Co-circulation of coxsackieviruses A-6, A-10, and A-16 causes hand, foot, and mouth disease in Guangzhou city, China

Hand, foot, and mouth disease (HFMD), a common contagious disorder in children under 5 years of age, is characterised by a brief, generally mild, febrile illness with multiple oral ulcers, and eruption of vesiculo-papular rashes over the hands, feet, mouth, and buttocks [1‐3]. Some cases can develop severe neurological and systemic complications, such as aseptic meningitis, acute flaccid paralysis, encephalitis, cardiorespiratory failure, and severe pulmonary oedema, and can even lead to death [4, 5]. HFMD is mainly caused by human enteroviruses (EVs) of the family Picornaviridae. Over 100 recognised EV serotypes exist, classified into four groups, namely, EV-A to EV-D [6]. Their genome comprises one open reading frame, which encodes four structural viral proteins (VP1, VP2, VP3, and VP4) and seven non-structural proteins (2A-2C and 3A-3D) [7]. VP1, the main region encoding neutralising epitopes, contains virulence determinants and also provides conclusive evidence regarding the phylogeny and genotype of these viruses [8].

Since the first HFMD case was described in 1959 [9], outbreaks or sporadic cases have frequently occurred globally [10‐13]. In the recent decades, more and more outbreaks of HFMD had reported from Asia-Pacific region, including Vietnam, Malaysia, Thailand, Japan, India, and China [10, 13‐17]. In China, HFMD was recognised as a C-class notifiable disease after an outbreak occurred in Fuyang in early 2008 [17, 18]. According to the Chinese national enhanced surveillance system, HFMD rates ranked first among all notifiable diseases and over 1.2/1000 in children are affected annually [3]. Enterovirus A71 (EV-A71) and coxsackievirus A16 (CVA-16), belonging to EV-A, have been identified as the predominant causative pathogens in the nationwide surveillance. However, other serotypes had gradually played an important role in the HFMD outbreaks. Since a Coxsackievirus A6 (CVA-6) associated HFMD outbroke in Finland in 2008, several outbreaks caused by CVA-6 were reported in Europe, North America, and Asia [12, 19‐22]. Moreover, Coxsackievirus A10 (CVA-10) had also been identified as the responsible pathogen for a series of HFMD outbreaks [20, 23, 24]. Guangzhou city is one of the most serious cities in HFMD with high incidence, and the HFMD pathogen spectrum are changing according to previous studies [22, 25‐27]. Therefore, the change of HFMD pathogen spectrum has become a big challenge for prevention and control of HFMD.

In this study, the clinical and epidemiological characteristics of HFMD were analysed in Guangzhou, 2018, and the evolutionary dynamics were also explicated based on entire VP1 sequences.

Currently, EV-A71 and CVA-16 are still the main known causative agents of HFMD. However, CVA-6 and CVA-10 were frequently detected in HFMD outbreaks in recent years [19, 32, 35], which implied that the epidemiological features of HFMD are changing. In our study, we reported for the first time a new HFMD epidemic pattern in Guangzhou in 2018 characterised by co-circulation of CVA-6, CVA-10, and CVA-16. Additionally, seasonally circulating diversity in serotypes was observed in this study, which provided a scientific basis for prevention and control of HFMD in the future.

In this study, our results showed that the co-circulation of three main EV serotypes (CVA-6, CVA-10, and CVA-16) caused the HFMD outbreak in Guangzhou, which was clearly different with the epidemic characteristics described before. In previous study, EV-A71 and CVA-16 had been reported to the predominant pathogens of HFMD in Guangzhou between 2008 and 2010 [26, 36]. However, the number of CVA-6 has been increased dramatically since 2010, and CVA-6 has become the major pathogen in 2017 [22, 25]. In 2018, CVA-6, CVA-10, and CVA-16 had been co-circulating in the HFMD outbreak in Guangzhou. Based on the limited co-circulating information, we suspected it was associated with genetic variation [19, 37], and further studies were needed to verified it. According to previous studies, EV-A71 was frequently detected in China and considered to be closely associated with fatal case due to HFMD outbreak worldwide [4, 36, 38, 39]. Our results indicated that 26 EV-A71-positive HFMD cases were identified, and the ratio of HFMD severe was significantly higher in the group of EV-A71. The relatively lower viral diversity of EV-A71 and successful application of EV-A71 vaccine may be responsible for the reduced incidence of EV-A71 [40‐42]. Moreover, we still need to keep our eyes on the HFMD caused by EV-A71 even the number of it was low. Up to date, only CVA-16 and EV-A71 were identified as the main pathogens in the Chinese nationwide surveillance of HFMD. The molecular epidemiological data of this study demonstrated that multi-serotypes circulated concomitantly, which might become a HFMD epidemic trend in the future. Notably, several polioviruses (PV) were detected in this study. PV type 1–3, belonging to EV-C, is notable as the pathogen of poliomyelitis (polio) [17, 43]. We observed that all the patients associated with PV presented typical clinical manifestation of HFMD, such as skin rashes on hands and feet, and the age with serotypes of the affected associated with the PV inoculation [44, 45]. However, the vaccination records of eight patients were unclear and further studies are needed. In addition, our data indicated that apart from CVA-6, CVA-10, and CVA-16, a total of 18 enterovirus serotypes were associated with HFMD cases, which had been described in previous studies [32, 46]. However, most of these serotypes have been rarely detected in clinical laboratories. Therefore, the development of a detection kit which is capable of detecting more enterovirus serotypes is crucial for clinical diagnosis of HFMD. Further, it is vital for researchers and the Centre for Disease Control and Prevention to pay attention to other EVs [47, 48]. This is especially important as only the commercial EV-A71 vaccine, which lacks cross immunity protection for patients infected with other EVs, has been successfully applied to children [47]. Our result implicated that the development of multi-serotype vaccines will be helpful for decreasing the morbidity associated with HFMD.

A seasonal pattern of HFMD was observed in this study. There were two peaks when the majority of cases were admitted to the hospital, and one major peak in May and a small peak in October. This phenomenon was consistent with some earlier studies reported in Guangzhou and other southern regions, including Thailand and Taiwan [26, 38, 49‐51]. The exact reason was still unclear. But climate and geographical location were thought to be involved in the seasonal change of HFMD to a certain extent. High temperature and high humidity had increased the incidence of HFMD, and it had also been found that atmospheric pressure and wind speed were associated with HFMD occurrence [51]. Additionally, different serotypes of EV were distinct in their monthly distribution. Significantly, CVA-6 was the predominant pathogen of the small HFMD peak in autumn, and CVA-10 and CVA-16 were prevalent in the beginning of summer (the peak in May), which was similar to that observed in Shanghai and southern Vietnam to a certain extent [10, 52]. The discrepancy in the monthly distribution of CVA-6 and CVA-10 in different regions may be attributed to the adaptive changes in the pathogens. Hence, it is crucial to develop reasonable preventive measures to combat the HFMD outbreak caused by various serotypes in different seasons.

In this study, EVs have an obvious predilection in patients. The majority of patients were under 5 years of age in this study, which is possibly associated with the incomplete immunity of children [3]. The incidence rate in boys with HFMD was higher than that of girls, with 1.4:1 sex ratio, which was similar to the results of previous studies [10, 49, 52]. We suspected that poor hygiene, more outdoor activities, and host factors may be related to that phenomenon [22]. Moreover, our results did not show any age or gender predilection in different serotypes. Compared to the group of CVA-10 and CVA-16, patients infected with CVA-6 were likely to have the rising hsCRP. Moreover, the group of CVA-10 had an obvious increase in WBC, which was caused by the rising of neutrophils. However, further studies regarding this are required as the existing information is limited.

Based on the entire VP1 sequence, low intra-typic divergence was observed among CVA-6, CVA-10, and CVA-16 detected from Guangzhou. The first reported Chinese CVA-6 strain was grouped to genogroup B. Subsequently, the sub-genogroup E2, which replaced genogroups B, C, D, and E1, has gradually become the predominant strain circulating worldwide since 2008 [12]. The CVA-6 Guangzhou were further segregated into two groups within sub-genotype E2. One cluster was close to the subtypes detected in Yunnan, Guangdong, and Guangxi, while the other was closely related to strains from Hong Kong and Fujian. Both CVA-6 clusters were similar to the strains in adjacent provinces of Guangzhou, which indicated that the subtypes of CVA-6 identified in Guangzhou had a common ancestor in nearby regions and are prevalent in the south of China. Of the CVA-10, Guangzhou sequences were identified to genogroup E, which was prevalent not only in Guangzhou, but also in Xiamen, Yunnan, and Jiangxi. The evolutionary characteristics of sub-genotype E are still not known and further studies are required to elucidate the increase in CVA-10 prevalence in Guangzhou during 2018. The CVA-16 phylogenetic dendrogram showed that genogroup B, consisting of the majority of CVA-16, was classified into B1a, B1b, and B1c. Furthermore, sub-genogroups B1a and B1b associated with outbreaks of HFMD in Nanjing, Fujian, and Yantai, were recognised as the predominant subtypes circulating around the mainland [39, 53‐55], which was consistent with the results of the present study in Guangzhou. The CVA-16 phylogenetic dendrogram indicated that sub-genogroup B1b, including 19 Guangzhou CVA-16, was dominant in our study, similar to the results of previous reports on other regions [53, 55]. The Guangzhou CVA-16 located in the B1b group showed high similarity to the strain isolated in Shenzhen, a city adjacent to the Guangdong province. Additionally, four CVA-16 sequences belonging to the B1a sub-genogroup were closely related to strains circulating in Shenzhen and Yunnan. Therefore, we hypothesised that compared to B1a, the sub-genogroup B1b shares a common ancestor with strains circulating in Shenzhen, which might become endemic strains of CVA-16 in the future. Intriguingly, one sequence was located in the sub-genogroup B1c, which has been reported only in Malaysia, France, and India [16, 54]. Hence, we believed that one located in B1c was obtained from other countries via import transmission [55]. Comprehensive and long-term surveillance of HFMD is critical for understanding the trend of the epidemic and emergence of new genotypes, and for controlling EV infection in the future.

Our study has several limitations. For example, this study was not a multi-center and multi-year research, and some laboratory diagnosis lacked in this study, which may cause a bias to the different serotypes in laboratory data.

We found that a total of 21 EV serotypes were identified in 1220 HFMD patients, and CVA-6, CVA-10, and CVA-16 were the most commonly detected during 2018. Co-circulation of three serotypes, CVA-6, CVA-10, and CVA-16, was the dominant reason for the outbreak of HFMD in Guangzhou in 2018. In this study, we presented a comprehensive and detailed investigation regarding the outbreak of HFMD in Guangzhou based on clinical manifestations and phylogenetic analysis, which we believe will be beneficial for understanding and controlling the disease in the near future.