Global CVB3 epidemiology
With the rapid development of molecular biology technology, a cDNA copy covering two-thirds of the CVB3 genome was cloned in Sweden in 1984 [22]. In 1985, the biologically active CVB3 virus (Nancy strain) cDNA, including the entire genome, was synthesized in Germany [23]. When the full-length genome of CVB3 was first reported in 1987, the number of CVB3 isolates significantly increased. CVB3 related outbreaks have been reported frequently, such as an outbreak in southern Louisiana, USA in 1959 (51 patients) [24], an outbreak in South Africa in 1984 with a variety of clinical manifestations [25], an outbreak of herpangina in Japan in 1987[26] (22 patients), an outbreak in Thailand in 1988 [27], an October 1992 outbreak of infant infection in a hospital in Beijing (35 patients) [28], a June 2005 outbreak of neonatal infections in Taiwan, China [29], 2008 outbreaks of aseptic meningitis in Shandong Province, China (81 CVB3 isolates) [30], and Hong kong, China (69 CVB3 isolates) [31], an outbreak of HFMD in Hebei in 2012 (35 cases), and an outbreak of HFMD in Shandong in 2016 (42 patients) [8]. During the EV-D68 outbreak in 2014 in the United States, CVB3 was the most commonly identified type of EV infection [32].
To date, CVB3 strains are associated with various diseases, and the manifestations vary from mild respiratory [33], gastrointestinal infections [34, 35], herpangina [36], and hand, foot, and mouth disease [7, 8, 37], to more severe diseases such as heart disease (including viral myocarditis [6, 38], pericarditis [39], and acute myocardial infarction [40]), CNS involvement (meningitis [30, 36, 41–43], meningo-cerebellitis [11], encephalitis [44], and AFP [45–47]), and pancreatic interrelated disease (pancreatitis [48], diabetes [49]). More seriously, the virus can cause neonatal sepsis-like illness and sudden death in infected infants [38, 50] and adults with low immune function.
Summary Of Cvb3 Datasets
In total, 319 entire VP1 sequences (before 2021 August 31th) were collected for the CVB3 genotyping and clinical manifestations summary. The CVB3 strains isolated between 1949 (the prototype strains) and 2020 from 17 countries, included countries in Asia ( China, Japan, India, Thailand, Uzbekistan, Indonesia), Europe (Germany, France, UK, Poland, Denmark, Russia, Romania, Moldova), North America (USA), Oceania (Australia), and Africa (Madagascar), representing wide temporal and regional distributions. In total, 84 CVB3 whole genome sequences (near the whole genome) (this study, n = 26; GenBank, n = 58) were used for the analysis. The sequence information is summarized in Additional file 1: Table S1. In total, 44 CVB3 sequences were included from mainland of China from 2006 to 2020, in 17 provinces, municipalities, and autonomous regions, representing seven administrative regions of China: Southeast China (Shandong, Jiangsu, Shanghai, Anhui, and Fujian), Central China (Hunan), South China (Guangdong), North China (Beijing and Tianjin), Northwest China (Shaanxi, Gansu, and Xinjiang), Southwest China (Sichuan, Yunnan, and Tibet), and Northeast China (Jilin) (Fig. 1). CVB3 strains isolated between 1949 (the prototype strains) and 2020 were from 11 countries and regions, including mainland of China.
Five groups were assigned based on the complete VP1 coding region of CVB3 isolates
In total, 319 entire VP1 sequences were collected from mainland China (n = 167), Taiwan, China (n = 46), Japan (n = 3), India (n = 17), Thailand(n = 2), Uzbekistan (n = 1), Indonesia (n = 1), Germany (n = 27), France (n = 19), UK (n = 1), Poland (n = 4), Denmark (n = 1), Russia (n = 3), Romania (n = 2), Moldova (n = 1), USA (n = 8), Australia (n = 14), and Madagascar (n = 2). We divided the global CVB3 into five groups (A–E) based on the entire VP1 sequence (Fig. 2A). The group mean distances varied from 16.9% (groups C to D) to 21.5% (groups A to C). The mean genetic distance within the group varied from 0.6% (group A) to 12.0% (group E), indicating the reliability of genotyping. The prototype strain Nancy, which was isolated in the USA in 1949, clustered with the strains isolated in Australia, the USA, Germany, and mainland of China to form Group A. Group B included strains isolated in the USA in 1956 and in Germany in 1999. Most Indian strains, one strain from Uzbekistan, and two from Madagascar formed group C. Most Group D strains were isolated in China. Group E comprised strains isolated from 13 countries, suggesting that group E might be transmitted globally. Notably, the Chinese isolates fell into groups A, D, and E. Group D CVB3 was first isolated from cases of acute flaccid paralysis in 1990 and has persistently circulated in mainland of China from 1993 to 2017, suggesting strong transmission potential. We found that the dominant group D had a wide temporal and geographical distribution, whereas group A disappeared after 2014. Emerging group E was only detected among HFMD cases in 2020 in one province and was possibly imported from other countries. The basic data for each group are summarized in Table 1.
Table 1
Information of 5 group CVB3s relying on entire VP1 sequences
Group | Sequences number | Country of isolation | Year | Intra-group distances(%) |
A | 12 | Australia, USA, Germany, China | 1949-2014 | 0.6 |
B | 2 | USA, Germany | 1956-1999 | 3.6 |
C | 20 | India, Uzbekistan, Madagascar | 1999-2011 | 5.2 |
D | 204 | China, Thailand, Russia, Japan, France | 1990-2017 | 8.3 |
E | 81 | China, Thailand, Indonesia, Japan, Moldova, Russia, Romania, Poland, Germany, France, Denmark, the UK, Australia, USA | 1989-2020 | 12.0 |
To investigate the evolutionary history of CVB3, MCC trees (Fig. 2B) were constructed based on the entire P1 nucleotide sequence of CVB3 strains (n = 77). The correlation coefficient was 0.74 and the R2 value was 0.54. A positive correlation was observed between genetic divergence and sampling time. The average nucleotide substitution rate for the P1 coding region in all CVB3 strains worldwide was 4.82×
10−3 substitutions/site/year (95% HPD, 3.51 × 10−3–6.05 × 10−3), which is slightly higher compared to the P1 evolutionary rate of CV-A6 reported by others [1], indicating its rapid spreading rate. The topological structure of the MCC tree constructed using BEAST was nearly identical to that of the ML tree constructed using MEGA software. Based on our analysis, the estimated TMRCA of CVB3 was in the early 1900s, corresponding to at least 40 years before the first reported detection of CVB3 in 1949. Global CVB3 strains isolated since 1903 formed two branches. Branch 1 included only group A, which arose in 1946, including the prototype strain Nancy. Branch 2 included group B (differentiated in 1927), group C with a tMRCA that emerged in 1990, group D (emerged in approximately 1994), and group E emerging in 1982.
We also focused on the clinical manifestations of different groups. Myocarditis (41.7%) was more common in group A than that in the other groups. Group C (40%) and group D (31.4%) presented more cases of AFP compared to the other groups. In addition, while comparing the numbers (n = 65) and proportions (31.9%) of HFMD cases among the five groups in this study, we found that group D may be more likely to cause HFMD. The most common diseases in group E were HFMD (9.9%), diabetes (9.9%), and aseptic meningitis (8.6%) (Fig. 2C).
Full-length genome analysis of CVB3 exhibited diverse recombination lineages in China and worldwide
Phylogenetic trees were constructed based on the entire P1, P2, and P3 region nucleotide sequences of CVB3 strains along with the prototype of EV-B from GenBank database. Consistent with the phylogenetic tree of the VP1 coding region, the P1 phylogenetic tree (Fig. 3A) indicated the existence of the five CVB3 groups in the world as expected, verifying the primary genotyping results. Unlike the P1 phylogenetic trees, those of the P2 and P3 non-structural regions showed that several independent lineages clustered with the prototype strains of other EV-B prototypes rather than with the prototype of CVB3, suggesting the occurrence of recombination between CVB3 and other EV-B serotypes (Fig. 3B, 3C).The CVB3 whole genomes displayed significant genetic diversity in the non-structural regions, and a total of twenty-three recombinant lineages were detected (Table 2). We named the recombinant lineages based on their first isolation time. Groups A and B had only one lineage each, consistent with the VP1 region phylogenic tree. Group C contained three lineages: lineage F, lineage O1, and lineage O2. Group D isolates were differentiated into lineage G, lineage K, lineage L, lineage M, lineage R, lineage S1, lineage S2, lineage T, and lineage U, suggesting that at least nine different CVB3 recombinant lineages are circulating in China. Group E was separated into independent lineages by other EV-B prototype strains, including lineages C, D, E, H, I, J, N, P, and Q. Some recombinant lineages, such as lineages B–F, are ancient lineages from the last century and tend to be absent. The recombinant lineage J was associated with group E, which clearly covered several countries and was spread extensively. We also used similarity scanning to certify the twenty-three diverse recombinant lineages using ORF1 sequences (Fig. 3D). The intra-lineage nucleotide similarity of lineages A, I, J, K, L, O1, O2, R, S, U, and T was between 95.8–99.9%, indicating that all the strains in the same lineage displayed high intra-lineage homology throughout the ORF1 sequences. The sliding window analysis performed across intra-lineage sequences revealed low pairwise genetic diversity; however, when all the lineages were considered together, the diversity was obviously higher in the non-capsid regions (Fig. 3E).
Table 2 Information of 23 recombinant lineages of CVB3s based on P2 and P3 non-structural region sequences
Lineage
|
Group
|
Sequence number
|
Nucleotide mean distances(%)
|
Isolated countries/regions
|
Isolated year
|
A
|
A
|
10
|
0.46
|
USA, China
|
1949-2014
|
B
|
B
|
1
|
NA
|
USA
|
1956
|
C
|
E
|
1
|
NA
|
Romania
|
1989
|
D
|
E
|
1
|
NA
|
France
|
1993
|
E
|
E
|
1
|
NA
|
Romania
|
1995
|
F
|
C
|
1
|
NA
|
Uzbekistan
|
1999
|
G
|
D
|
1
|
NA
|
China
|
2001
|
H
|
E
|
1
|
NA
|
Moldova
|
2002
|
I
|
E
|
1
|
NA
|
USA
|
2005
|
J
|
E
|
11
|
4.14
|
Australia, the UK, USA, China
|
2006-2020
|
K
|
D
|
9
|
4.24
|
China
|
2006-2013
|
L
|
D
|
10
|
2.80
|
China, Thailand
|
2008-2012
|
M
|
D
|
1
|
NA
|
China
|
2009
|
N
|
E
|
1
|
NA
|
Australia
|
2009
|
O1
|
C
|
11
|
1.39
|
India
|
2009
|
O2
|
C
|
1
|
NA
|
India
|
2009
|
P
|
E
|
1
|
NA
|
Thailand
|
2010
|
Q
|
E
|
1
|
NA
|
Denmark
|
2010
|
R
|
D
|
8
|
2.65
|
China
|
2012-2016
|
S1
|
D
|
2
|
0.10
|
China
|
2016
|
S2
|
D
|
2
|
0.43
|
China
|
2016
|
T
|
D
|
2
|
1.84
|
China
|
2016
|
U
|
D
|
6
|
2.30
|
China
|
2016-2017
|
Previous studies have shown that EV-Bs are more susceptible to recombination compared to EV-As [51]. Regarding CVB3, many recombinant lineages, which have persisted for many years and were widespread, are still globally active. For example, lineage J included 11 strains showing high sequence identity with each other. The P2 and P3 non-structural coding region of lineage J, which clustered with the E18 (MG720260) and E30 (EF066392) strains, exhibit recombination activities in the evolutionary process of CVB3 (Fig. 4A). Furthermore, the non-structural genomic region of lineage L was found to have recombination breakpoints with three different EV-B serotypes: CVB5 (MN749140, JX843811, JN695051), E25 (KJ957190), and E30 (KF878942) strains; in contrast, the P1 coding region showed higher similarity with the CVB3 prototype strain (Fig. 4B). Lineage O1 showed a complex recombination with the E11 (MK791152), E25 (MF678298), EVB75 (MW183139), and EVB88 (MH144607, MH118025) strains in the whole P3 region (Fig. 4C). Lineage U showed obvious recombination events, corresponding to the CVB5 (JN695051), E25 (KJ957190), and E30 (KF878942) strains (Fig. 4D). Therefore, the other serotypes of EV-Bs, such as echovirus (E11, E18, E25, and E30), CVB5, and novel EV-B75 and EV-B88 play important roles in the natural recombination of CVB3. These results indicate that inter-serotype recombination events occurred in the non-structural coding region of CVB3. The current circulating CVB3 strains are recombinants; however, the exact source of recombinant fragments was not identified; therefore, further studies are still needed to determine the EV serotype circulating simultaneously.