Complete genome analysis of echovirus 30 strains isolated from hand-foot-and-mouth disease in Yunnan province, China

Hand-foot-and-mouth disease (HFMD) is a kind of childhood infectious disease. As one of the class C infectious diseases in China, HFMD is common in children under 5 years old, while the main symptoms usually include mouth sores and small blisters on the hands and feet, and may be accompanied by low-grade fever, anorexia, etc. Most cases heal spontaneously in about one week, however, some develop rapidly, leading to severe complications such as aseptic meningoencephalitis, viral myocarditis, etc., and even death. HFMD is caused by more than 20 enteroviruses, among which, enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the most common causes [1‐3].

Enteroviruses belong to the genus Enterovirus, Picornaviridae. The main enteroviruses associated with human diseases are EV-A, EV-B, EV-C, and EV-D, of them, Echovirus 30 (E30) belongs to EV-B [2]. In multiple retrospective studies of clinical samples of feces, serum, and cerebrospinal fluid from several countries, E30 infection is the most common causative agent of aseptic meningitis outbreaks in Asia, Europe, and the Americas in recent years [4‐7]. In addition, serious infections of E30 have been documented in Zhejiang, Fujian, Jiangsu, and Shandong in China, inflicting an immense burden on families worldwide [8]. In this study, four E30 strains were isolated from fecal samples of children with HFMD, and the whole genome characteristics were analyzed, providing a certain reference for the prevention and control of HFMD.

HFMD can be caused by a variety of enteroviruses, and EV71 and CVA16 are previously thought to be the major pathogens in Asia [7, 8, 17, 18]. However, with the introduction of the inactivated EV71 vaccine, the proportion of HFMD caused by EV enteroviruses has changed, with other EV serotypes instead of EV71 becoming the predominant etiologic agent [19‐21], such as CVA6, CVA10, CVB3, CVB5, etc [1, 2, 9, 22‐24]. As E30 is frequently detected in HFMD cases, E30 also has the potential to cause pandemics, suggesting the value of continuous, extensive surveillance of other enterovirus serotypes in addition to the main causative viruses for HFMD [6].

The VP1 coding region of enteroviruses contains the main antigenic neutralization sites and lots of important structural domains. It is used as the basis for molecular typing of enterovirus and plays an important role in the binding and entry process of the virus into the host cells [25‐30]. BC and DE loops of the VP1 coding region are important regions which closely related to the antigenicity of the virus, the binding affinity of corresponding specific neutralizing antibodies, and the ability of the virion to enter host cells [31‐34]. The phylogenetic tree of complete genome sequences reflects that the four isolates formed a separate evolutionary branch with the 2017 US strain, the 2016 Inner Mongolia, China strain, the 2019 Wuhan, China strain, and the 2021 Jiangsu, China strain, which indicated that domestic and international travel can lead to the exchange of viral strains from different countries and regions and a new epidemic lineage is constructed through recombination, genetic mutation, and other changes together.

Selection pressure analysis revealed that the 133rd loci was detected by all these three methods. The 133rd codon loci of the VP1 sequence is located in the DE loop structural region where equipped with the crucial antigen determinant of virion particles. Compared with prototype strain Bastianni, 15K3 and 9K3 mutated from R to T at the 133rd site, while 52R3 and 32R3 mutated from R to A at the same place. However, 15K3 and 9K3 were isolated from KMB-17 cells, 52R3 and 32R3 were isolated from RD cells, which indicated that E30 evolved with environmental changes, according to the law of adaptive mutation in the process of virus evolution, and this substitution may affect viral adaptation in different cells. In addition, T80A, A84V, and D87E were found in the BC loop, while N132T, R133T, and R133A were found in the DE loop. The mutations may affect the evolution of the virus, but further research is needed.

E30 outbreaks present a periodic pattern of three to five years, lasting one to two years, which are usually associated with the rapid spread of different strains [4, 35]. It has been reported that recombination is common for EV-B, and the non-structural protein (NSP) region is the hot spot of recombination and leads to the emergence of new epidemic strains [36‐39].

Recombination is a major driver in the evolution of enteroviruses, especially for EV-B [37]. Recombination analysis of four new E30 strains in this study indicated that recombination events may occur in P2, P3, and 3’-UTR regions. The recombinant donor may be E6 strain P735/CHN/2013, E18 strains (S5758/CHN/2018, S6064/CHN/2018, and S6363/CHN/2018), E3 strain 123-R2/CHN/2018, and E9 strain KM812/CHN/2010, and these possible recombinant donors have been all isolated from China. E6 strain P735/CHN/2013 was isolated from fecal samples of children with HFMD in China in 2013.The E3 strain 123-R2/CHN/2018 and 3 E18 strains, were separated in 2018 and E9 strain KM812/CHN/2010 was separated in Kunming, Yunnan Province, China in 2010. These viruses are frequently found to cocirculate among patients with HFMD, which increases the probability of recombination. The limitation of this study is that the samples collected were merely stool samples. Taking multiple samples at the same time may be helpful to the diagnosis and treatment of the disease

In summary, this study extends the whole genome sequence of E30 in GenBank, in which mutations and recombinations have driven the evolution of E30 and further improved and enriched the genetic characteristics of E30, providing fundamental data for the prevention and control of diseases caused by E30. Furthermore, we demonstrated the value of continuous and extensive surveillance of enterovirus serotypes other than the major HFMD-causing viruses.