Evolutionary phylogeography and transmission pattern of echovirus 14: an exploration of spatiotemporal dynamics based on the 26-year acute flaccid paralysis surveillance in Shandong, China

Enteroviruses (EVs) are common human pathogens belonging to the genus Enterovirus, family Picornaviridae. Most EV infections are usually asymptomatic, but sometimes they are associated with diverse clinical syndromes ranging from minor febrile illness to severe, potentially fatal diseases such as encephalitis, paralysis, myocarditis and neonatal enteroviral sepsis [1, 2]. EVs have traditionally been detected based on virus isolation in cell cultures and subsequently serotyped by serum neutralization test, and were classified into echoviruses (E), coxsackieviruses (CV) group A and B, and polioviruses. Currently, based on the phylogenetic relationships in multiple genome regions, International Committee on Taxonomy of Viruses reclassified EVs into 12 species: EV-A to EV-H, EV-J and rhinovirus A, B and C [3]. Of these species, seven (EV-A to EV-D and rhinovirus A, B, C) of the EV species infect humans while remaining infect non-human animals.

Echovirus 14 (E-14) was first isolated from a hospitalized patient suffering from aseptic meningitis syndrome in 1954 [4] and had been classified as a member of EV-B. In contrast to other common EVs such as EV-A71 and CV-A16, both of which are categorized as EV-A and are major etiological agents for the widely spread hand, foot and mouth disease (HFMD) [5–7], E-14 is considered relatively rare as it less likely to cause worldwide occurrences of clusters and outbreaks. However, epidemics of E-14 infection had been observed in Europe and Asia in the 1960s [8–11]. Similarly, association of E-14 with many clinical recognized syndromes such as gastrointestinal syndrome [8–10], acute serous meningitis [12], paralysis [13], fatal neonatal hepatic necrosis [14], aseptic meningitis [11, 15] has been reported previously. In recent years, E-14 is more than detected in an outbreak or epidemic caused by other pathogens [16, 17] or exhibited sporadic infection in conventional virological surveillance [18–20].

As a coastal province, Shandong has a population of about 96 million and major ports that could potentially serve as portals for importation of exogenous viruses. The acute flaccid paralysis (AFP) surveillance, developed for polio eradication program, was launched in Shandong in 1988. Currently, the AFP surveillance has been conducted in all 138 counties of Shandong province and more than 600 sentinel hospitals were included. AFP surveillance can obtain non-polio enterovirus (NPEV) strains as a side benefit and produce baseline data of local NPEV distribution and a genetic overview [21]. Our previous study demonstrated continuous AFP surveillance provided valuable information on the circulation and emergence of different EV types in the context of limited EV surveillance in China [20]. In this present study, we investigated the genetic characterization of E-14 strains isolated from Shandong AFP surveillance during 1988–2013. Phylogenetic and phylogeographic analysis were combined to elucidate the evolution relationship and spatiotemporal dynamics of E-14 worldwide.

Compared with many EV serotypes which can lead to serious illness, E-14 is considered relatively rare as it less likely to cause worldwide occurrences of clusters or outbreaks. To the best of our knowledge, no detailed molecular epidemiology study was carried out based on a large number of E-14 sequences. Owning to the launch of the Global Poliomyelitis Eradication Initiative, we had accumulated a significant number of E-14 sequences associated with AFP surveillance which was developed for casting a wider net for poliovirus detection in Shandong since 1988. For the detection of circulating EVs, AFP surveillance has also provided a major source of information in China, as specialized EV surveillance based on other approaches or populations was still very limited. In addition, studies conducted in other countries [18, 22–27] also described the NPEV serotypes identified from the AFP surveillance, and the information had attracted more attention of the scholars worldwide.

Although E-14 may cause mild to severe neurological disorders as reported from Russia [12], France [9, 10], Italy [8] and Japan [11, 13, 15], unfortunately, no clinical information, molecular epidemiology and disease correlation of this serotype have been reported. This study, which was performed a comprehensive molecular epidemiologic analysis of Shandong E-14 isolates, was feasible because of the large number of AFP database during the last 26 years in Shandong, China. During 1990–2013, a total of 10322 AFP cases and contacts were reported, and 958 NPEV isolates were obtained from stool specimens in Shandong AFP surveillance [20]. A total of 53 NPEV serotypes were identified, serotypes such as E-6, E-14 and EV-A71 were the most commonly reported. It should also be notified that no EV-D68 strains were detected in our AFP surveillance, although this EV serotype was associated with AFP and other neurological complications. The proportion of E-14 isolated in this study was high (7.6%) as compared to other identified NPEV serotypes [20]. Additionally, E-14 was also found a prevalent EV serotype with AFP surveillance in other regions of the world [18, 19, 28]. Consequently, all these findings suggest E-14 as the possible causative agent of paralysis, and the molecular epidemiologic analysis of E-14 is still necessary.

EVs are well known for their high genome plasticity, due to both high mutation and recombination rates. In this study, we analyzed the global E-14 genetic diversity, of 73 isolates from Shandong and 54 strains isolated in twelve different countries. The phylogenetic tree (Fig. 2) revealed that E-14 strains clustered in both time and regional distributions. For E-14 isolated in China, strains in group C and B were from 1993 to 2006 and 2000 to 2010, respectively and there were 10 and 6 years had passed since the last isolation in these two groups. The isolation years of group A spanned from 1993 to 2014, revealing a long time circulation of this group in Shandong region. Moreover, it is obvious that E-14 isolates belonging to group B and C may have been disappeared after circulating for a period of time and was replaced by group A. Viruses in group A may have become the dominant group circulating in China. Alternatively, the information on Chinese E-14 isolates is limited, and more sequences are needed to determine domestic E-14 phylogenetic relationships.

We used a phylogenetic molecular clock approach to further infer the historical background and geographic diffusion of global E-14 strains. The MCC tree (Fig. 3) constructed was congruent with the results observed in phylogenetic analysis (Fig. 2). It was perceived by Bayesian MCMC evolutionary analysis that E-14 strains were circulated for about 80 years in the world. Moreover, E-14 strains isolated in China may be the product of geographic segregation and has evolved independently in China for a long time. Two observations suggest that the common ancestor of global E-14 lineages existed in the USA. First, USA was the first country reported to detect E-14 [11] and second, the USA strain is phylogenetically more diverse within the phylogenetic tree (Fig. 2) and the MCC tree (Fig. 3) than those from elsewhere. However, these observations may reflect differences in AFP surveillance intensity among countries and more information is required before we can conclude that E-14 was introduced to the USA multiple times from other regions. Interestingly, our results indicate that circulation of E-14 in Australia is very dynamic, as the virus likely to be the reservoir for evolutionary origin of E-14 in the world. The results that the nucleotide substitution rate of E-14 VP1 gene was similar to other EV serotypes [29–34] also suggested the evolutionary pressure acting on E-14 existed no significant difference with other serotype strains.

A particular focus of this study was to determine the phylogeographic relationship between E-14 collected from Shandong province and compare these with those from other countries. To our knowledge, the current study is the first applying the Bayesian phylogeographic reconstruction approach to gain insight into the emergence and spread of E-14 strains worldwide [35]. The phylogeographic analysis revealed that the transmission of E-14 in China likely originated from the Australia, and then the endemic circulation was observed without spreading to foreign countries. However, E-14 strains in China and Australia have no direct evolutionary relationship according to the phylogenetic analysis. The reason why the phylogenetic and phylogeographic analysis was inconsistent between E-14 strains in China and Australia remains unclear, and analysis of more sequences with greater detail information will yield more precise diffusion process. In addition to spread from Australia to India, E-14 strains seem to have been reintroduced into Australia from India, and the same scene occurred among Shandong province with Guangdong and Yunnan provinces in China. We believe that large-scale patterns in human mobility between these locations will provide a more plausible explanation on E-14 emergence and introduction. Overall, as a well documented origin from the USA, E-14 in this area has become silent over the past few decades. Australia may be responsible for the transmission of E-14 in recent years, and two regional transmissions were mainly detected, with one located between Europe and Africa countries and the other was in the Asia-Pacific region.

The results in this study are subject to a selection bias. Sequences from GenBank are limited, especially for the complete VP1 genes of E-14. In addition, the first E-14 sequence represents a strain isolated in 1954 in the USA, whereas the next sequences can be obtained from GenBank originated in 1980. Therefore, partial VP1 sequences were used for the ananlysis of the phylogenetic relationships among global E-14 strains. Due to availability of limited gene information of global E-14, further collection and analysis, together with detailed epidemiological information is required to understand the spatiotemporal dynamics of E-14 worldwide. All these indicate that EV surveillance in most countries remains to be strengthened and the systematic surveillance are necessary in investigating the EV circulation and understanding the social burden of EV infection.

In this study, we collected the complete VP1 sequences of E-14 based on the 26-year acute flaccid paralysis surveillance in Shandong province, and inferred the historical background and geographic diffusion of global E-14 strains. The estimated nucleotide substitution rate for global E-14 was 9.17 × 10⁻³ substitutions/site/year, and the estimated t _MRCA was August 1928. Our research confirmed that the origin of E-14 could be in the USA, however, regional transmissions were detected with one located between Europe and Africa countries and the other was in the Asia-Pacific region. Australia may be responsible for the spread of E-14 in recent years.