Discussion
From 2008-2012, EV-A71 was the major pathogen causing HFMD and was responsible for most severe and fatal cases in mainland China (1). Our previous studies have shown that locally circulating EV-A71 strains belong to the C4a sub-genotype, which is a dominant genotype causing most of severe cases and death in mainland China during 2008-2012 (2, 17-19). However, the pathogen spectrum of HFMD substantially changed as CVA6 increasingly replaced EV-A71 to be predominant in many cities of China, particularly after the launch of EV-A71 vaccination since 2016 (20). We formerly observed that the proportion of EV-A71 continued to decrease as vaccination rates increased, at 22.5% in 2017, 0.5% in 2018 and disappeared from 2019, suggesting that immune protection in children is quite efficient after programmed EV-A71 vaccination (12). Although prevalence and phylogenetic analysis of the predominant strains were extensively explored, the virus-virus interactions (VVIs) of EVs were not well studied.
VVI is a measurable difference in the course of infection of one virus as a result of a concurrent or prior infection by a different species or strain of virus (21). In this study, we demonstrated the presence of VVI in HFMD cases at the epidemiological level by 13-year continuous surveillance of EV serotypes. To our knowledge, this study provides the first-hand evidence of different types of VVIs among the main causative agents CVA6, CVA16 and EV-A71. It’s appealing to find that CVA16 and EV-A71 interacted in a positive way, which in turn boosted the large concentrated HFMD outbreaks with resonance of EV-A71 and CVA16 infection during the second quarter of each year during 2010-2016 (Figure 6C). As frequent CVA6 outbreaks occurred after 2013 in the city of Nanchang, and it prevailed in a staggered epidemic pattern negatively interacted with CVA16 and EV-A71. To our knowledge, EV-A71 and CVA16 share the same entry receptor SCARB2, while KREMEN1 was proven as an entry receptor for most of the coxsackie type A viruses including CVA2-CVA6, CVA10, and CVA12 (22, 23). Whether the VVIs among EVs are interlinked with the entry receptors is unknown (Supplementary Figure 3A). Therefore, it will be appealing to explore the potential mechanism behind it in vitro and in vivo in the future.
In this study, impact of EV-A71 vaccination on the interactions between CVA6 and CVA16 was not observed, suggesting little potential cross-immunization between EV-A71 and CVA6 or CVA16 (Supplementary Figure 3B). However, during the past three local COVID-19 outbreaks spanning 3 years, the VVIs were interfered in the coming months of each outbreak and subsequently the epidemic peak of HFMD was restrained (Figure 2A-2B). This result was also supported by a national-scale (covering 31 provincial capitals in mainland China) impact of NPIs on EV transmission in 2020 (13). It will be more convincing to use computer simulations to obtain improved understanding of how the epidemiology of viral infections is interlinked, which can help improve disease forecasting and evaluation of HFMD control interventions in the future.
Children age less than 5 years are still highest-risk population of EV infection regardless of serotypes and pathogen spectrum fluctuation, and male cases were more common than females in all serotypes of EVs. It’s still unclear what is behind the proneness of gender. Formalin-inactivated EV71 vaccines are currently available for children of 6-59 months in China and substantially mitigated EV-A71 transmission (12, 24). The ARIMA models based on HFMD cases and EV-A71 cases effectively present the protective effect of EV-A71 vaccines against EV-71 infections in 2017, leading to lower incidence of HFMD and EV-A71-infection in Nanchang (Supplementary Figure 4A-B). However, these vaccines fail to confer cross-protection against CVA16 (Supplementary Figure 4C), highlighting the necessity of developing a multivalent HFMD vaccine.  Although access to EV-A71 vaccine is convenient in Nanchang, we observed a downward trend of EV-A71 vaccination that was likely due to the COVID-19 intervention (Supplementary Figure 5). However, follow-up of vaccination rate and public health education are necessary to consolidate the achievements of elimination of EV-A71 infection. Although CVA6, CVA10, CVA16 and EV-A71 are routinely detected for suspected HFMD cases, there is still a quite proportion (24.8%) of UEV-associated HFMD. We first clarified serotypes and identified top four causative agents CVA4, CVA2, CVA5 and CVB3 from local UEV cases, which helps us get a clear view on the vicissitude of pathogen spectrum. Whether these strains epidemiologically interlink with each other or not remains unknow (Supplementary Figure 3C). Previous evidence suggests that CVA6 began sporadically spreading in China from late 2012 before turning dominant in 2013 (7, 15, 25, 26). Despite the lack of publicly available CV-A6 surveillance data after 2015, our survey observed a dominant trend of CV-A6 in Nanchang (Supplementary Figure 2). Nevertheless, CVA16 has been sustaining a stable proportion and low-volatility pattern since 2010. Thus, we assume that CVA6 and CVA16 will regain the negative VVI in the coming years without external intervention such as vaccines or NPIs. Moreover, an enhanced monitoring is necessary for neglected serotypes CVA2, CVA4, CVA5 and CVB3 that were “hidden” in UEV under current surveillance system. Therefore, serum neutralizing antibody assay for pre-school children will be helpful to comprehensively understand the etiology features of HFMD.