Echoviruses are currently associated with a variety of human diseases, including asymptomatic infections, febrile illness, AM, and severe diseases in newborns. E-30 is one of the main pathogens that cause acute aseptic encephalitis, AM, and acute myocarditis and exhibits strong infectivity [12, 38, 40]. E-30 has shown wide circulation, including to the United States, Canada, France, Italy, Germany, England, Japan, South Korea, India, and China, and has demonstrated a highly epidemic trend in the previous 10 years [12, 40–44]. Therefore, understanding its pathogenesis and molecular epidemiology has important public health implications. During the period from June to August 2016, the number of hospitalized patients with AM increased sharply at Tongliao People’s Hospital, thereby surpassing the baseline of previous cases with AM in Tongliao city, Inner Mongolia.
We identified 363 patients during this time period in Tongliao city, Inner Mongolia, belonging to three townships that were affected by this outbreak, with Kailu and Naimanqi townships accounting for the greatest proportion of cases (n = 331, 91.2%). Compared with the homochromous numbers of AM cases in 2015, the number of AM patients increased significantly from June to August 2016 accompanied by a higher morbidity relative to other months. Children aged 6 to 12 years constituted the greatest proportion of all cases, consistent with previous reports [9, 39]. According to previous studies, viral encephalitis outbreaks caused by E-30 usually occurred from June to August in local regions and at the peak of the local enterovirus infection [6, 11, 13, 39, 44]. The epidemic then spread to other villages and towns, resulting in an outbreak within a short time period [6, 9, 13, 39, 45].
Outbreaks of aseptic meningitis caused by E-30 occur mostly in densely populated eastern coastal areas, such as Jiangsu, Zhejiang, Shandong, Fujian, and Guangdong [6, 7, 9, 10, 12, 13, 39]. The outbreak in the present study was caused by Lineage 2, which showed a close phylogenetic relationship with the strains isolated from Zhejiang, Jiangsu, Shandong, Sichuan, and Yunnan provinces of China. Using the Bayesian method, we observed the turnover of E-30 diffusion in China, implying its complex diffusion dynamic. The branches of E-30 isolated from the different provinces aggregated together, and E-30 spread simultaneously in several provinces simultaneously. E-30 is widespread in China and has caused a large number of AM outbreaks. Moreover, we observed the evolution of E-30 along the date clue, indicating gradual break outs in different provinces. The active status of E-30 in China has facilitated its evolution and transmission, and the accumulation of genomic variants might play a significant role in local outbreaks of AM.
Mapping of transmission links through Bayesian inference showed that Sichuan and Gansu provided more outward migrations, whereas Fujian, Henan, Guangxi, and Inner Mongolia had more inward migrations. Additionally, more emigration events were found in Gansu, Sichuan, Guangxi, and Yunnan provinces, with high PP and BF support (See Supplementary Table S1, Additional File 1). The outbreak in the present study possibly originated from these E-30-migration events; however, we were unable to directly locate the accurate source of E-30 due to low PP and BF support for Inner Mongolia (data not shown). However, the strains isolated from Sichuan province shared the closest phylogenetic relationships with those from Inner Mongolia (this study). Further, the high PP and BF support verified the transmission events from Gansu to Sichuan provinces, suggesting that the two outbreaks (Sichuan and Inner Mongolia) possibly possessed the same origin (See Supplementary Fig. S6 and Table S1, Additional File 1). Moreover, Gansu province could play an important role in E-30 outbreaks and spread in Sichuan and Inner Mongolia based on the highest PP and BF support (See Supplementary Table S1, Additional File 1). Furthermore, Gansu, Sichuan, and Inner Mongolia are neighbors in terms of their geographic distribution in China, suggesting local outbreaks and spread events among different provinces of China. We also observed that Fujian, Shandong, Taiwan, and Zhejiang provinces showed higher Markov rewards values as compared with other provinces, indicating that they played significant roles in E-30 evolution and circulation over time in China. The eastern provinces of China were primary regions experiencing E-30 infection and played an important role for further nationwide diffusion.
We then assessed the relative genetic diversity following the date clue, which showed fluctuant progression. Thus, the genetic diversity peaked ~ 2001 and ~ 2008, and different lineages showed polymorphic characteristics. The relative genetic diversity of Lineage 1 showed a similar fluctuant progression with that of all genomes before 2002, whereas that of Lineage 2 showed a similar fluctuant progression with that of all genomes after 2002. Therefore, Lineage 2 contributed to the enhanced genetic diversity of E-30 after 2002, whereas Lineage 1 was important for the genetic diversity of E-30 before 2002. This explains the contribution of fluctuant genetic diversity through the switch between different lineages.
The nucleotide and amino acid sequences in the P2 and P3 regions are highly conserved within an enterovirus species, and the P2 and P3 sequences do not correlate with EV serotypes due to frequent recombination; however, these sequences clearly distinguish different EV species [46]. In the present study, we identified overt evidence of inter-serotype recombination events. After screening several recombination signals, strain MN337405.1_Echovirus_E18_LJ/0601/2019 was detected as the putative recombination donor. This strain provided the raw recombination materials in the P2 and P3 coding regions for other recombinants. Frequent recombination and mutations in enteroviruses are recognized as the main mechanisms associated with their evolution, enabling their rapid response and adaption to new environments [46]. Accumulation of inter-species and intra-species recombination events is regarded as a strong driver for emergence and disappearance of certain enterovirus serotypes. Some studies have confirmed the ease of intra-species recombination events, and that EV-B is more susceptible to recombination [47–49]. The recombination events identified in the present study (i.e., the recombination donor was isolated from the CSF of an adult with severe meningitis in 2019 in China) verifies this. The recombination signals imply a vital role for E-30 evolution and might be related to E-30 pathogenicity and transmission.