Based on the analysis of 9676 samples, this study reveals the epidemiological trends of EV infections in children in Hangzhou and identifies the predominant serotypes. Furthermore, it confirms that enteroviruses are one of the common etiological agents of viral encephalitis in children. EV related diseases are one of the more common infectious diseases in humans, which can cause a variety of acute infectious diseases and often lead to sudden public health events¹⁴. From 2008 to 2012, most of the HFMD cases in China were caused by EV-A71 or CV-A16 infection, and more than 90% of the death cases were caused by the co-infection of both¹⁵. From 2008 to 2015, about 570,000 confirmed cases of HFMD were reported in China, and the positive rates of EV-A71, CV-A16, and other EVs were 44%, 25%, and 31%, respectively. EV-A71 infection was the primary infection in severe cases and deaths¹⁶.

The monovalent EV-A71 vaccine was approved in China in 2016, following which the number of severe cases and deaths of HFMD caused by EV-A71 showed a decreasing trend. The test results of the samples in this study showed that the detection rate of EV-A71 and CV-A16 decreased year by year from 2018 to 2023, and the detection rate of the above two types of enterovirus reached 0% in the past two years. However, due to the absence of cross-protection between EV serotypes, the monovalent EV-A71 vaccine does not protect against infection from other EV serotypes, as evidenced by the similar number of EV infection cases after the monovalent EV-A71 vaccine was launched. At the end of 2019, due to the normalization of prevention and control measures after the outbreak of novel coronavirus and the enhancement of public awareness of infectious disease prevention, the number of EV nucleic acid cases decreased significantly. Although the number of samples has decreased compared with 2018 and 2019, the detection rate of EV has not decreased but has shown an increasing trend. In terms of virus type, CV-A6 and other EV types have surpassed EV-A71 and CV-A16 to become the main pathogens of EV-related infectious diseases, which is consistent with the epidemiological survey data reported in other regions of China^{17,18,19,20,21,22}.

Fever symptoms are almost always present in the acute stage of EV infection. Comprehensive analysis is conducted based on the presence of other accompanying symptoms, such as rashes (considering the time, place, shape, etc.), respiratory system, central nervous system, or circulatory system-related symptoms. However, considering the large number of EV serotypes, different serotypes of EVs can cause a variety of clinical symptoms. Hence, making a correct diagnosis based solely on clinical manifestations is challenging²³. The results of this study found 2863 cases of hand-foot-mouth disease, 1520 cases of viral encephalitis, 1176 cases of herpetic angina, 174 cases of upper respiratory tract infection, 60 cases of lower respiratory tract infection, and 45 cases of digestive tract infection among 5838 EV-positive children. The results revealed that EV nucleic acid detection of different specimens in children with suspected EV infection, especially in children with viral encephalitis, can improve the detection rate of EV-related disease pathogens. Due to the large number of EV serotypes, further analyses are required to determine the EV serotypes.

Considering the lack of effective antiviral drugs for EV infection, vaccines remain the most economical and effective measure for the prevention and control of infection. In 2016, the monovalent inactivated EV-A71 vaccine developed by three domestic manufacturers was approved in China, showing protection rates against HFMD caused by EV-A71 of over 90% for all three monovalent EV-A71 inactivated vaccines^24,25. The vaccines effectively reduced the number of deaths of HFMD caused by EV-A71. The EV-A71 vaccination rate among children in Zhejiang Province was 24.1%, playing a critical role in preventing severe HFMD²⁶. With the increase in EV-A71 vaccination, the cases of EV-A71 infection decreased significantly, but the cases of other EV infections demonstrated no significant decrease but showed an increasing trend. Due to the continuous changes in the pathogen spectrum of EV infection, developing multivalent vaccines is particularly important²⁷.

In conclusion, based on the findings of this study, we have observed that pediatric enterovirus infections can manifest with a diverse array of clinical presentations. Relying solely on clinical symptoms for diagnosis can easily lead to misdiagnosis, which in turn may adversely affect treatment outcomes. Furthermore, there are no significant distinguishing features in the clinical manifestations, cerebrospinal fluid cytology, or biochemical markers among cases of viral encephalitis caused by different viruses. Given that the severity of encephalitis can vary depending on the causative pathogen, performing nucleic acid testing on multiple specimens, particularly EV nucleic acid testing and virus typing, is of paramount importance for children with suspected encephalitis.

Nevertheless, the limitations of this study should be acknowledged. First, only limited EV serotypes such as CV-A16, CV-A10, CV-A6, and EV-A71 were tested. Many EVs in the test results were unclassified, and accurate etiological diagnosis of EV-related diseases could not be made. In response to the above problems, more effective detection methods should be developed, such as the application of metagenomic sequencing technology for pathogenic nucleic acid detection, which can effectively identify unknown viruses. The different types of CV and related imaging findings can be further explored.