the pathogens was illustrated in Fig. 2, the epidemic pathogen in diferent months varied greatly in 7 years. From April to May and September 2010, CV-A16 was the predominant pathogen, and the rest of 2010 and the whole year of 2011 and 2012, the main epidemic pathogen was EV-A71. From January 2013 to January 2014, other EVs became the dominant pathogen. In February and May to August 2014, the epidemic pathogen was still predominant in EV-A71, but in the peri- ods of March to April and September to October, EV-A71 and CV-A16 were the co-epidemic pathogens. From November 2014 to March 2016, other EVs became the dominant pathogen again, and EV-A71 returned to be the main epidemic pathogen in the rest of 2016. From 2010 to 2016, most of the cases of HFMD in Shaanxi Province were reported by Xi’an City, Xianyang City and Weinan City. Te number of cases in the three cities accounted for 40.95%, 16.88% and 12.02% of the total cases, respectively. Te proportion of pathogens detected in 11 cities was diferenced (Fig. 3). EV-A71 was SCIENTIFIC REPORTS | (2020) 10:989 | https://doi.org/10.1038/s41598-020-57807-z 2 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 3. Geographic distribution of pathogen proportion of HFMD confrmed cases in Shaanxi Province, China, 2010–2016. Te map shows the proportion of EVA71, CVA16 and other EVs detected in the confrmed HFMD cases in each city. Te proportion of pathogens detected in 11 cities has diference. the main pathogen causing HFMD in Xi’an City, Weinan City, Yulin City and Yangling District. In Xianyang City, Hanzhong City, Ankang City, Tongchuan City and Yan’an City, the dominant pathogen of HFMD was other EVs, while in Baoji City and Shangluo City, EV-A71 and other EVs were the co-epidemic pathogens. Among the HFMD positive cases confrmed in laboratory during 2010–2016, there were 10244 males and 6588 females, with an average annual male to female ratio of 1.55:1. Te proportion of pathogens in the gen- der was shown in Table 1, there was no signifcant diference in the pathogen proportion of diferent gender (χ2 = 0.133, P > 0.05). HFMD positive cases mainly concentrated in children aged 1 to 3 years old, accounting for 73.12%, of which the proportion of 1- year-old group was the highest (31.99%). Tere was signifcant diference in the proportion of pathogens in diferent age groups(χ2 = 231.765, P <0.001). Of all laboratory-confrmed cases during 2010–2016, there were 13,882 mild cases, 2886 severe cases and 64 deaths. Te positive rates of EV-A71, CV-A16 and other EVs in mild cases were 36.62%, 23.71% and 39.67%, respectively. In severe cases, the positive rate of EV-A71 was 65.70%, while CV-A16 and other EVs were 6.27% and 28.03% respectively. Furthermore, the positive rate of EV-A71 reached 98.43% in all the deaths except 1 cases of other EVs infection (Fig. 4). Tere was signifcant diference in the proportion of pathogens in diferent case types. EV-A71 was more frequently detected in deaths and severe cases than in mild cases, while CV-A16 was more frequently detected in mild cases than in severe cases, no CV-A16 was detected in deaths. Serotypes of other EVs. In this study, a total of 709 other EVs were identifed during 2010–2016, includ- ing 19 serotypes, belonging to species A and B of EVs. Among the other EVs, the most frequently detected was CV-A6, accounting for 41.47%, followed by CV-A10 (26.66%), CV-A2 (2.54%), CV-A4 (1.55%), Echo6 (1.16%) and CV-B1 (1.13%), respectively.