Journal of General Virology (2014), 95, 868–873
Short Communication
DOI 10.1099/vir.0.060004-0
Recombination strategies and evolutionary dynamics of the Human enterovirus A global gene pool Alexander N. Lukashev,1 Elena Yu. Shumilina,1 Ilya S. Belalov,1 Olga E. Ivanova,1 Tatiana P. Eremeeva,1 Vadim I. Reznik,2 O. E. Trotsenko,3 Jan Felix Drexler4 and Christian Drosten4
Correspondence
1
Alexander N. Lukashev
2
[email protected]
Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia Center of Hygiene and Epidemiology in Khabarovsk Region, Khabarovsk, Russia
3
Khabarovsk Institute of Epidemiology and Microbiology, Khabarovsk, Russia
4
Institute of Virology, University of Bonn Medical Centre, Bonn, Germany
Received 1 October 2013 Accepted 14 January 2014
We analysed natural recombination in 79 Human enterovirus A strains representing 13 serotypes by sequencing of VP1, 2C and 3D genome regions. The half-life of a non-recombinant tree node in coxsackieviruses 2, 4 and 10 was only 3.5 years, and never more than 9 years. All coxsackieviruses that differed by more than 7 % of the nucleotide sequence in any genome region were recombinants relative to each other. Enterovirus 71 (EV71), on the contrary, displayed remarkable genetic stability. Three major EV71 clades were stable for 19–29 years, with a half-life of non-recombinant viruses between 13 and 18.5 years in different clades. Only five EV71 strains out of over 150 recently acquired non-structural genome regions from coxsackieviruses, while none of 80 contemporary coxsackieviruses had non-structural genes transferred from the three EV71 clades. In contrast to earlier observations, recombination between VP1 and 2C genome regions was not more frequent than between 2C and 3D regions.
Human enteroviruses are members of the genus Enterovirus within the family Picornaviridae. These small, non-enveloped RNA viruses have a single-stranded non-segmented genome of positive polarity of about 7500 nt. The genome encodes a single large polyprotein comprising four structural proteins, VP1–VP4 (collectively termed the structural genome region), and seven non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C and 3D (the non-structural genome region) (Racaniello, 2007). Human enteroviruses comprise four species, Human enterovirus A–D (HEV-A–D). Enterovirus 71 (EV71) is the best studied non-polio enterovirus, and it was further classified into subtypes A, B0–B5, C1–C5 and D (Brown et al., 1999; McMinn, 2012). Enteroviruses are highly prevalent in the human population, especially in infants (Witsø et al., 2006). Infection with HEVs is usually subclinical, but can occasionally result in single cases and outbreaks of meningitis, sepsis-like infection and myocarditis. Infection with the three poliovirus serotypes (members of the HEV-C species) and EV71 can produce severe neurological lesions (Pallansch & Roos, 2007). Global GenBank accession numbers for identified sequences are KC879327– KC879556. Two supplementary tables are available with the online version of this paper.
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prevalence of enteroviruses and high infection rates in infants greatly facilitate natural recombination. Recombination is apparently most prevalent in the HEV-B species (Simmonds & Welch, 2006). However, it is common also in HEV-A (Huang et al., 2009; Oberste et al., 2004; Simmonds & Welch, 2006) and HEV-C (Brown et al., 2003). As a result, enterovirus species exist as highly dynamic global gene pools (reviewed by Lukashev, 2005). It has been shown that the half-life of recombinant forms (combinations of discrete VP1 and 3D genes without further signs of recombination) in circulation may vary from 1.3 to 9.8 years between different HEV-B serotypes (McWilliam Leitch et al., 2010). Recombination in HEV-A was investigated in a number of studies, but these were confined by limited geographical coverage (Simmonds & Welch, 2006), sampling restricted to a few prototype strains (Oberste et al., 2004), or by analysing only the neurovirulent EV71, but no other contemporary HEV-A (Chen et al., 2010; Huang et al., 2008; Mirand et al., 2010; van der Sanden et al., 2011). The aim of this study was to determine recombination patterns in HEV-A on a whole species level with an extensive geographical and temporal coverage. Seventy-eight HEV-A strains were isolated and identified according to a standard World Health Organization (WHO) protocol (WHO, 1992) in RD cell culture in the course of the WHO polio surveillance programme and enterovirus 060004 G 2014 SGM Printed in Great Britain
Recombination in Enterovirus A
surveillance in Russia and New Independent States in 2001– 2011 (Table S1, available in the online Supplementary Material). According to the national regulations, informed consent is not required for anonymized surveillance studies. Three genome regions were amplified by PCR, namely the complete VP1 and partial 2C and 3D. PCR primers are listed in Table S2. All viruses used for the study differed by at least 1 % nucleotide sequence in their VP1 genome region; less divergent sequences were excluded upon preliminary analysis. GenBank accession numbers for sequences obtained here are KC879327–KC879556. All complete or nearly complete HEV-A sequences available in GenBank were aligned using CLUSTAL W (Thompson et al., 1994). Redundant sequences sharing more than 97 % overall nucleotide sequence identity were then omitted, yielding 76 unique reference sequences. Sequence handling was performed with BioEdit v.7.0.5.2 software (Hall, 1999). Phylogenies were calculated using a maximum-likelihood tree algorithm with the Tajima–Nei substitution model in the MEGA 5.2 software package (Tamura et al., 2011). In the VP1 genome region (nt 2439–3329 in the prototype EV71 strain BrCr, GenBank accession number U22521), phylogenetic grouping expectedly corresponded to serotypes (Fig. 1). A Bayesian likelihood-based algorithm implemented in BEAST version 1.7.4 (Drummond & Rambaut, 2007) was then used to date tree nodes that were conserved across all three genome regions. Bayesian molecular dating was performed separately for distinct serotypes, because analysis of the VP1 alignment that included multiple serotypes consistently yielded substitution rates incompatible with those reported in other studies and those resulting from analysis of individual serotypes (data not shown). The SRD06 substitution model optimized for coding sequences (Shapiro et al., 2006) was used with a lognormal relaxed clock setting, which was preferred over assumption of a strict clock upon Bayes factor testing (log10 Bayes factor of tree likelihood .10), while the exponential clock setting was not significantly superior to the lognormal clock. Each analysis was run over 10 000 000 generations, and trees were sampled every 1000 generations, resulting in 10 000 trees. Trees were annotated with TreeAnnotator v.1.4.8 using a burn-in of 2000 trees. Substitution rates in different serotypes (expressed as 1023) were 4.05 (95 % confidence interval 3.23–4.93) substitutions per site year21 in EV71, 8.32 (5.35–11.6) in coxsackievirus A (CVA)2, 5.53 (3.57–7.57) in CVA4 and 14.1 (4.01– 24.2) in CVA10. The substitution rate for EV71 was remarkably similar to previously published estimates of 4.2–4.6 (Tee et al., 2010) and 3.7 (Mirand et al., 2010), but differed from the substitution rate of 7.2 reported in another work (McWilliam Leitch et al., 2012). Importantly, in the latter work, lower substitution rates were observed within individual EV71 subgenotypes (3.2–7.3), implying that the overall rate of 7.2 may be inaccurate. While there have been no published studies of substitution rates in other HEV-A types, the observed variation is compatible with the range of substitution rates in HEV-B serotypes (McWilliam http://vir.sgmjournals.org
Leitch et al., 2010). The time of the most recent common ancestor (tMRCA) of coxsackievirus serotypes (CVA2, 69 years; CVA4, 66 years; CVA10, 72 years) was lower than tMRCA of EV71 (91 years); therefore, EV71 may be a cause of an emerging infection, but according to the phylogenetic evidence, it cannot be deemed an emerging virus. In the 2C (nt 4350–4766) and 3D (nt 5973–6602) genome regions, distribution of pairwise distances was relatively even, and did not allow assigning recombinant forms as was done earlier (McWilliam Leitch et al., 2012). Phylogenetic grouping generally did not correspond to the VP1 genome region (Fig. 1), implying common recombination. All coxsackieviruses except for type 16 were extensively shuffled in the non-structural genome regions. This kind of phylogenetic relationship is best exemplified by group I (Fig. 1) consisting of 23 viruses of 11 serotypes in the 2C genome region or group Ia that comprised 29 viruses of nine serotypes in the 3D genome region. The other extremity of recombination frequency was represented by EV71 and CVA16. In the 2C and 3D genome regions, most EV71 strains fell into three groups (groups II–IV, Table 1, Fig. 1), which corresponded to subgenotypes B4–B5, C1– C3 and C4, respectively. While grouping of all EV71 B subgenotypes was conserved throughout the genome in our study, it is known that recombination occurred within that group; therefore we regarded only the group comprising subgenotypes B4 and B5, which was also conserved in previous studies with a more extensive sampling of EV71 (McWilliam Leitch et al., 2012). There were few EV71 sequences with alternative 2C (four out of 48 sequences, two of B4 and two of C2 genotype) and 3D genome regions (five sequences, the four mentioned above and one EV71C4). However, the three EV71 groups present in the 2C and 3D genome regions (a total of 51 and 50 sequences, respectively) did not contain a single sequence from another serotype. Within these EV71 groups, there was also no significant evidence of recombination (change of tree topology supported by bootstrap values over 70) between the three genome regions. Similarly, all 14 CVA16 isolates sequenced here formed a single phylogenetic group without signs of recombination between VP1, 2C and 3D genome regions (Fig. 1). These three groups, which were characterized by limited recombination, had a tMRCA of around two decades. Median ages of non-recombinant viruses from the root of their respective group (roughly equivalent to half-lives of non-recombinant viruses reported by McWilliam Leitch et al., 2012) were between 13 and 18.5 years (Table 1). These numbers make EV71 the least recombining enterovirus compared with echovirus 9 (E9), E30 and E11 that featured recombinant form half-lives of 1.3, 3.1 and 9.8 years, respectively (McWilliam Leitch et al., 2010). The difference from numbers reported for EV71 by McWilliam Leitch et al. (2012) (6 and 9 years for GtB and GtC, respectively) may be explained by the aforementioned uncertainty of the substitution rate and by limited sampling of GtB sequences (by either quantity or geographical coverage). The sampling of CVA16 was confined to Russian 869
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89
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96
60
99
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III
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EU703812_EV71-C4_CHN_2008 HQ426649_EV71-C4_CHN_2010 GQ994990_EV71-C4_CHN_2009 GQ892830_EV71-C4_CHN_2008 41785_EV71_RUS_2011 42266_EV71_RUS_2011 HQ325852_EV71-C4 CHN_2010 EU864507_EV71-C4_CHN_2008 GQ279370_EV71-C4_CHN_2008 95 FJ194965_EV71-C4_CHN_2008 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 FJ713137_EV71-C4_CHN_2009 81 HM003207_EV71-C4_CHN_2008 GQ279369_EV71-C4_CHN_2008 FJ607337_EV71-C4_CHN_2008 GU196833_EV71-C4_CHN_2009 HQ129932_EV71-C4_CHN_2006 75 FJ194964_EV71-C4_CHN_2008 80 GQ994989_EV71-C4_CHN_2009 FJ357374_EV71-C4_TW_2005 86 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AF302996_EV71-C4_CHN_1998 90 HQ188292_EV71-C4_CHN_2008 95 DQ341361_EV71-C1_AUS_2000 79 99 EU414335_EV71-C1_CHN_2006 DQ452074_EV71-C1_NOR_2003 98 DQ341358_EV71-C1_MAL_2000 91 27578_EV71-C1_RUS_2007 DQ341359_EV71-C1_MAL_1998 EU527983_EV71-C5_TW_2007 89 DQ341356_EV71-C3_KOR_2000 AF304459_EV71-C2_TW_1998 13384_EV71-C2_RUS_2001 DQ381846_EV71-C2_AUS_1999 97 HM622392_EV71-C2_TW_2008 29256_EV71-C2_RUS_2007 DQ341357_EV71-C2_AUS_1999 37731_EV71_UKR_2010 40190_EV71_RUS_2010 FJ172159_EV71-C2_CHN_2008 97 32896_EV71-C2_RUS_2008 86 38555_EV71_KGZ_2010 34020_EV71-C2_RUS_2009 99 41464_EV71_RUS_2011 71 AB482183_EV71-B1_JPN_1973 98 HQ189392_EV71-B1_HUN_1978 FJ357382_EV71-B2_TW_1986 99 99 FJ357384_EV71-B2_TW_1986 99 DQ341354_EV71-B4_CHN_1998 99 DQ341368_EV71-B4_MAL_1997 96 84 DQ341365_EV71-B4_MAL_2001 FJ357377_EV71-B4_TW_2000 FJ357375_EV71-B4_TW_1999 DQ341364_EV71-B5_CHN_2000 FJ357378_EV71-B5_TW_2003 97 FJ461781_EV71-B5_CHN_2008 94 EU527985_EV71-B5_TW_2007 89 HM622390_EV71-B5_TW_2009 97 U22521_EV71-A_BrCr_USA_1969 U05876_CVA16_SOA_1951 34950_CVA16_RUS_2009 41555_CVA16_KAZ_2011 99 38394_CVA16_RUS_2010 99 42234_CVA16_RUS_2011 37323_CVA16_UKR_2010 99 GQ279368_CVA16_CHN_2008 99 89 40188_CVA16_RUS_2004 AF177911_CVA16_TW_1998 FJ198212_CVA16_CHN_2008 99 GQ279371_CVA16_CHN_2008 97 JN590244_CVA16_CHN_2010 29072_CVA16_RUS_2007 31758_CVA16_RUS_2008 34351_CVA16_RUS_2009 99 94 39156_CVA16_RUS_2010 99 24315_CVA14_RUS_2005 AY421769_CVA14_USA_1950 99 34082_CVA7_RUS_2009 37183_CVA7_TKR_2010 GU942820_CVA7_CAN_1958 99 30009_CVA7_RUS_2007 75 80 AY421765_CVA7_USA_1949 99 21364_EV90_RUS_2003 AY697460_EV90_BAN_1999 AY697461_EV91_BAN_2000 AY697459_EV89_BAN_2000 41631_EV76_TJK_2011 AY697458_EV76_FRA_1991 99 41755_EV76_KGZ_2011 99 40961_EV76_TJK_2011 92 JF905564_EV76_CHN_2004 70 37699_CVA2_KAZ_2010 99 42096_CVA2_RUS_2011 93 41963_CVA2_RUS_2011 24004_CVA2_RUS_2005 97 32898_CVA2_RUS_2008 92 40179_CVA2_RUS_2010 99 41149_CVA2_RUS_2011 99 42115_CVA2_RUS_2011 40879_CVA2_RUS_2011 31793_CVA2_UKR_2008 99 96 32007_CVA2_RUS_2008 HQ728259_CVA2_SD_CHN_2009 AY421760_CVA2_USA_1947 70 39633_CVA4_RUS_2010 86 39797_CVA4_RUS_2010 37966_CVA4_RUS_2010 32900_CVA4_RUS_2008 36191_CVA4_RUS_2009 99 36240_CVA4_RUS_2010 92 72 41413_CVA4_RUS_2011 41423_CVA4_RUS_2011 29518_CVA4_RUS_2007 99 94 30482_CVA4_RUS_2007 23187_CVA4_AZE_2004 42351_CVA4_TKM_2011 27203_CVA4_RUS_2006 98 40194_CVA4_RUS_2010 99 40238_CVA4_RUS_2011 99 HQ728260_CVA4_SZ_CHN_2009 AY421762_CVA4_USA_1948 76 28297_CVA6_UZB_2007 40428_CVA6_TKM_2011 99 AY421764_CVA6_USA_1949 41156_CVA6_RUS_2011 40180_CVA6_RUS_2010 98 42593_CVA6_RUS_2011 70 29055_CVA5_RUS_2007 85 30204_CVA5_RUS_2007 99 HQ728261_CVA5_SD_CHN_2009 99 41143_CVA5_RUS_2011 99 AY421763_CVA5_USA_1950 AY421768_CVA12_USA_1948 99 22809_CVA8_RUS_2004 99 24267_CVA8_RUS_2005 71 22468_CVA8_ARM_2004 99 29089_CVA8_TKM_2007 99 AY421766_CVA8_USA_1949 26412_CVA3_KGZ_2006 98 AY421761_CVA3_USA_1948 AY421767_CVA10_USA_1950 99 22205_CVA10_TAJ_2004 22888_CVA10_RUS_2004 40181_CVA10_RUS_2004 99 HQ728262_CVA10_SD_CHN_2009 21462_CVA10_GEO_2003 27190_CVA10_RUS_2006 99 99 31228_CVA10_RUS_2008 34266_CVA10_RUS_2009 99 97 40191_CVA10_RUS_2010 83 36053_CVA10_RUS_2010 38905_CVA10_RUS_2009 97 40184_CVA10_RUS_2010
0.05
81
2C
I
61
30
83
III
IV
II
42266_EV71_RUS_2011 HQ325852_EV71-C4_CHN_2010 EU703812_EV71-C4_CHN_2008 HQ426649_EV71-C4_CHN_2010 41785_EV71_RUS_2011 GQ892830_EV71-C4_CHN_2008 GQ994990_EV71-C4_CHN_2009 HM003207_EV71-C4_CHN_2008 FJ713137_EV71-C4_CHN_2009 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 GQ279370_EV71-C4_CHN_2008 EU864507_EV71-C4_CHN_2008 FJ194965_EV71-C4_CHN_2008 86 GU196833_EV71-C4_CHN_2009 FJ607337_EV71-C4_CHN_2008 HQ129932_EV71-C4_CHN_2006 GQ279369_EV71-C4_CHN_2008 FJ194964_EV71-C4_CHN_2008 FJ357374_EV71-C4_TW_2005 GQ994989_EV71-C4_CHN_2009 HQ188292_EV71-C4_CHN_2008 99 AF302996_EV71-C4_CHN_1998 87 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AY421763_CVA5_USA_1950 30009_CVA7_RUS_2007 DQ341354_EV71-B4_CHN_1998 DQ341368_EV71-B4_MAL_1997 99 AB482183_EV71-B1_JPN_1973 96 HQ189392_EV71-B1_HUN_1978 FJ357382_EV71-B2_TW_1986 99 FJ357384_EV71-B2_TW_1986 94 DQ341365_EV71-B4_MAL_2001 92 FJ357377_EV71-B4_TW_2000 FJ357375_EV71-B4_TW_1999 FJ461781_EV71-B5_CHN_2008 99 DQ341364_EV71-B5_CHN_2000 FJ357378_EV71-B5_TW_2003 73 EU527985_EV71-B5_TW_2007 HM622390_EV71-B5_TW_2009 AY421762_CVA4_USA_1948 U05876_CVA16_SOA_1951 AY421769_CVA14_USA_1950 29256_EV71-C2_RUS_2007 99 28297_CVA6_UZB_2007 34082_CVA7_RUS_2009 72 26412_CVA3_KGZ_2006 36240_CVA4_RUS_2010 29089_CVA8_TKM_2007 40181_CVA10_RUS_2004 84 40180_CVA6_RUS_2010 98 42593_CVA6_RUS_2011 41156_CVA6_RUS_2011 41143_CVA5_RUS_2011 42115_CVA2_RUS_2011 95 37183_CVA7_TKR_2010 40428_CVA6_TKM_2011 42351_CVA4_TKM_2011 22888_CVA10_RUS_2004 24315_CVA14_RUS_2005 22205_CVA10_TAJ_2004 24004_CVA2_RUS_2005 27203_CVA4_RUS_2006 99 22809_CVA8_RUS_2004 24267_CVA8_RUS_2005 99 31793_CVA2_UKR_2008 32007_CVA2_RUS_2008 37699_CVA2_KAZ_2010 73 97 42093_CVA2_RUS_2011 84 41963_CVA2_RUS_2011 41149_CVA2_RUS_2011 79 32898_CVA2_RUS_2008 40194_CVA4_RUS_2010 23187_CVA4_AZE_2004 88 HQ728259_CVA2_SD_CHN_2009 75 40238_CVA4_RUS_2011 89 HQ728260_CVA4_SZ_CHN_2009 99 21462_CVA10_GEO_2003 27190_CVA10_RUS_2006 22468_CVA8_ARM_2004 HM622392_EV71-C2_TW_2008 40179_CVA2_RUS_2010 40879_CVA2_RUS_2011 41413_CVA4_RUS_2011 96 32900_CVA4_RUS_2008 97 37966_CVA4_RUS_2010 78 39633_CVA4_RUS_2010 39797_CVA4_RUS_2010 32896_EV71-C2_2008 FJ172159_EV71-C2_CHN_2008 38555_EV71_KGZ_2010 34020_EV71-C2_RUS_2009 72 40190_EV71-C2_RUS_2010 41464_EV71_RUS_2011 37731_EV71_UKR_2010 92 AF304459_EV71-C2_TW_1998 DQ341357_EV71-C2_AUS_1999 13384_EV71-C2_RUS_2001 DQ381846_EV71-C2_AUS_1999 DQ341356_EV71-C3_KOR_2000 DQ341359_EV71-C1_MAL_1998 DQ452074_EV71-C1_NOR_2003 77 85 27578_EV71-C1_RUS_2007 81 EU414335_EV71-C1_CHN_2006 DQ341358_EV71-C1_MAL_2000 98 DQ341361_EV71-C1_AUS_2000 EU527983_EV71-C5_TW_2007 AY421766_CVA8_USA_1949 21364_EV90_RUS_2003 AY697460_EV90_BAN_1999 99 AY697461_EV91_BAN_2000 75 AY697459_EV89_BAN_2000 AY697458_EV76_FRA_1991 99 74 40961_TJK_2011 JF905564_EV76_04360/SD_CHN_2004 41631_EV76_TJK_2011 41755_EV76_KGZ_2011 98 36053_CVA10_RUS_2010 82 40191_CVA10_RUS_2010 38905_CVA10_RUS_2009 98 77 40184_CVA10_RUS_2010 95 34266_CVA10_RUS_2009 31228_CVA10_RUS_2008 95 71 36191_CVA4_RUS_2009 41423_CVA4_RUS_2011 29518_CVA4_RUS_2007 99 30482_CVA4_RUS_2008 GU942820_CVA7_CAN_1958 HQ728261_CVA5_SD_CHN_2009 29055_CVA5_RUS_2007 99 83 30204_CVA5_RUS_2007 HQ728262_CVA10_SD_CHN_2009 AY421760_CVA2_USA_1947 59 AY421761_CVA3_USA_1948 AY421764_CVA6_USA_1949 AY421767_CVA10_USA_1950 99 U22521_EV71-A_BrCr_USA_1969 AY421768_CVA12_USA_1948 AY421765_CVA7_USA_1949 31758_CVA16_RUS_2008 99 34351_CVA16_RUS_2009 39156_CVA16_RUS_2010 34950_CVA16_RUS_2009 99 38394_CVA16_RUS_2010 42234_CVA16_RUS_2011 98 41555_CVA16_KAZ_2011 99 GQ279371_CVA16_CHN_2008 84 JN590244_CVA16_CHN_2010 29072_CVA16_RUS_2007 FJ198212_CVA16_CHN_2008 99 37323_CVA16_UKR_2010 GQ279368_CVA16_CHN_2008 40188_CVA16_RUS_2004 AF177911_CVA16_TW_1998 60
90
0.05
87
la
99
3D
99
81
72
99
90
99
III
IV
II
41785_EV71_RUS_2011 HQ426649_EV71-C4_CHN_2010 FJ194965_EV71-C4_CHN_2008 HQ325852_EV71-C4_CHN_2010 42266_EV71_RUS_2011 EU703812_EV71-C4_CHN_2008 GQ892830_EV71-C4_CHN_2008 GQ994990_EV71-C4_CHN_2009 80 EU864507_EV71-C4_CHN_2008 GQ279370_EV71-C4_CHN_2008 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 FJ357374_EV71-C4_TW_2005 FJ713137_EV71-C4_CHN_2009 HM003207_EV71-C4_CHN_2008 GQ279369_EV71-C4_CHN_2008 GU196833_EV71-C4_CHN_2009 99 HQ129932_EV71-C4_CHN_2006 FJ194964_EV71-C4_CHN_2008 GQ994989_EV71-C4_CHN_2009 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AF302996_EV71-C4_CHN_1998 HQ188292_EV71-C4_CHN_2008 U05876_CVA16_SOA_1951 AY421762_CVA4_USA_1948 AY421769_CVA14_USA_1950 94 32898_CVA2_RUS_2008 24004_CVA2_RUS_2005 29256_EV71-C2_RUS_2007 99 22888_CVA10_RUS_2004 26412_CVA3_KGZ_2006 DQ341354_EV71-B4_CHN_1998 96 99 DQ341368_EV71-B4_MAL_1997 22468_CVA8_ARM_2004 41156_CVA6_RUS_2011 31793_CVA2_UKR_2008 40879_CVA2_RUS_2011 99 27190_CVA10_RUS_2006 32007_CVA2_RUS_2008 HM622392_EV71-C2_TW_2008 41413_CVA4_RUS_2011 32900_CVA4_RUS_2008 99 37966_CVA4_RUS_2010 91 99 39633_CVA4_RUS_2010 87 39797_CVA4_RUS_2010 99 40238_CVA4_RUS_2011 HQ728260_CVA4_SZ_CHN_2009 HQ728259_CVA2_SD_CHN_2009 30009_CVA7_2007 FJ607337_EV71-C4_CHN_2008 40181_CVA10_RUS_2004 42593_CVA6_RUS_2011 40180_CVA6_RUS_2010 24315_CVA14_RUS_2005 42351_CVA4_TKM_2011 36240_CVA4_RUS_2010 40179_CVA2_RUS_2010 93 29089_CVA8_TKM_2007 42096_CVA2_RUS_2011 24267_CVA8_RUS_2005 22205_CVA10_TAJ_2004 40194_CVA4_RUS_2010 88 23187_CVA4_AZE_2004 37699_CVA2_KAZ_2010 27203_CVA4_RUS_2006 79 28297_CVA6_UZB_2007 96 34082_CVA7_RUS_2009 22809_CVA8_RUS_2004 40428_CVA6_TKM_2011 87 42115_CVA2_RUS_2011 41149_CVA2_RUS_2011 99 41963_CVA2_RUS_2011 37183_CVA7_TKR_2010 41143_CVA5_RUS_2011 98 34020_EV71-C2_RUS_2009 41464_EV71_RUS_2011 38555_EV71_KGZ_2010 98 32896_EV71-C2_2009 FJ172159_EV71-C2_CHN_2008 40190_EV71_RUS_2010 90 37731_EV71_UKR_2010 AF304459_EV71-C2_TW_1998 13384_EV71-C2_RUS_2001 DQ341357_EV71-C2_AUS_1999 DQ381846_EV71-C2_AUS_1999 99 DQ341356_EV71-C3_KOR_2000 DQ341359_EV71-C1_MAL_1998 27578_EV71-C1_RUS_2007 93 99 DQ341358_EV71-C1_MAL_2000 90 EU414335_EV71-C1_CHN_2006 70 DQ341361_EV71-C1_AUS_2000 DQ452074_EV71-C1_NOR_2003 AY421766_CVA8_USA_1949 EU527983_EV71-C5_TW_2007 AY421765_CVA7_USA_1949 99 EU527985_EV71-B5_TW_2007 HM622390_EV71-B5_TW_2009 76 FJ461781_EV71-B5_CHN_2008 FJ357378_EV71-B5_TW_2003 DQ341364_EV71-B5_CHN_2000 FJ357375_EV71-B4_TW_1999 99 DQ341365_EV71-B4_MAL_2001 89 FJ357377_EV71-B4_TW_2000 94 AB482183_EV71-B1_JPN_1973 HQ189392_EV71-B1_HUN_1978 99 FJ357382_EV71-B2_TW_1986 FJ357384_EV71-B2_TW_1986 99 AY421763_CVA5_USA_1950 HQ728262_CVA10_SD_CHN_2009 HQ728261_CVA5_SD_CHN_2009 93 41423_CVA4_RUS_2011 99 29055_CVA5_RUS_2007 86 21462_CVA10_2003 30204_CVA5_RUS_2007 34950_CVA16_RUS_2009 41555_CVA16_KAZ_2011 99 38394_CVA16_RUS_2010 42234_CVA16_RUS_2011 31758_CVA16_RUS_2008 34351_CVA16_RUS_2009 99 39156_CVA16_RUS_2010 FJ198212_CVA16_CHN_2008 40188_CVA16_RUS_2004 85 AF177911_CVA16_TW_1998 29072_CVA16_RUS_2007 99 GQ279371_CVA16_CHN_2008 74 JN590244_CVA16_CHN_2010 99 37323_CVA16_UKR_2010 GQ279368_CVA16_CHN_2008 93 86 AY421760_CVA2_USA_1947 76 AY421767_CVA10_USA_1950 AY421764_CVA6_USA_1949 AY421768_CVA12_USA_1948 88 AY421761_CVA3_USA_1948 U22521_EV71-A_BrCr_USA_1969 GU942820_CVA7_CAN_1958 99 29518_CVA4_RUS_2007 99 30482_CVA4_RUS_2007 31228_CVA10_RUS_2008 99 36191_CVA4_RUS_2009 93 34266_CVA10_RUS_2009 36053_CVA10_RUS_2010 99 40191_CVA10_RUS_2010 86 38905_CVA10_RUS_2009 95 40184_CVA10_RUS_2010 AY697458_EV76_FRA_1991 21364_EV90_RUS_2003 99 AY697459_EV89_BAN_2000 93 AY697460_EV90_BAN_1999 83 AY697461_EV91_BAN_2000 40961_EV76_TJK_2011 71 JF905564_EV76_SD_CHN_2004 41631_EV76_TJK_2011 41755_EV76_KGZ_2011 98
A. N. Lukashev and others
Fig. 1. Phylogenetic trees of HEV-A in three genome regions. Strains were colour-coded according to their type. Scale bars indicate maximum likelihood distances. Numbers at tree nodes indicate bootstrap support values. Roman numbers indicate groups referred to in the text.
Journal of General Virology 95
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Table 1. Features of conserved HEV-A groups Group II III IV –
Serotypes
Number of members in VP1/2C/3D
Median non-recombinant virus age from node
EV71 genotypes B4–B5 EV71 subtypes C1–C3 EV71 subtype C4 CVA2, 4, 10 (7 conserved groups)
10/8/8 20/18/18 25/25/24 22/22/22
13 18.5 16 3.5
Group tMRCA in VP1 (years)* 22 29 19 3.5
*Time of tMRCA was calculated as node height minus age of the most recent isolate of a group.
strains as opposed to the globally representative sampling of EV71. Therefore, it is not possible to conclude if this serotype is indeed less involved in recombination within HEV-A, especially as diverse recombinant forms of CVA16 were described in the study by McWilliam Leitch et al. (2012). In accordance with earlier reports on genetic identity of novel HEV-A serotypes (Oberste et al., 2005), three isolates of EV76 and one of EV90 did not have signs of recombination with classical HEV-A serotypes. However, these viruses displayed multiple phylogenetic conflicts involving EV76, EV89, EV90 and EV91, supporting that these types might represent a subspecies of HEV-A. All other coxsackievirus A types, except for CVA16, were highly involved into recombination within the global gene pool. Bayesian dating was done for three types with significant sampling, namely CVA2 (13 viruses), CVA4 (17 viruses) and CVA10 (13 viruses). The median age of tree nodes that were conserved across the three genome regions (reliably supported by bootstrap values above 70 in one genome region and intact, but not necessarily well supported, in another region) was just 3.6 years, and never more than 8.7 years, which is several-fold less than the age of non-recombinant EV71 groups that had tMRCAs of 19– 29 years. The median age of non-recombinant isolates was 3.5 years, and never exceeded 9 years. To further illustrate two distinct recombination patterns in EV71, CVA16 and other coxsackieviruses, pairwise genetic distances in three genome regions were plotted separately within each of these two groups (Fig. 2). In EV71 and CVA16, the pairwise distances in three genome regions correlated very well up to a genetic distance of about 0.14 (Fig. 2a, region a), indicating long-term circulation of viruses in the absence of recombination. There were discordant pairwise distances (low in VP1 and high in 2C and 3D, Fig. 2a, b region) that corresponded to the few EV71 strains that acquired an ‘alien’ 2C and 3D genome region (see above). There were no viruses with low pairwise distances in 2C/3D and divergent VP1 genome regions (Fig. 2a, c region), which agreed with the absence of such viruses on phylogenetic trees. The coxsackievirus group also included viruses without apparent signs of recombination (with concordant pairwise http://vir.sgmjournals.org
distances), but only with genetic distances up to approximately 0.07 (Fig. 2b, a region). Recombination events apparent as discordant genetic distances beginning from as low as 0.01 involved all genome regions (Fig. 2b, regions b, c). In viruses that differed by over 0.07 of their RNA sequence in any genome region, pairwise sequence distances were largely discordant between all genome regions,
(a) 0.30
β
δ
0.20
α
0.10
γ 0.10 (b)
0.30
0.20
β
0.30
0.40
0.50
δ
0.20
0.10
α
γ
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0.20
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0.50
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Fig. 2. Plot of pairwise distances among EV71 and CVA16 (a) and among other coxsackieviruses of species HEV-A (b) in three genome regions. Each point represents a pair of pairwise distances between the same strains in two genome regions that correspond to the symbol description. The uncorrected nucleotide sequence distance in the first genome region is plotted on the x-axis and that in the second region of the pair on the y-axis. Circled areas are described in the text. 871
A. N. Lukashev and others
indicating common shuffling of genome fragments by recombination (Fig. 2b, d region). This observation corresponds well with the maximum age of conserved tree nodes for coxsackieviruses (8.7 years) multiplied by the estimated substitution rates (5.561023–14.161023 substitutions per site year21). The number of conserved groups was the highest between the VP1 and 2C genome regions (14 groups), and about the same between the VP1 and 3D, and the 2C and 3D regions (seven and eight groups, respectively), while the distance between these genome regions is about equal (750 nt between VP1 and 2C and 870 nt between 2C and 3D). This observation contradicts the reports of an apparent recombination hot-spot on the border of structural and non-structural genes in enteroviruses and other picornaviruses (Benschop et al., 2010; Heath et al., 2006; Lukashev, 2005, 2010). It is likely that the apparent recombination hot-spot reported previously at the VP1– 2A junction may only correspond to an artefact due to a stronger phylogenetic signal in the P1 genome region. This strong signal would result from the absence of recombination between capsid genes of different serotypes, higher variability of the structural genome region and, consequently, a better-resolved phylogeny. As a result, it is easier to detect a switch of phylogeny on the border of this region. The non-structural genes are arbitrarily shuffled between serotypes every few years, so the phylogenetic signal is quickly degraded and fewer recombination events can be detected with sufficient statistical support. Before this study, EV71 comprised over 80 % of all HEV-A complete genomic sequences in GenBank and was the sole serotype used in many studies. Recombination was implied by the emergence of several EV71 subtypes (McWilliam Leitch et al., 2012; Yoke-Fun & AbuBakar, 2006). The recombination partners in these recombination events remained unknown because there were no available sequences of contemporary HEV-A strains. Our study indicates that the recombination events leading to the emergence of EV71 subtypes involved members of the global HEV-A gene pool and not only EV71. This is evident because the three EV71 groups II–IV were evenly interspersed on the phylogenetic trees for the 2C and 3D genome regions and did not originate from a shared common ancestor. Surprisingly, EV71 subtypes almost ceased recombining with the rest of the HEV-A gene pool upon their emergence. Perfect correspondence of EV71 phylogenies in different genome regions was reported earlier (Bible et al., 2008; McWilliam Leitch et al., 2012; van der Sanden et al., 2011). This study highlights the degree of genetic conservation in EV71, which apparently recombines about five times less frequently than its peers in the HEV-A species CVA2, CVA4 and CVA10. Moreover, the high half-life of non-recombinant EV71 strains indicates that they are the most conserved types in terms of recombination frequency among enteroviruses that have been closely studied so far. 872
Several factors could explain the low apparent recombination in EV71. First, this could be due to reproductive isolation at the level of permissible/preferred cell types. However, EV71 and some other HEV-A members share the same putative cellular receptors PSGL-1 and SCARB2 (Nishimura & Shimizu, 2012). Also, use of different receptors by HEV-B serotypes (Racaniello, 2007) did not hamper promiscuous recombination (Lukashev et al., 2003; Simmonds & Welch, 2006), and poliovirus recombines freely with other HEV-C members that do not use the poliovirus receptor (Brown et al., 2003; Jegouic et al., 2009). Another factor that could contribute to the low recombination rate in EV71 could be adaptation of nonstructural genes to the EV71 capsid. Such adaptation of 2C and VP3 proteins was described in HEV-C species (Liu et al., 2010). Yet another possibility is that the lower substitution rate in EV71 (which may result from either the polymerase error rate or a less tight bottleneck at each infection cycle) does not require such frequent recombination to recover deleterious genomes that are constantly generated within a quasispecies. As a result, EV71 can ‘afford’ less frequent recombination within a quasispecies, which also results in lower frequency of recombination with coxsackieviruses. Finally, the long apparent circulation of non-recombinant EV71 variants can be the result of less pronounced expansion and extinction cycles in this type. It has been noted that the apparent recombination rate is lower in the ‘endemic’ echovirus 11 than in ‘epidemic’ echoviruses 9 and 30 (McWilliam Leitch et al., 2010). EV71 is an ‘endemic’ type as revealed by the annual isolation pattern (Khetsuriani et al., 2006), which corresponds well to a low apparent recombination rate. Paradoxically, EV71, which causes the most clinically relevant epidemics, displays the recombination phenotype of an ‘endemic’ enterovirus.
Acknowledgements This study was supported by the Global Polio Eradication Initiative of the WHO through the WHO European Office and by the Government of the Russian Federation by the budgetary allocations for the implementation of activities under the Global Polio Eradication Initiative (Government act 1771-p of 14.10.2010). We would like to thank the staff of the following regional laboratories for technical assistance in collection of stool samples and virus isolation: Centres of Hygiene and Epidemiology in Moscow, Omsk region, Stavropol region; Regional Centre of Epidemiological Surveillance for Poliomyelitis, Pasteur Institute, St Petersburg; National Poliomyelitis Laboratories in Armenia, Georgia, Kazakhstan and Ukraine. The study was partially funded by the EU FP7 project EVA (contract number 228292) and by infrastructural support from the German Centre for Infection Research (DZIF).
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Short Communication
DOI 10.1099/vir.0.060004-0
Recombination strategies and evolutionary dynamics of the Human enterovirus A global gene pool Alexander N. Lukashev,1 Elena Yu. Shumilina,1 Ilya S. Belalov,1 Olga E. Ivanova,1 Tatiana P. Eremeeva,1 Vadim I. Reznik,2 O. E. Trotsenko,3 Jan Felix Drexler4 and Christian Drosten4
Correspondence
1
Alexander N. Lukashev
2
[email protected]
Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia Center of Hygiene and Epidemiology in Khabarovsk Region, Khabarovsk, Russia
3
Khabarovsk Institute of Epidemiology and Microbiology, Khabarovsk, Russia
4
Institute of Virology, University of Bonn Medical Centre, Bonn, Germany
Received 1 October 2013 Accepted 14 January 2014
We analysed natural recombination in 79 Human enterovirus A strains representing 13 serotypes by sequencing of VP1, 2C and 3D genome regions. The half-life of a non-recombinant tree node in coxsackieviruses 2, 4 and 10 was only 3.5 years, and never more than 9 years. All coxsackieviruses that differed by more than 7 % of the nucleotide sequence in any genome region were recombinants relative to each other. Enterovirus 71 (EV71), on the contrary, displayed remarkable genetic stability. Three major EV71 clades were stable for 19–29 years, with a half-life of non-recombinant viruses between 13 and 18.5 years in different clades. Only five EV71 strains out of over 150 recently acquired non-structural genome regions from coxsackieviruses, while none of 80 contemporary coxsackieviruses had non-structural genes transferred from the three EV71 clades. In contrast to earlier observations, recombination between VP1 and 2C genome regions was not more frequent than between 2C and 3D regions.
Human enteroviruses are members of the genus Enterovirus within the family Picornaviridae. These small, non-enveloped RNA viruses have a single-stranded non-segmented genome of positive polarity of about 7500 nt. The genome encodes a single large polyprotein comprising four structural proteins, VP1–VP4 (collectively termed the structural genome region), and seven non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C and 3D (the non-structural genome region) (Racaniello, 2007). Human enteroviruses comprise four species, Human enterovirus A–D (HEV-A–D). Enterovirus 71 (EV71) is the best studied non-polio enterovirus, and it was further classified into subtypes A, B0–B5, C1–C5 and D (Brown et al., 1999; McMinn, 2012). Enteroviruses are highly prevalent in the human population, especially in infants (Witsø et al., 2006). Infection with HEVs is usually subclinical, but can occasionally result in single cases and outbreaks of meningitis, sepsis-like infection and myocarditis. Infection with the three poliovirus serotypes (members of the HEV-C species) and EV71 can produce severe neurological lesions (Pallansch & Roos, 2007). Global GenBank accession numbers for identified sequences are KC879327– KC879556. Two supplementary tables are available with the online version of this paper.
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prevalence of enteroviruses and high infection rates in infants greatly facilitate natural recombination. Recombination is apparently most prevalent in the HEV-B species (Simmonds & Welch, 2006). However, it is common also in HEV-A (Huang et al., 2009; Oberste et al., 2004; Simmonds & Welch, 2006) and HEV-C (Brown et al., 2003). As a result, enterovirus species exist as highly dynamic global gene pools (reviewed by Lukashev, 2005). It has been shown that the half-life of recombinant forms (combinations of discrete VP1 and 3D genes without further signs of recombination) in circulation may vary from 1.3 to 9.8 years between different HEV-B serotypes (McWilliam Leitch et al., 2010). Recombination in HEV-A was investigated in a number of studies, but these were confined by limited geographical coverage (Simmonds & Welch, 2006), sampling restricted to a few prototype strains (Oberste et al., 2004), or by analysing only the neurovirulent EV71, but no other contemporary HEV-A (Chen et al., 2010; Huang et al., 2008; Mirand et al., 2010; van der Sanden et al., 2011). The aim of this study was to determine recombination patterns in HEV-A on a whole species level with an extensive geographical and temporal coverage. Seventy-eight HEV-A strains were isolated and identified according to a standard World Health Organization (WHO) protocol (WHO, 1992) in RD cell culture in the course of the WHO polio surveillance programme and enterovirus 060004 G 2014 SGM Printed in Great Britain
Recombination in Enterovirus A
surveillance in Russia and New Independent States in 2001– 2011 (Table S1, available in the online Supplementary Material). According to the national regulations, informed consent is not required for anonymized surveillance studies. Three genome regions were amplified by PCR, namely the complete VP1 and partial 2C and 3D. PCR primers are listed in Table S2. All viruses used for the study differed by at least 1 % nucleotide sequence in their VP1 genome region; less divergent sequences were excluded upon preliminary analysis. GenBank accession numbers for sequences obtained here are KC879327–KC879556. All complete or nearly complete HEV-A sequences available in GenBank were aligned using CLUSTAL W (Thompson et al., 1994). Redundant sequences sharing more than 97 % overall nucleotide sequence identity were then omitted, yielding 76 unique reference sequences. Sequence handling was performed with BioEdit v.7.0.5.2 software (Hall, 1999). Phylogenies were calculated using a maximum-likelihood tree algorithm with the Tajima–Nei substitution model in the MEGA 5.2 software package (Tamura et al., 2011). In the VP1 genome region (nt 2439–3329 in the prototype EV71 strain BrCr, GenBank accession number U22521), phylogenetic grouping expectedly corresponded to serotypes (Fig. 1). A Bayesian likelihood-based algorithm implemented in BEAST version 1.7.4 (Drummond & Rambaut, 2007) was then used to date tree nodes that were conserved across all three genome regions. Bayesian molecular dating was performed separately for distinct serotypes, because analysis of the VP1 alignment that included multiple serotypes consistently yielded substitution rates incompatible with those reported in other studies and those resulting from analysis of individual serotypes (data not shown). The SRD06 substitution model optimized for coding sequences (Shapiro et al., 2006) was used with a lognormal relaxed clock setting, which was preferred over assumption of a strict clock upon Bayes factor testing (log10 Bayes factor of tree likelihood .10), while the exponential clock setting was not significantly superior to the lognormal clock. Each analysis was run over 10 000 000 generations, and trees were sampled every 1000 generations, resulting in 10 000 trees. Trees were annotated with TreeAnnotator v.1.4.8 using a burn-in of 2000 trees. Substitution rates in different serotypes (expressed as 1023) were 4.05 (95 % confidence interval 3.23–4.93) substitutions per site year21 in EV71, 8.32 (5.35–11.6) in coxsackievirus A (CVA)2, 5.53 (3.57–7.57) in CVA4 and 14.1 (4.01– 24.2) in CVA10. The substitution rate for EV71 was remarkably similar to previously published estimates of 4.2–4.6 (Tee et al., 2010) and 3.7 (Mirand et al., 2010), but differed from the substitution rate of 7.2 reported in another work (McWilliam Leitch et al., 2012). Importantly, in the latter work, lower substitution rates were observed within individual EV71 subgenotypes (3.2–7.3), implying that the overall rate of 7.2 may be inaccurate. While there have been no published studies of substitution rates in other HEV-A types, the observed variation is compatible with the range of substitution rates in HEV-B serotypes (McWilliam http://vir.sgmjournals.org
Leitch et al., 2010). The time of the most recent common ancestor (tMRCA) of coxsackievirus serotypes (CVA2, 69 years; CVA4, 66 years; CVA10, 72 years) was lower than tMRCA of EV71 (91 years); therefore, EV71 may be a cause of an emerging infection, but according to the phylogenetic evidence, it cannot be deemed an emerging virus. In the 2C (nt 4350–4766) and 3D (nt 5973–6602) genome regions, distribution of pairwise distances was relatively even, and did not allow assigning recombinant forms as was done earlier (McWilliam Leitch et al., 2012). Phylogenetic grouping generally did not correspond to the VP1 genome region (Fig. 1), implying common recombination. All coxsackieviruses except for type 16 were extensively shuffled in the non-structural genome regions. This kind of phylogenetic relationship is best exemplified by group I (Fig. 1) consisting of 23 viruses of 11 serotypes in the 2C genome region or group Ia that comprised 29 viruses of nine serotypes in the 3D genome region. The other extremity of recombination frequency was represented by EV71 and CVA16. In the 2C and 3D genome regions, most EV71 strains fell into three groups (groups II–IV, Table 1, Fig. 1), which corresponded to subgenotypes B4–B5, C1– C3 and C4, respectively. While grouping of all EV71 B subgenotypes was conserved throughout the genome in our study, it is known that recombination occurred within that group; therefore we regarded only the group comprising subgenotypes B4 and B5, which was also conserved in previous studies with a more extensive sampling of EV71 (McWilliam Leitch et al., 2012). There were few EV71 sequences with alternative 2C (four out of 48 sequences, two of B4 and two of C2 genotype) and 3D genome regions (five sequences, the four mentioned above and one EV71C4). However, the three EV71 groups present in the 2C and 3D genome regions (a total of 51 and 50 sequences, respectively) did not contain a single sequence from another serotype. Within these EV71 groups, there was also no significant evidence of recombination (change of tree topology supported by bootstrap values over 70) between the three genome regions. Similarly, all 14 CVA16 isolates sequenced here formed a single phylogenetic group without signs of recombination between VP1, 2C and 3D genome regions (Fig. 1). These three groups, which were characterized by limited recombination, had a tMRCA of around two decades. Median ages of non-recombinant viruses from the root of their respective group (roughly equivalent to half-lives of non-recombinant viruses reported by McWilliam Leitch et al., 2012) were between 13 and 18.5 years (Table 1). These numbers make EV71 the least recombining enterovirus compared with echovirus 9 (E9), E30 and E11 that featured recombinant form half-lives of 1.3, 3.1 and 9.8 years, respectively (McWilliam Leitch et al., 2010). The difference from numbers reported for EV71 by McWilliam Leitch et al. (2012) (6 and 9 years for GtB and GtC, respectively) may be explained by the aforementioned uncertainty of the substitution rate and by limited sampling of GtB sequences (by either quantity or geographical coverage). The sampling of CVA16 was confined to Russian 869
93
870
80
99
0.1
89
67
96
60
99
VP1
III
IV
II
EU703812_EV71-C4_CHN_2008 HQ426649_EV71-C4_CHN_2010 GQ994990_EV71-C4_CHN_2009 GQ892830_EV71-C4_CHN_2008 41785_EV71_RUS_2011 42266_EV71_RUS_2011 HQ325852_EV71-C4 CHN_2010 EU864507_EV71-C4_CHN_2008 GQ279370_EV71-C4_CHN_2008 95 FJ194965_EV71-C4_CHN_2008 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 FJ713137_EV71-C4_CHN_2009 81 HM003207_EV71-C4_CHN_2008 GQ279369_EV71-C4_CHN_2008 FJ607337_EV71-C4_CHN_2008 GU196833_EV71-C4_CHN_2009 HQ129932_EV71-C4_CHN_2006 75 FJ194964_EV71-C4_CHN_2008 80 GQ994989_EV71-C4_CHN_2009 FJ357374_EV71-C4_TW_2005 86 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AF302996_EV71-C4_CHN_1998 90 HQ188292_EV71-C4_CHN_2008 95 DQ341361_EV71-C1_AUS_2000 79 99 EU414335_EV71-C1_CHN_2006 DQ452074_EV71-C1_NOR_2003 98 DQ341358_EV71-C1_MAL_2000 91 27578_EV71-C1_RUS_2007 DQ341359_EV71-C1_MAL_1998 EU527983_EV71-C5_TW_2007 89 DQ341356_EV71-C3_KOR_2000 AF304459_EV71-C2_TW_1998 13384_EV71-C2_RUS_2001 DQ381846_EV71-C2_AUS_1999 97 HM622392_EV71-C2_TW_2008 29256_EV71-C2_RUS_2007 DQ341357_EV71-C2_AUS_1999 37731_EV71_UKR_2010 40190_EV71_RUS_2010 FJ172159_EV71-C2_CHN_2008 97 32896_EV71-C2_RUS_2008 86 38555_EV71_KGZ_2010 34020_EV71-C2_RUS_2009 99 41464_EV71_RUS_2011 71 AB482183_EV71-B1_JPN_1973 98 HQ189392_EV71-B1_HUN_1978 FJ357382_EV71-B2_TW_1986 99 99 FJ357384_EV71-B2_TW_1986 99 DQ341354_EV71-B4_CHN_1998 99 DQ341368_EV71-B4_MAL_1997 96 84 DQ341365_EV71-B4_MAL_2001 FJ357377_EV71-B4_TW_2000 FJ357375_EV71-B4_TW_1999 DQ341364_EV71-B5_CHN_2000 FJ357378_EV71-B5_TW_2003 97 FJ461781_EV71-B5_CHN_2008 94 EU527985_EV71-B5_TW_2007 89 HM622390_EV71-B5_TW_2009 97 U22521_EV71-A_BrCr_USA_1969 U05876_CVA16_SOA_1951 34950_CVA16_RUS_2009 41555_CVA16_KAZ_2011 99 38394_CVA16_RUS_2010 99 42234_CVA16_RUS_2011 37323_CVA16_UKR_2010 99 GQ279368_CVA16_CHN_2008 99 89 40188_CVA16_RUS_2004 AF177911_CVA16_TW_1998 FJ198212_CVA16_CHN_2008 99 GQ279371_CVA16_CHN_2008 97 JN590244_CVA16_CHN_2010 29072_CVA16_RUS_2007 31758_CVA16_RUS_2008 34351_CVA16_RUS_2009 99 94 39156_CVA16_RUS_2010 99 24315_CVA14_RUS_2005 AY421769_CVA14_USA_1950 99 34082_CVA7_RUS_2009 37183_CVA7_TKR_2010 GU942820_CVA7_CAN_1958 99 30009_CVA7_RUS_2007 75 80 AY421765_CVA7_USA_1949 99 21364_EV90_RUS_2003 AY697460_EV90_BAN_1999 AY697461_EV91_BAN_2000 AY697459_EV89_BAN_2000 41631_EV76_TJK_2011 AY697458_EV76_FRA_1991 99 41755_EV76_KGZ_2011 99 40961_EV76_TJK_2011 92 JF905564_EV76_CHN_2004 70 37699_CVA2_KAZ_2010 99 42096_CVA2_RUS_2011 93 41963_CVA2_RUS_2011 24004_CVA2_RUS_2005 97 32898_CVA2_RUS_2008 92 40179_CVA2_RUS_2010 99 41149_CVA2_RUS_2011 99 42115_CVA2_RUS_2011 40879_CVA2_RUS_2011 31793_CVA2_UKR_2008 99 96 32007_CVA2_RUS_2008 HQ728259_CVA2_SD_CHN_2009 AY421760_CVA2_USA_1947 70 39633_CVA4_RUS_2010 86 39797_CVA4_RUS_2010 37966_CVA4_RUS_2010 32900_CVA4_RUS_2008 36191_CVA4_RUS_2009 99 36240_CVA4_RUS_2010 92 72 41413_CVA4_RUS_2011 41423_CVA4_RUS_2011 29518_CVA4_RUS_2007 99 94 30482_CVA4_RUS_2007 23187_CVA4_AZE_2004 42351_CVA4_TKM_2011 27203_CVA4_RUS_2006 98 40194_CVA4_RUS_2010 99 40238_CVA4_RUS_2011 99 HQ728260_CVA4_SZ_CHN_2009 AY421762_CVA4_USA_1948 76 28297_CVA6_UZB_2007 40428_CVA6_TKM_2011 99 AY421764_CVA6_USA_1949 41156_CVA6_RUS_2011 40180_CVA6_RUS_2010 98 42593_CVA6_RUS_2011 70 29055_CVA5_RUS_2007 85 30204_CVA5_RUS_2007 99 HQ728261_CVA5_SD_CHN_2009 99 41143_CVA5_RUS_2011 99 AY421763_CVA5_USA_1950 AY421768_CVA12_USA_1948 99 22809_CVA8_RUS_2004 99 24267_CVA8_RUS_2005 71 22468_CVA8_ARM_2004 99 29089_CVA8_TKM_2007 99 AY421766_CVA8_USA_1949 26412_CVA3_KGZ_2006 98 AY421761_CVA3_USA_1948 AY421767_CVA10_USA_1950 99 22205_CVA10_TAJ_2004 22888_CVA10_RUS_2004 40181_CVA10_RUS_2004 99 HQ728262_CVA10_SD_CHN_2009 21462_CVA10_GEO_2003 27190_CVA10_RUS_2006 99 99 31228_CVA10_RUS_2008 34266_CVA10_RUS_2009 99 97 40191_CVA10_RUS_2010 83 36053_CVA10_RUS_2010 38905_CVA10_RUS_2009 97 40184_CVA10_RUS_2010
0.05
81
2C
I
61
30
83
III
IV
II
42266_EV71_RUS_2011 HQ325852_EV71-C4_CHN_2010 EU703812_EV71-C4_CHN_2008 HQ426649_EV71-C4_CHN_2010 41785_EV71_RUS_2011 GQ892830_EV71-C4_CHN_2008 GQ994990_EV71-C4_CHN_2009 HM003207_EV71-C4_CHN_2008 FJ713137_EV71-C4_CHN_2009 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 GQ279370_EV71-C4_CHN_2008 EU864507_EV71-C4_CHN_2008 FJ194965_EV71-C4_CHN_2008 86 GU196833_EV71-C4_CHN_2009 FJ607337_EV71-C4_CHN_2008 HQ129932_EV71-C4_CHN_2006 GQ279369_EV71-C4_CHN_2008 FJ194964_EV71-C4_CHN_2008 FJ357374_EV71-C4_TW_2005 GQ994989_EV71-C4_CHN_2009 HQ188292_EV71-C4_CHN_2008 99 AF302996_EV71-C4_CHN_1998 87 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AY421763_CVA5_USA_1950 30009_CVA7_RUS_2007 DQ341354_EV71-B4_CHN_1998 DQ341368_EV71-B4_MAL_1997 99 AB482183_EV71-B1_JPN_1973 96 HQ189392_EV71-B1_HUN_1978 FJ357382_EV71-B2_TW_1986 99 FJ357384_EV71-B2_TW_1986 94 DQ341365_EV71-B4_MAL_2001 92 FJ357377_EV71-B4_TW_2000 FJ357375_EV71-B4_TW_1999 FJ461781_EV71-B5_CHN_2008 99 DQ341364_EV71-B5_CHN_2000 FJ357378_EV71-B5_TW_2003 73 EU527985_EV71-B5_TW_2007 HM622390_EV71-B5_TW_2009 AY421762_CVA4_USA_1948 U05876_CVA16_SOA_1951 AY421769_CVA14_USA_1950 29256_EV71-C2_RUS_2007 99 28297_CVA6_UZB_2007 34082_CVA7_RUS_2009 72 26412_CVA3_KGZ_2006 36240_CVA4_RUS_2010 29089_CVA8_TKM_2007 40181_CVA10_RUS_2004 84 40180_CVA6_RUS_2010 98 42593_CVA6_RUS_2011 41156_CVA6_RUS_2011 41143_CVA5_RUS_2011 42115_CVA2_RUS_2011 95 37183_CVA7_TKR_2010 40428_CVA6_TKM_2011 42351_CVA4_TKM_2011 22888_CVA10_RUS_2004 24315_CVA14_RUS_2005 22205_CVA10_TAJ_2004 24004_CVA2_RUS_2005 27203_CVA4_RUS_2006 99 22809_CVA8_RUS_2004 24267_CVA8_RUS_2005 99 31793_CVA2_UKR_2008 32007_CVA2_RUS_2008 37699_CVA2_KAZ_2010 73 97 42093_CVA2_RUS_2011 84 41963_CVA2_RUS_2011 41149_CVA2_RUS_2011 79 32898_CVA2_RUS_2008 40194_CVA4_RUS_2010 23187_CVA4_AZE_2004 88 HQ728259_CVA2_SD_CHN_2009 75 40238_CVA4_RUS_2011 89 HQ728260_CVA4_SZ_CHN_2009 99 21462_CVA10_GEO_2003 27190_CVA10_RUS_2006 22468_CVA8_ARM_2004 HM622392_EV71-C2_TW_2008 40179_CVA2_RUS_2010 40879_CVA2_RUS_2011 41413_CVA4_RUS_2011 96 32900_CVA4_RUS_2008 97 37966_CVA4_RUS_2010 78 39633_CVA4_RUS_2010 39797_CVA4_RUS_2010 32896_EV71-C2_2008 FJ172159_EV71-C2_CHN_2008 38555_EV71_KGZ_2010 34020_EV71-C2_RUS_2009 72 40190_EV71-C2_RUS_2010 41464_EV71_RUS_2011 37731_EV71_UKR_2010 92 AF304459_EV71-C2_TW_1998 DQ341357_EV71-C2_AUS_1999 13384_EV71-C2_RUS_2001 DQ381846_EV71-C2_AUS_1999 DQ341356_EV71-C3_KOR_2000 DQ341359_EV71-C1_MAL_1998 DQ452074_EV71-C1_NOR_2003 77 85 27578_EV71-C1_RUS_2007 81 EU414335_EV71-C1_CHN_2006 DQ341358_EV71-C1_MAL_2000 98 DQ341361_EV71-C1_AUS_2000 EU527983_EV71-C5_TW_2007 AY421766_CVA8_USA_1949 21364_EV90_RUS_2003 AY697460_EV90_BAN_1999 99 AY697461_EV91_BAN_2000 75 AY697459_EV89_BAN_2000 AY697458_EV76_FRA_1991 99 74 40961_TJK_2011 JF905564_EV76_04360/SD_CHN_2004 41631_EV76_TJK_2011 41755_EV76_KGZ_2011 98 36053_CVA10_RUS_2010 82 40191_CVA10_RUS_2010 38905_CVA10_RUS_2009 98 77 40184_CVA10_RUS_2010 95 34266_CVA10_RUS_2009 31228_CVA10_RUS_2008 95 71 36191_CVA4_RUS_2009 41423_CVA4_RUS_2011 29518_CVA4_RUS_2007 99 30482_CVA4_RUS_2008 GU942820_CVA7_CAN_1958 HQ728261_CVA5_SD_CHN_2009 29055_CVA5_RUS_2007 99 83 30204_CVA5_RUS_2007 HQ728262_CVA10_SD_CHN_2009 AY421760_CVA2_USA_1947 59 AY421761_CVA3_USA_1948 AY421764_CVA6_USA_1949 AY421767_CVA10_USA_1950 99 U22521_EV71-A_BrCr_USA_1969 AY421768_CVA12_USA_1948 AY421765_CVA7_USA_1949 31758_CVA16_RUS_2008 99 34351_CVA16_RUS_2009 39156_CVA16_RUS_2010 34950_CVA16_RUS_2009 99 38394_CVA16_RUS_2010 42234_CVA16_RUS_2011 98 41555_CVA16_KAZ_2011 99 GQ279371_CVA16_CHN_2008 84 JN590244_CVA16_CHN_2010 29072_CVA16_RUS_2007 FJ198212_CVA16_CHN_2008 99 37323_CVA16_UKR_2010 GQ279368_CVA16_CHN_2008 40188_CVA16_RUS_2004 AF177911_CVA16_TW_1998 60
90
0.05
87
la
99
3D
99
81
72
99
90
99
III
IV
II
41785_EV71_RUS_2011 HQ426649_EV71-C4_CHN_2010 FJ194965_EV71-C4_CHN_2008 HQ325852_EV71-C4_CHN_2010 42266_EV71_RUS_2011 EU703812_EV71-C4_CHN_2008 GQ892830_EV71-C4_CHN_2008 GQ994990_EV71-C4_CHN_2009 80 EU864507_EV71-C4_CHN_2008 GQ279370_EV71-C4_CHN_2008 FJ360544_EV71-C4_CHN_2008 GQ994991_EV71-C4_CHN_2009 FJ357374_EV71-C4_TW_2005 FJ713137_EV71-C4_CHN_2009 HM003207_EV71-C4_CHN_2008 GQ279369_EV71-C4_CHN_2008 GU196833_EV71-C4_CHN_2009 99 HQ129932_EV71-C4_CHN_2006 FJ194964_EV71-C4_CHN_2008 GQ994989_EV71-C4_CHN_2009 AY465356_EV71-C4_CHN_2003 EU131776_EV71-C4_TW_2002 AF302996_EV71-C4_CHN_1998 HQ188292_EV71-C4_CHN_2008 U05876_CVA16_SOA_1951 AY421762_CVA4_USA_1948 AY421769_CVA14_USA_1950 94 32898_CVA2_RUS_2008 24004_CVA2_RUS_2005 29256_EV71-C2_RUS_2007 99 22888_CVA10_RUS_2004 26412_CVA3_KGZ_2006 DQ341354_EV71-B4_CHN_1998 96 99 DQ341368_EV71-B4_MAL_1997 22468_CVA8_ARM_2004 41156_CVA6_RUS_2011 31793_CVA2_UKR_2008 40879_CVA2_RUS_2011 99 27190_CVA10_RUS_2006 32007_CVA2_RUS_2008 HM622392_EV71-C2_TW_2008 41413_CVA4_RUS_2011 32900_CVA4_RUS_2008 99 37966_CVA4_RUS_2010 91 99 39633_CVA4_RUS_2010 87 39797_CVA4_RUS_2010 99 40238_CVA4_RUS_2011 HQ728260_CVA4_SZ_CHN_2009 HQ728259_CVA2_SD_CHN_2009 30009_CVA7_2007 FJ607337_EV71-C4_CHN_2008 40181_CVA10_RUS_2004 42593_CVA6_RUS_2011 40180_CVA6_RUS_2010 24315_CVA14_RUS_2005 42351_CVA4_TKM_2011 36240_CVA4_RUS_2010 40179_CVA2_RUS_2010 93 29089_CVA8_TKM_2007 42096_CVA2_RUS_2011 24267_CVA8_RUS_2005 22205_CVA10_TAJ_2004 40194_CVA4_RUS_2010 88 23187_CVA4_AZE_2004 37699_CVA2_KAZ_2010 27203_CVA4_RUS_2006 79 28297_CVA6_UZB_2007 96 34082_CVA7_RUS_2009 22809_CVA8_RUS_2004 40428_CVA6_TKM_2011 87 42115_CVA2_RUS_2011 41149_CVA2_RUS_2011 99 41963_CVA2_RUS_2011 37183_CVA7_TKR_2010 41143_CVA5_RUS_2011 98 34020_EV71-C2_RUS_2009 41464_EV71_RUS_2011 38555_EV71_KGZ_2010 98 32896_EV71-C2_2009 FJ172159_EV71-C2_CHN_2008 40190_EV71_RUS_2010 90 37731_EV71_UKR_2010 AF304459_EV71-C2_TW_1998 13384_EV71-C2_RUS_2001 DQ341357_EV71-C2_AUS_1999 DQ381846_EV71-C2_AUS_1999 99 DQ341356_EV71-C3_KOR_2000 DQ341359_EV71-C1_MAL_1998 27578_EV71-C1_RUS_2007 93 99 DQ341358_EV71-C1_MAL_2000 90 EU414335_EV71-C1_CHN_2006 70 DQ341361_EV71-C1_AUS_2000 DQ452074_EV71-C1_NOR_2003 AY421766_CVA8_USA_1949 EU527983_EV71-C5_TW_2007 AY421765_CVA7_USA_1949 99 EU527985_EV71-B5_TW_2007 HM622390_EV71-B5_TW_2009 76 FJ461781_EV71-B5_CHN_2008 FJ357378_EV71-B5_TW_2003 DQ341364_EV71-B5_CHN_2000 FJ357375_EV71-B4_TW_1999 99 DQ341365_EV71-B4_MAL_2001 89 FJ357377_EV71-B4_TW_2000 94 AB482183_EV71-B1_JPN_1973 HQ189392_EV71-B1_HUN_1978 99 FJ357382_EV71-B2_TW_1986 FJ357384_EV71-B2_TW_1986 99 AY421763_CVA5_USA_1950 HQ728262_CVA10_SD_CHN_2009 HQ728261_CVA5_SD_CHN_2009 93 41423_CVA4_RUS_2011 99 29055_CVA5_RUS_2007 86 21462_CVA10_2003 30204_CVA5_RUS_2007 34950_CVA16_RUS_2009 41555_CVA16_KAZ_2011 99 38394_CVA16_RUS_2010 42234_CVA16_RUS_2011 31758_CVA16_RUS_2008 34351_CVA16_RUS_2009 99 39156_CVA16_RUS_2010 FJ198212_CVA16_CHN_2008 40188_CVA16_RUS_2004 85 AF177911_CVA16_TW_1998 29072_CVA16_RUS_2007 99 GQ279371_CVA16_CHN_2008 74 JN590244_CVA16_CHN_2010 99 37323_CVA16_UKR_2010 GQ279368_CVA16_CHN_2008 93 86 AY421760_CVA2_USA_1947 76 AY421767_CVA10_USA_1950 AY421764_CVA6_USA_1949 AY421768_CVA12_USA_1948 88 AY421761_CVA3_USA_1948 U22521_EV71-A_BrCr_USA_1969 GU942820_CVA7_CAN_1958 99 29518_CVA4_RUS_2007 99 30482_CVA4_RUS_2007 31228_CVA10_RUS_2008 99 36191_CVA4_RUS_2009 93 34266_CVA10_RUS_2009 36053_CVA10_RUS_2010 99 40191_CVA10_RUS_2010 86 38905_CVA10_RUS_2009 95 40184_CVA10_RUS_2010 AY697458_EV76_FRA_1991 21364_EV90_RUS_2003 99 AY697459_EV89_BAN_2000 93 AY697460_EV90_BAN_1999 83 AY697461_EV91_BAN_2000 40961_EV76_TJK_2011 71 JF905564_EV76_SD_CHN_2004 41631_EV76_TJK_2011 41755_EV76_KGZ_2011 98
A. N. Lukashev and others
Fig. 1. Phylogenetic trees of HEV-A in three genome regions. Strains were colour-coded according to their type. Scale bars indicate maximum likelihood distances. Numbers at tree nodes indicate bootstrap support values. Roman numbers indicate groups referred to in the text.
Journal of General Virology 95
Recombination in Enterovirus A
Table 1. Features of conserved HEV-A groups Group II III IV –
Serotypes
Number of members in VP1/2C/3D
Median non-recombinant virus age from node
EV71 genotypes B4–B5 EV71 subtypes C1–C3 EV71 subtype C4 CVA2, 4, 10 (7 conserved groups)
10/8/8 20/18/18 25/25/24 22/22/22
13 18.5 16 3.5
Group tMRCA in VP1 (years)* 22 29 19 3.5
*Time of tMRCA was calculated as node height minus age of the most recent isolate of a group.
strains as opposed to the globally representative sampling of EV71. Therefore, it is not possible to conclude if this serotype is indeed less involved in recombination within HEV-A, especially as diverse recombinant forms of CVA16 were described in the study by McWilliam Leitch et al. (2012). In accordance with earlier reports on genetic identity of novel HEV-A serotypes (Oberste et al., 2005), three isolates of EV76 and one of EV90 did not have signs of recombination with classical HEV-A serotypes. However, these viruses displayed multiple phylogenetic conflicts involving EV76, EV89, EV90 and EV91, supporting that these types might represent a subspecies of HEV-A. All other coxsackievirus A types, except for CVA16, were highly involved into recombination within the global gene pool. Bayesian dating was done for three types with significant sampling, namely CVA2 (13 viruses), CVA4 (17 viruses) and CVA10 (13 viruses). The median age of tree nodes that were conserved across the three genome regions (reliably supported by bootstrap values above 70 in one genome region and intact, but not necessarily well supported, in another region) was just 3.6 years, and never more than 8.7 years, which is several-fold less than the age of non-recombinant EV71 groups that had tMRCAs of 19– 29 years. The median age of non-recombinant isolates was 3.5 years, and never exceeded 9 years. To further illustrate two distinct recombination patterns in EV71, CVA16 and other coxsackieviruses, pairwise genetic distances in three genome regions were plotted separately within each of these two groups (Fig. 2). In EV71 and CVA16, the pairwise distances in three genome regions correlated very well up to a genetic distance of about 0.14 (Fig. 2a, region a), indicating long-term circulation of viruses in the absence of recombination. There were discordant pairwise distances (low in VP1 and high in 2C and 3D, Fig. 2a, b region) that corresponded to the few EV71 strains that acquired an ‘alien’ 2C and 3D genome region (see above). There were no viruses with low pairwise distances in 2C/3D and divergent VP1 genome regions (Fig. 2a, c region), which agreed with the absence of such viruses on phylogenetic trees. The coxsackievirus group also included viruses without apparent signs of recombination (with concordant pairwise http://vir.sgmjournals.org
distances), but only with genetic distances up to approximately 0.07 (Fig. 2b, a region). Recombination events apparent as discordant genetic distances beginning from as low as 0.01 involved all genome regions (Fig. 2b, regions b, c). In viruses that differed by over 0.07 of their RNA sequence in any genome region, pairwise sequence distances were largely discordant between all genome regions,
(a) 0.30
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Fig. 2. Plot of pairwise distances among EV71 and CVA16 (a) and among other coxsackieviruses of species HEV-A (b) in three genome regions. Each point represents a pair of pairwise distances between the same strains in two genome regions that correspond to the symbol description. The uncorrected nucleotide sequence distance in the first genome region is plotted on the x-axis and that in the second region of the pair on the y-axis. Circled areas are described in the text. 871
A. N. Lukashev and others
indicating common shuffling of genome fragments by recombination (Fig. 2b, d region). This observation corresponds well with the maximum age of conserved tree nodes for coxsackieviruses (8.7 years) multiplied by the estimated substitution rates (5.561023–14.161023 substitutions per site year21). The number of conserved groups was the highest between the VP1 and 2C genome regions (14 groups), and about the same between the VP1 and 3D, and the 2C and 3D regions (seven and eight groups, respectively), while the distance between these genome regions is about equal (750 nt between VP1 and 2C and 870 nt between 2C and 3D). This observation contradicts the reports of an apparent recombination hot-spot on the border of structural and non-structural genes in enteroviruses and other picornaviruses (Benschop et al., 2010; Heath et al., 2006; Lukashev, 2005, 2010). It is likely that the apparent recombination hot-spot reported previously at the VP1– 2A junction may only correspond to an artefact due to a stronger phylogenetic signal in the P1 genome region. This strong signal would result from the absence of recombination between capsid genes of different serotypes, higher variability of the structural genome region and, consequently, a better-resolved phylogeny. As a result, it is easier to detect a switch of phylogeny on the border of this region. The non-structural genes are arbitrarily shuffled between serotypes every few years, so the phylogenetic signal is quickly degraded and fewer recombination events can be detected with sufficient statistical support. Before this study, EV71 comprised over 80 % of all HEV-A complete genomic sequences in GenBank and was the sole serotype used in many studies. Recombination was implied by the emergence of several EV71 subtypes (McWilliam Leitch et al., 2012; Yoke-Fun & AbuBakar, 2006). The recombination partners in these recombination events remained unknown because there were no available sequences of contemporary HEV-A strains. Our study indicates that the recombination events leading to the emergence of EV71 subtypes involved members of the global HEV-A gene pool and not only EV71. This is evident because the three EV71 groups II–IV were evenly interspersed on the phylogenetic trees for the 2C and 3D genome regions and did not originate from a shared common ancestor. Surprisingly, EV71 subtypes almost ceased recombining with the rest of the HEV-A gene pool upon their emergence. Perfect correspondence of EV71 phylogenies in different genome regions was reported earlier (Bible et al., 2008; McWilliam Leitch et al., 2012; van der Sanden et al., 2011). This study highlights the degree of genetic conservation in EV71, which apparently recombines about five times less frequently than its peers in the HEV-A species CVA2, CVA4 and CVA10. Moreover, the high half-life of non-recombinant EV71 strains indicates that they are the most conserved types in terms of recombination frequency among enteroviruses that have been closely studied so far. 872
Several factors could explain the low apparent recombination in EV71. First, this could be due to reproductive isolation at the level of permissible/preferred cell types. However, EV71 and some other HEV-A members share the same putative cellular receptors PSGL-1 and SCARB2 (Nishimura & Shimizu, 2012). Also, use of different receptors by HEV-B serotypes (Racaniello, 2007) did not hamper promiscuous recombination (Lukashev et al., 2003; Simmonds & Welch, 2006), and poliovirus recombines freely with other HEV-C members that do not use the poliovirus receptor (Brown et al., 2003; Jegouic et al., 2009). Another factor that could contribute to the low recombination rate in EV71 could be adaptation of nonstructural genes to the EV71 capsid. Such adaptation of 2C and VP3 proteins was described in HEV-C species (Liu et al., 2010). Yet another possibility is that the lower substitution rate in EV71 (which may result from either the polymerase error rate or a less tight bottleneck at each infection cycle) does not require such frequent recombination to recover deleterious genomes that are constantly generated within a quasispecies. As a result, EV71 can ‘afford’ less frequent recombination within a quasispecies, which also results in lower frequency of recombination with coxsackieviruses. Finally, the long apparent circulation of non-recombinant EV71 variants can be the result of less pronounced expansion and extinction cycles in this type. It has been noted that the apparent recombination rate is lower in the ‘endemic’ echovirus 11 than in ‘epidemic’ echoviruses 9 and 30 (McWilliam Leitch et al., 2010). EV71 is an ‘endemic’ type as revealed by the annual isolation pattern (Khetsuriani et al., 2006), which corresponds well to a low apparent recombination rate. Paradoxically, EV71, which causes the most clinically relevant epidemics, displays the recombination phenotype of an ‘endemic’ enterovirus.
Acknowledgements This study was supported by the Global Polio Eradication Initiative of the WHO through the WHO European Office and by the Government of the Russian Federation by the budgetary allocations for the implementation of activities under the Global Polio Eradication Initiative (Government act 1771-p of 14.10.2010). We would like to thank the staff of the following regional laboratories for technical assistance in collection of stool samples and virus isolation: Centres of Hygiene and Epidemiology in Moscow, Omsk region, Stavropol region; Regional Centre of Epidemiological Surveillance for Poliomyelitis, Pasteur Institute, St Petersburg; National Poliomyelitis Laboratories in Armenia, Georgia, Kazakhstan and Ukraine. The study was partially funded by the EU FP7 project EVA (contract number 228292) and by infrastructural support from the German Centre for Infection Research (DZIF).
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