Virus Research 179 (2014) 212–219
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Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice Jian-Feng Han a,1 , Nan Yu b,1 , Yu-Xian Pan b , Si-Jie He b , Li-Juan Xu a , Rui-Yuan Cao a , Yue-Xiang Li a , Shun-Ya Zhu a , Yu Zhang a , E-De Qin a , Xiao-Yan Che b , Cheng-Feng Qin a,∗ a
Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Laboratory of Emerging Infectious Disease and Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, No. 253 Gong ye da dao zhong, Guangzhou, Guangdong 510282, China b
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 21 October 2013 Accepted 22 October 2013 Available online 6 November 2013 Keywords: Human coxsackievirus A16 Virulence Phylogenetics Homologous recombination
a b s t r a c t Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3 -untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human coxsackievirus A16 (CA16) is a member of the Enterovirus genus in the Picornaviridae family. CA16 has been recognized as one of the major pathogens causing hand, foot, and mouth disease (HFMD) in infants and young children, along with enterovirus 71 (EV71) and many other enteroviruses. Cocirculation of EV71 and CA16 has been described in HFMD outbreaks in China, Malaysia, and Taiwan (Yan et al., 2012; Chan et al., 2012; Chang et al., 1999). CA16 infection used to be associated with a milder outcome and fewer complications (Chang et al., 1999), and the neurological complications and fatalities during HFMD outbreaks were largely attributed to EV71 (Liu et al., 2000). However, damage to the central nervous system (CNS) is also a pathological feature of CA16 infection. Severe neurological diseases, including aseptic meningitis, encephalitis, and even fatal cases of CA16, have also been reported (Goto et al., 2009;
∗ Corresponding author. Tel.: +86 1066948631. E-mail address: [email protected] (C.-F. Qin). 1 These authors contributed equally to this work. 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.020
Wang et al., 2004). At present, the pathogenesis of CA16 infection in humans remains largely unknown. CA16 contains a single-stranded, positive-sense, polyadenylated RNA genome of approximately 7400 bases; the genome consists of structural and non-structural regions (namely, P1, P2 and P3) with untranslated regions (UTRs) at the termini (Knowles et al., 2011). The P1 region encodes four structural capsid proteins (VP4, VP2, VP3, and VP1), whereas P2 and P3 encode seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). The four capsid proteins form the icosahedral virion structure, with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. Among these capsid proteins, VP1 contains major antigenic and neutralization sites (Zhang et al., 2012; Shi et al., 2013) and therefore plays a critical role in the process of virus infection. Phylogenetic analysis grouped all CA16 strains into two genotypes (A and B) (Perera et al., 2007), and the B genotypes can be further divided into the B1 and B2 subgenotypes. Two clusters, B1a and B1b, were recently identified within subgenotype B1 (Zhang et al., 2010a). Both viral and host factors contribute to the pathogenicity of CA16, and virulence supposedly plays a critical role in the pathogenesis of severe neurological diseases caused by enteroviruses. For poliovirus, the major virulence determinants are located in the
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5 -UTR and VP1 genes (De Jesus, 2007). Mutations in the 5 -UTR, VP1 gene, and nonstructural 3D gene of EV71 result in attenuation in mice and cynomologus monkeys (Arita et al., 2005, 2008; Kung et al., 2010). Halim et al. identified a single amino acid residue (L129) in the VP1 protein as the critical determinant of virulence of coxsackievirus B (Halim and Ramsingh, 2000). Overall, CA16 virulence determinants have not been well studied, especially in viruses isolated directly from clinical samples. In this study, two CA16 strains with distinct virulence patterns were isolated from HFMD patients in Guangdong, China in 2009, which provided a unique opportunity to investigate potential virulence determinants by comparing the isolates’ phenotypic and genomic characterizations. 2. Materials and methods 2.1. Clinical samples, cells, and viruses Clinical samples were collected from two HFMD patients in Zhujiang hospital in Guangdong, China in 2009. Both patients were diagnosed with HFMD based on clinical manifestations and laboratory tests. Human rhabdomyosarcoma (RD) cells (ATCC, CCL-136TM ) and African green monkey kidney Vero cells (ATCC, CCL-81TM ) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a 5% CO2 atmosphere. The two CA16 viruses (GD09/24 and GD09/119) were isolated in Vero cells and passaged once before in vitro and in vivo characterization.
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antibody assay (IFA) was performed. RD cells were incubated with CA16 antisera for 1 h at 37 ◦ C. The cells were then incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG (ZSBIO) at a dilution of 1:300 in Evans Blue for another 30 min. Finally, the cells were rinsed again and visualized under a fluorescent microscope (Olympus) after being dried at room temperature.
2.5. Virus titration Virus titers were determined using a microtitration assay and are represented as 50% tissue culture infectious doses (TCID50 values) in tissue culture or plaque-forming units (PFUs) in plaque assays. Briefly, serially diluted virus samples were used to infect RD cells in 96-well plates, and quadruplicate samples were used for each dilution. The 96-well plates were incubated for 3 days at 37 ◦ C, and the TCID50 values were measured by determining the cytopathic effects. The TCID50 values were calculated using the Reed–Muench method (Reed and Muench, 1938). The plaque-forming assay in RD cells was performed as previously described (Han et al., 2011). Briefly, approximately 4 × 105 cells were incubated overnight and were infected with serially diluted virus suspensions. After adsorption for 1 h, the virus suspension was replaced with DMEM containing 2% FBS and low melting point agarose. The medium was removed 72 h post-infection, and the cells were fixed in 10% formaldehyde and subsequently stained with 1% crystal violet solution. The titer of the virus was expressed as PFU/ml.
2.2. RT-PCR and DNA sequencing To sequence the CA16 isolates, total RNA from CA16-infected RD cells was extracted using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. cDNA was produced by reverse transcription using a PrimeScript RT-PCR Kit (Takara) with an oligo (dT) primer. The overlapping DNA fragments were produced by polymerase chain reaction (PCR) with LA Taq DNA polymerase (Takara). All of the primers used in this study are listed in the supplemental Table. The cycling parameters were denaturation for 3 min at 94 ◦ C, followed by 30 cycles of 30 s at 94 ◦ C, 30 s at 55 ◦ C, and 1 min at 72 ◦ C, and a final extension for 7 min at 72 ◦ C. The PCR products were sequenced and assembled using SeqMan software. The UTRs were determined using a 5 /3 -Rapid Amplification of cDNA Ends (RACE) Kit (Roche). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2013.10.020. 2.3. Genome sequence analysis Homology analysis of the CA16 strains was performed using ClustalX and MegAlign software. A phylogenetic tree was generated using the Molecular Evolutionary Genetics Analysis (MEGA) program via the maximum likelihood and the neighbor-joining methods. The EV71 prototype strain BrCr was set as the outgroup. A total of 26 CA16 strains were collected from GenBank and included in the analysis. Homologous recombination was analyzed using Simplot software. RNA secondary structure prediction was performed using RNAfold, and the structures were drawn with RNAStructure. 2.4. IFA To identify the CA16-specific antigen protein, human RD cells were infected with the CA16 isolates, and an indirect fluorescent
2.6. One-step growth curve One-step growth curves of the CA16 isolates were performed as previously described (Ye et al., 2012). Briefly, RD cell monolayers in 6-well dishes were washed with DMEM and inoculated at an MOI (multiplicity of infection) of 1. After adsorption for 1 h, the cells were thoroughly washed to remove the unbound virus and were cultured at 37 ◦ C for 24, 48, and 72 h. The supernatants were collected, and the virus titers were determined as described above.
2.7. Animal experiments One-day-old BALB/c mice were obtained from the Beijing Laboratory Animal Center. All animal experiments were approved by and performed according to the guidelines of the Animal Experiment Committee of State Key Laboratory of Pathogen and Biosecurity. Groups of mice (n = 5) were inoculated with 10-fold serial viral dilutions via intracranial (i.c.) or intraperitoneal (i.p.) injection. All animals were then monitored for clinical symptoms, paralysis, and death for up to 14 days postinoculation. The survival curves were drawn using GraphPad software, and the 50% lethal doses (LD50 values) for each virus strain were calculated using the Reed–Muench method (Reed and Muench, 1938). For the dead mice, the intact brain was dissected out and rinsed with PBS three times before further assay.
2.8. Nucleotide sequence accession numbers The complete genomic sequences of the CA16/GD09/24 and CA16/GD09/119 isolates described in this study were deposited into GenBank under the accession numbers KC117317 and KC117318, respectively.
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Percent survival
100 80 60 40
GD09/119 GD09/24
20 0
0
2
4
6
8
10
12
14
Days post-inoculation Fig. 2. Mice survival curves of the CA16 strains via intracranial (i.c.) injection. Oneday-old BALB/c mice were intracranially administered 100 PFU virus, and mortality was monitored for 14 days. The survival curves were created using GraphPad software (version 5.0).
Fig. 1. In vitro plaque phenotypes and propagation characteristics of the CA16 isolates. (a) Plaque morphology of the CA16 isolates. A standard plaque-forming assay was performed in RD cells as described elsewhere. (b) One-step growth curve analysis of the CA16 strains in RD cells. Virus supernatants were collected after 24, 48, and 72 h, and the virus titers are expressed as TCID50 /ml.
3. Results
significantly higher compared to that of GD09/24. All of the mice died within 5 days of infection with GD09/119, while 60% of the mice challenged with an equal dose of GD09/24 survived. Virus recovery and genome sequencing of the brains of dead mice confirmed the CA16 infection (data not shown). Importantly, mice receiving i.p. inoculation with varying doses of the CA16 isolates also developed the typical central nervous system (CNS) clinical manifestations, similar to the i.c. infection. The mortality and average survival times of the mice infected with GD09/119 and GD09/24 are shown in Table 1. According to the LD50 of each virus, GD09/119 was approximately 100-fold more virulent than GD09/24. Furthermore, CA16-specific genes could be detected by RT-PCR in the brain and skeletal muscles of dead mice (data not shown), and CA16 was isolated from the brains of dead mice. Collectively, these results demonstrated that although the two clinical CA16 isolates presented similar growth characteristics in vitro, GD09/119 was more virulent than GD09/24 in mice.
3.1. In vitro characterization of the CA16 clinical isolates Two CA16 strains (GD09/24 and GD09/119) were isolated using Vero cells from anal swab samples of HFMD patients. IFA with human convalescent phase sera (1:100 dilution) from confirmed patients with CA16 infections revealed that the CA16-specific viral proteins were expressed in both GD09/24- and GD09/119-infected cells (data not shown). Then, both strains were inoculated into CA16-susceptive RD cells, and the typical cytopathic effects, such as cell rounding, aggregation, and fall-off, appeared 24 h post infection. The plaque-forming assay revealed that both isolates yielded similar small plaque morphologies (Fig. 1a). Furthermore, one-step growth curve analysis showed similar replication kinetics for both isolates in RD cells (Fig. 1b). Both viruses peaked at 72 h postinfection on RD cells, and the highest titer was approximately 6 log TCID50 /ml. 3.2. Virulence comparison of the CA16 isolates in mice The suckling mouse model of CA16 infection was recently described by Mao et al. (Mao et al., 2012). To further characterize the two CA16 clinical isolates, both viruses were inoculated into groups of one-day-old BALB/c mice via i.p. or i.c. routes. Following i.c. infection, both GD09/24 and GD09/119 were both virulent to suckling mice, resulting in the typical neurological symptoms, including significant weight loss, hind limb paralysis, moribund demeanor, and death. As shown in Fig. 2, the mortality of GD09/119 was
3.3. Phylogenetic and homologous recombination analyses Furthermore, to compare the genomic characteristics of these newly isolated CA16 strains, the complete genome sequences of GD09/119 and GD09/24 were determined and deposited in GenBank under the accession numbers KC117317 and KC117318, respectively. The genomic RNAs of both strains were 7409 bp in length, and the 5 -UTR was 745 bp, while the 3 -UTR was 82 bp. BLAST analysis indicated that the two isolates were most homologous with the CA16 strain SZ/HK08-7 (GQ279371), which was isolated in Shenzhen city, Guangdong, China in 2008, with nucleotide identities of 95.6% and 96.5% to GD09/119 and GD09/24, respectively. The nucleotide sequence identities among the different CA16 strains recorded in GenBank (a total of 28 strains) ranged from 78.8% to 96.5%. Maximal amino acid identity (>99.7%) of VP1 was noted among the isolates from the Guangdong region (including shzh05-1, GZ08, and SZ/HK08-7). Amino acid sequence homology analysis revealed 95.9% identity in the 3D proteins and 96.5% identity in the non-structural proteins among the isolates. Importantly, phylogenetic analysis was performed based on both the complete VP1 gene (Fig. 3a) and the complete genome (Fig. 3b) by either the neighbor joining method or the maximum likelihood method using the MEGA program. The results indicated that all CA16 strains circulating in China belonged to the B genotype; the two newly isolated strains, GD09/119 and GD09/24, were both within the B1b genotype (Fig. 3a and b).
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Table 1 Mice challenged with CA16 strains via i.p. route. Strain
Virus dose (PFU)
LD50 (PFU/ml)
AST (d)
GD09/24
104 103 102 10 1
5/5 (100) 2/5 (40) 1/5 (20) 0/5 (0) 0/5 (0)
Death/total mice (mortality, %)
1.48 × 103
6.2 11.6 12.6 14.0 14.0
GD09/119
104 103 102 10 1
5/5 (100) 5/5 (100) 5/5 (100) 2/5 (40) 0/5 (0)
14.8
6.6 7.4 9.0 12.6 14.0
AST, average survival time.
Homologous recombination events occur frequently within enterovirus species (Simmonds and Welch, 2006; Han et al., 2012). Thus, homologous recombination analysis of the two CA16 isolates with other human enterovirus species was performed using SimPlot software. Similarity scanning analysis using GD09/119 as a query sequence showed that most of the 5 half of the genome (nt 750–3600) had high similarity (77.4%) to the CA16 prototype strain G-10 and that the 3 half (nt 3600–7400) of the genome showed high similarity (83.5%) to the EV71 strain BrCr (Fig. 4a). Additionally, bootscanning results revealed that GD09/119 was most closely related to CA16 strain G-10 in the P1 region and to EV71 strain BrCr in the P2 and P3 regions (Fig. 4b). Genetic algorithms for the detection of recombination indicated that the putative breakpoints were
located within the 2A domain at approximately nt 3620. A similar recombination pattern was observed when using GD09/24 as a reference sequence (data not shown). The above analysis demonstrated that the CA16 strains were possibly progeny of intertypic and intratypic recombination events involving the EV71 and CA16 prototypes. 3.4. Genome comparison and virulence determinant analysis To reveal the potential relationship between the genome sequence and pathogenicity phenotypes of these newly isolated CA16 isolates, the complete genome sequences of GD09/119 and GD09/24 were compared in detail. Sequence homology analysis
Fig. 3. Phylogenetic analysis of selected human CA16 strains from different origins. The neighbor-joining tree was generated using MEGA 5.0 software, and prototype EV71 strains were used as the outgroup. (a) Phylogenetic tree based on complete VP1 sequences. (b) Phylogenetic tree based on complete genome sequences. Both GD09/24 and GD09/119 belonged to the B1b subtype.
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J.-F. Han et al. / Virus Research 179 (2014) 212–219 Table 2 Sequence comparison of 5 UTR between CA16 strains. Nucleotide position
Nucleotide change
56 63 71 91 94 100 104 123 153 158 199 232 265 270 275 419 513 517 659 669 671 686 703 719 726
GD09/24
GD09/119
C G T C C T C C A C C T T A A T A C C C G T C C C
T A C T T C T T G T T C C G G C G T T T A C T T T
Table 3 Amino acids sequence comparison between CA16 strains.
Fig. 4. Recombination analysis of CA16. (a) Similarity scanning analysis of CA16 with representative enterovirus strains on the basis of the full-length genome sequences. (b) Bootscanning analysis indicated recombination with EV71 within the nonstructural regions; bootscanning analysis was performed with a window size of 200 nt and a step of 20 nt. The breakpoint was at approximately nt 3620 in the 2A region.
revealed that the complete genome nucleotide identity between the new CA16 isolates was 95.7% and that the amino acid homology was 99.2%. The 3 -UTRs were highly conserved between the two strains, but there were 25 nucleotides that were different in the 5 -UTRs, as shown in Table 2. However, RNA secondary structure predictions using RNAfold software indicated little topological difference between the strains (data not shown). All of the amino acid differences within the open reading frames (ORFs) between the two strains are shown in Table 3. Regarding VP1, the nucleotide identity between GD09/119 and GD09/24 was 95.2%, and the amino acid identity was 99.7%. There were 43 nucleotide changes in the VP1 gene between the newly isolated CA16 strains, but only one amino acid substitution at position 220 (methionine for GD09/24 and leucine for GD09/119) was identified. Sequence alignment with other CA16 strains revealed that VP1L220 was highly conserved among the genotype B strains, except for GD09/24; meanwhile, all genotype A CA16 strains (G-10 and FY18) contained an alanine residue at VP1-220 (Fig. 5). According to the crystal structure of EV71, L220 is located on the GH loop, near the pocket factor-binding site of the VP1 protein. A total of 86 nucleotide changes were present in the VP4, VP2, and VP3 sequences, but all were silent mutations. There were only single amino acid substitutions in 2B (T/M), 3A (S/N), and 3C (N/T), and there were four amino acid substitutions in 2A. Additionally, a total of nine amino acid substitutions were identified within the 3D gene (Table 3). Interestingly, GD09/24 was consistent with the
Coding protein
Nucleotide position
VP4 VP2 VP3 VP1 2A
– – – 220 75 82 129 132 15 – 11 – 55 39 92 169 172 259 323 342 346 451
2B 2C 3A 3B 3C 3D
Amino acid change GD09/24
GD09/119
M T I N M T
L S V S V M
S
N
N K V R K P H L K Y
T E T K R S N S R F
genotype A strain FY18 and strain G-10 at the 3D positions 342(L) and 451(Y), while an amino acid substitution at K39E resulted in a change in polarity. The functional roles of these amino acids remain unknown. 4. Discussion In this study, two CA16 strains were isolated from HFMD patients in Guangdong, China. In vitro characterization confirmed their similar growth properties; however, in vivo tests revealed that these viruses had distinct virulence patterns in mice. These two CA16 strains were both from HFMD patients with mild clinical manifestations, and whether there is any relationship between their virulence phenotypes and disease severity remains unknown.
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Fig. 5. Sequence alignments of selected enterovirus VP1 genes. MegAlign software was used to align different sequences using the Clustal W method. Amino acid 220 of the VP1 gene of the GD09/24 strain was distinct from the other strains, with the exception of strains in subgenotype A.
Previous investigations have indicated no significant differences among the EV71 isolates from fatal and non-fatal cases (Shih et al., 2000; Singh et al., 2002), which is in agreement with our findings in this study. Meanwhile, the in vitro characteristics may be not associated with enterovirus virulence in vivo, which suggests the importance of in vivo phenotypic differences in pathogenesis. In mainland China, CA16 has been recognized as the major causative agent of HFMD during the last century (Zhang et al., 2010a), and CA16 subgenotype B1 predominantly circulates in mainland China (Li et al., 2005; Zong et al., 2011). Our results indicated that the newly isolated CA16 strains belong to genotype B1b and that these strains are closely related to other CA16 strains isolated in mainland China (Fig. 4). Both genotypes B1a and B1b have been identified in Guangdong, China. These results demonstrated that the CA16 strains have been endemic in the Guangdong region for a long time. Recombination among the different enteroviruses has been well documented (Zhang et al., 2010a, 2010b; Hu et al., 2011; Zhao et al., 2011; Chen et al., 2010). Phylogenetic and recombination analysis of the CA16 strains circulating in China revealed that the current CA16 strains were progeny of homologous recombination within enterovirus subgroup A, especially with EV71 (Zhao et al., 2011). Our study revealed that recombination between CA16 and EV71 may be a frequent phenomenon among HFMD pathogens and may be a possible reason for the variation in virulence.
Virulence investigation usually involves the comparison of viral genome sequences using different virulent phenotypes or origins (Oberste, 2008). Complete genome comparison revealed that the virulence-associated sites of the enteroviruses were present in both the UTRs and the polyprotein-coding regions. Li et al. performed comparative genomic analysis of EV71 strains from severe cases and mild cases; their analysis indicated the critical roles of G/Q/R710 and E729 in VP1, K930 in protease 2A, and three nucleotide changes in the 5 -UTR (Li et al., 2011). Previously, Arita et al. suggested that a single mutation in the VP1 (G145E) gene of EV71 was essential for the mouse-adapted phenotype in NOD/SCID mice (Arita et al., 2008). Zaini et al. recently demonstrated that a single mutation in the VP1 (Q145E) gene could generate a virulent phenotype in fiveday-old BALB/c mice (Zaini and McMinn, 2012). Another amino acid residue in VP1 (K244E) was recently identified as a critical determinant of mouse adaptation and virulence through comparison with full-length genomes sequenced directly from different tissues and blood specimens (Cordey et al., 2012). Currently, little is known about the virulent determinants of CA16, and the crystal structure of the CA16 virion remains unknown. Recently, a comparison study of the CA16 strains isolated from Guangxi, China in 2010 suggested that nucleotide variations in the UTR regions and amino acid substitutions within the VP1 gene may be responsible for differences in virulence (Yang et al., 2012). In our study, L220 in the VP1
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gene was highly conserved among most CA16 genotype B strains, except for GD09/24, while the CA16 genotype A strains (G-10 and FY18) contained A220 at the same position (Fig. 5). In particular, residue 220 of the VP1 protein is located within the neutralization epitope (Shi et al., 2013), and passive transfer of antisera against this epitope conferred significant protection against either EV71 or CA16 challenge in mice (Foo et al., 2007; Tian et al., 2012). Recently, the mouse-adapted virulent CA16 strain MAV was developed (Cai et al., 2013); at residue 220, the MAV strain (L) was identical to GD09/119 (L) but different from GD09/24 (M). The crystal structure of EV71 indicated that L220 of VP1 is located in the GH loop and is exposed on the surface of the mature virus, near the pocket factor-binding site (Wang et al., 2012). The functional importance of this amino acid merits further investigation. The enterovirus 3D protein acts as a viral RNA-dependent RNA polymerase (RdRp) and plays a major role in viral RNA synthesis and uridylylation. In our study, a total of nine amino acid substitutions in the 3D protein were identified between the two CA16 strains; however, none of these substitutions were associated with RNA synthesis and uridylylation. The association of these differences with virulence in mice remains unknown. Previous studies suggested that the low fidelity of the 3D polymerase is essential for the high rate of mutation, which aids in the survival of the virus population in the presence of selective pressure (Pfeiffer and Kirkegaard, 2005). Mutations in the 3D region of poliovirus resulted in temperature susceptibility and viral attenuation in mice (Diamond and Kirkegaard, 1994). Comparison of the amino acid sequences of the 3D polymerases of EV71 clinical isolates revealed that the I251T substitution resulted in a strong temperature-sensitive phenotype, which might contribute to virulence attenuation (Kung, 2010). Both biochemical and reverse genetic studies are needed to clarify the critical roles of these amino acid substitutions in the CA16 strains (Liu et al., 2011). In conclusion, two Chinese CA16 strains from HFMD patients in Guangdong, China were isolated and characterized in vitro and in vivo. Phylogenetic and homologous recombinant analyses indicated that the CA16 isolates circulating in mainland China belonged to the genotype B1b and are likely the progeny of CA16 G-10. Combined with whole genome sequences and virulence in mice, potential associations revealed possible virulence-related candidate sites in the two strains. These findings provide insight into the evolutionary relationships among the CA16 isolates in China and provide useful templates for virulence variation research. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81000721 and 31270195) and the Natural Science Foundation of Beijing (Nos. 7112108 and 7122129). CFQ was supported by the Beijing Nova Program of Science and Technology (No. 2010B041). References Arita, M., Ami, Y., Wakita, T., Shimizu, H., 2008. Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. J. Virol. 82 (4), 1787–1797. Arita, M., Shimizu, H., Nagata, N., Ami, Y., Suzaki, Y., Sata, T., Iwasaki, T., Miyamura, T., 2005. Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys. J. Gen. Virol. 86 (Pt 5), 1391–1401. Cai, Y., Liu, Q., Huang, X., Li, D., Ku, Z., Zhang, Y., Huang, Z., 2013. Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection. Vaccine 31 (18), 2215–2221. Chan, Y.F., Wee, K.L., Chiam, C.W., Khor, C.S., Chan, S.Y., Amalina, W., Sam, M.Z.I.C., 2012. Comparative genetic analysis of VP4 VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997–2008. Trop. Biomed. 29 (3), 451–466.
Chang, L.Y., Lin, T.Y., Huang, Y.C., Tsao, K.C., Shih, S.R., Kuo, M.L., Ning, H.C., Chung, P.W., Kang, C.M., 1999. Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998. Pediatr. Infect. Dis. J. 18 (12), 1092–1096. Chen, X., Zhang, Q., Li, J., Cao, W., Zhang, J.X., Zhang, L., Zhang, W., Shao, Z.J., Yan, Y., 2010. Analysis of recombination and natural selection in human enterovirus 71. Virology 398 (2), 251–261. Cordey, S., Petty, T.J., Schibler, M., Martinez, Y., Gerlach, D., van Belle, S., Turin, L., Zdobnov, E., Kaiser, L., Tapparel, C., 2012. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog. 8 (7), e1002826. De Jesus, N.H., 2007. Epidemics to eradication: the modern history of poliomyelitis. Virol. J. 4, 70. Diamond, S.E., Kirkegaard, K., 1994. Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperaturesensitive mutants defective in RNA synthesis. J. Virol. 68 (2), 863–876. Foo, D.G., Alonso, S., Chow, V.T., Poh, C.L., 2007. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect. 9 (11), 1299–1306. Goto, K., Sanefuji, M., Kusuhara, K., Nishimura, Y., Shimizu, H., Kira, R., Torisu, H., Hara, T., 2009. Rhombencephalitis and coxsackievirus A16. Emerg. Infect. Dis. 15, 1689–1691. Halim, S., Ramsingh, A.I., 2000. A point mutation in VP1 of coxsackievirus B4 alters antigenicity. Virology 269 (1), 86–94. Han, J.F., Cao, R.Y., Deng, Y.Q., Tian, X., Jiang, T., Qin, E.D., Qin, C.F., 2011. Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo. Virol. J. 8, 106. Han, J.F., Jiang, T., Fan, X.L., Yang, L.M., Yu, M., Cao, R.Y., Wang, J.Z., Qin, E.D., Qin, C.F., 2012. Recombination of human coxsackievirus B5 in hand, foot, and mouth disease patients, China. Emerg. Infect. Dis. 18 (2), 351–353. Hu, Y.F., Yang, F., Du, J., Dong, J., Zhang, T., Wu, Z.Q., Xue, Y., Jin, Q., 2011. Complete genome analysis of coxsackievirus A2, A4 A5, and A10 strains isolated from hand, foot, and mouth disease patients in China revealing frequent recombination of human enterovirus A. J. Clin. Microbiol. 49 (7), 2426–2434. Knowles, N.J., et al., 2011. Picornaviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, CA, pp. 855–880. Kung, Y.H., Huang, S.W., Kuo, P.H., Kiang, D., Ho, M.S., Liu, C.C., Yu, C.K., Su, I.J., Wang, J.R., 2010. Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase. Virology 396 (1), 1–9. Li, L., He, Y., Yang, H., Zhu, J., Xu, X., Dong, J., Zhu, Y., Jin, Q., 2005. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People’s Republic of China. J. Clin. Microbiol. 43, 3835–3839. Li, R., Zou, Q., Chen, L., Zhang, H., Wang, Y., 2011. Molecular analysis of virulent determinants of enterovirus 71. PLoS One 6 (10), e26237. Liu, C.C., Tseng, H.W., Wang, S.M., Wang, J.R., Su, I.J., 2000. An outbreak of enterovirus 71 infection in Taiwan 1998 epidemiologic and clinical manifestations. J. Clin. Virol. 17 (1), 23–30. Liu, F., Liu, Q., Cai, Y., Leng, Q., Huang, Z., 2011. Construction and characterization of an infectious clone of coxsackievirus A16. Virol. J. 8, 534. Mao, Q., Wang, Y., Gao, R., Shao, J., Yao, X., Lang, S., Wang, C., Mao, P., Liang, Z., Wang, J., 2012. A neonatal mouse model of coxsackievirus a16 for vaccine evaluation. J. Virol. 86 (22), 11967–11976. Oberste, M.S., 2008. Comparative genomics of the coxsackie B viruses and related enteroviruses. Curr. Top. Microbiol. Immunol. 323, 33–47. Perera, D., Yusof, M.A., Podin, Y., Ooi, M.H., Thao, N.T., Wong, K.K., Zaki, A., Chua, K.B., Malik, Y.A., Tu, P.V., Tien, N.T., Puthavathana, P., McMinn, P.C., Cardosa, M.J., 2007. Molecular phylogeny of modern coxsackievirus A16. Arch. Virol. 152 (6), 1201–1208. Pfeiffer, J.K., Kirkegaard, K., 2005. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog. 1 (2), e11. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497. Shi, J., Huang, X., Liu, Q., Huang, Z., 2013. Identification of conserved neutralizing linear epitopes within the VP1 protein of coxsackievirus A16. Vaccine 31 (17), 2130–2136. Shih, S.R., Ho, M.S., Lin, K.H., Wu, S.L., Chen, Y.T., Wu, C.N., Lin, T.Y., Chang, L.Y., Tsao, K.C., Ning, H.C., Chang, P.Y., Jung, S.M., Hsueh, C., Chang, K.S., 2000. Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res. 68 (2), 127–136. Simmonds, P., Welch, J., 2006. Frequency and dynamics of recombination within different species of human enteroviruses. J. Virol. 80 (1), 483–493. Singh, S., Poh, C.L., Chow, V.T., 2002. Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000). Microbiol. Immunol. 46 (11), 801–808. Tian, X., Su, X., Li, X., Li, H., Li, T., Zhou, Z., Zhong, T., Zhou, R., 2012. Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon. PLoS One 7 (7), e41381. Wang, C.Y., Li, Lu.F., Wu, M.H., Lee, C.Y., Huang, L.M., 2004. Fatal coxsackievirus A16 infection. Pediatr. Infect. Dis. J. 23 (3), 275–276. Wang, X., Peng, W., Ren, J., Hu, Z., Xu, J., Lou, Z., Li, X., Yin, W., Shen, X., Porta, C., Walter, T.S., Evans, G., Axford, D., Owen, R., Rowlands, D.J., Wang, J., Stuart, D.I., Fry, E.E., Rao, Z., 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19 (4), 424–429.
J.-F. Han et al. / Virus Research 179 (2014) 212–219 Yan, X.F., Gao, S., Xia, J.F., Ye, R., Yu, H., Long, J.E., 2012. Epidemic characteristics of hand, foot, and mouth disease in Shanghai from 2009 to 2010: enterovirus 71 subgenotype C4 as the primary causative agent and a high incidence of mixed infections with coxsackievirus A16. Scand. J. Infect. Dis. 44 (4), 297–305. Yang, E.X., Zhao, H., Zhang, Y., et al., 2012. A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B). Sci. China Life Sci. 55, 336–342. Ye, Q., Li, X.F., Zhao, H., Li, S.H., Deng, Y.Q., Cao, R.Y., Song, K.Y., Wang, H.J., Hua, R.H., Yu, Y.X., Zhou, X., Qin, E.D., Qin, C.F., 2012. A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1’ formation and contributes to attenuation. J. Gen. Virol. 93 (Pt 9), 1959–1964. Zaini, Z., McMinn, P., 2012. A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype. J. Gen. Virol. 93 (Pt 9), 1935–1940. Zhang, J., Dong, M., Jiang, B., Dai, X., Meng, J., 2012. Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71. Virus Res. 167 (2), 337–342.
219
Zhang, Y., Wang, D., Yan, D., Zhu, S., Liu, J., Wang, H., Zhao, S., Yu, D., Nan, L., An, J., Chen, L., An, H., Xu, A., Xu, W., 2010a. Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China. J. Clin. Microbiol. 48 (2), 619–622. Zhang, Y., Zhu, Z., Yang, W., Ren, J., Tan, X., Wang, Y., Mao, N., Xu, S., Zhu, S., Cui, A., Zhang, Y., Yan, D., Li, Q., Dong, X., Zhang, J., Zhao, Y., Wan, J., Feng, Z., Sun, J., Wang, S., Li, D., Xu, W., 2010b. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol. J. 7, 94. Zhao, K., Han, X., Wang, G., Hu, W., Zhang, W., Yu, X.F., 2011. Circulating coxsackievirus a16 identified as recombinant type a human enterovirus, China. Emerg. Infect. Dis. 17 (8), 1537–1540. Zong, W., He, Y., Yu, S., Yang, H., Xian, H., Liao, Y., Hu, G., 2011. Molecular phylogeny of Coxsackievirus A16 in Shenzhen China, from 2005 to 2009. J. Clin. Microbiol. 49 (4), 1659–1661.
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Phenotypic and genomic characterization of human coxsackievirus A16 strains with distinct virulence in mice Jian-Feng Han a,1 , Nan Yu b,1 , Yu-Xian Pan b , Si-Jie He b , Li-Juan Xu a , Rui-Yuan Cao a , Yue-Xiang Li a , Shun-Ya Zhu a , Yu Zhang a , E-De Qin a , Xiao-Yan Che b , Cheng-Feng Qin a,∗ a
Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China Laboratory of Emerging Infectious Disease and Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, No. 253 Gong ye da dao zhong, Guangzhou, Guangdong 510282, China b
a r t i c l e
i n f o
Article history: Received 8 May 2013 Received in revised form 21 October 2013 Accepted 22 October 2013 Available online 6 November 2013 Keywords: Human coxsackievirus A16 Virulence Phylogenetics Homologous recombination
a b s t r a c t Human coxsackievirus A16 (CA16) infection results in hand, foot, and mouth disease (HFMD) along with other severe neurological diseases in children and poses an important public health threat in Asian countries. During an HFMD epidemic in 2009 in Guangdong, China, two CA16 strains (GD09/119 and GD09/24) were isolated and characterized. Although both strains were similar in plaque morphology and growth properties in vitro, the two isolates exhibited distinct pathogenicity in neonatal mice upon intraperitoneal or intracranial injection. Complete genome sequences of both CA16 strains were determined, and the possible virulence determinants were analyzed and predicted. Phylogenetic analysis revealed that these CA16 isolates from Guangdong belonged to the B1b genotype and were closely related to other recent CA16 strains isolated in mainland China. Similarity and bootscanning analyses of these CA16 strains detected homologous recombination with the EV71 prototype strain BrCr in the non-structural gene regions and the 3 -untranslated regions. Together, the phenotypic and genomic characterizations of the two clinical CA16 isolates circulating in China were compared in detail, and the potential amino acid residues responsible for CA16 virulence in mice were predicted. These findings will help explain the evolutionary relationship of the CA16 strains circulating in China, warranting future studies investigating enterovirus virulence. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Human coxsackievirus A16 (CA16) is a member of the Enterovirus genus in the Picornaviridae family. CA16 has been recognized as one of the major pathogens causing hand, foot, and mouth disease (HFMD) in infants and young children, along with enterovirus 71 (EV71) and many other enteroviruses. Cocirculation of EV71 and CA16 has been described in HFMD outbreaks in China, Malaysia, and Taiwan (Yan et al., 2012; Chan et al., 2012; Chang et al., 1999). CA16 infection used to be associated with a milder outcome and fewer complications (Chang et al., 1999), and the neurological complications and fatalities during HFMD outbreaks were largely attributed to EV71 (Liu et al., 2000). However, damage to the central nervous system (CNS) is also a pathological feature of CA16 infection. Severe neurological diseases, including aseptic meningitis, encephalitis, and even fatal cases of CA16, have also been reported (Goto et al., 2009;
∗ Corresponding author. Tel.: +86 1066948631. E-mail address: [email protected] (C.-F. Qin). 1 These authors contributed equally to this work. 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.020
Wang et al., 2004). At present, the pathogenesis of CA16 infection in humans remains largely unknown. CA16 contains a single-stranded, positive-sense, polyadenylated RNA genome of approximately 7400 bases; the genome consists of structural and non-structural regions (namely, P1, P2 and P3) with untranslated regions (UTRs) at the termini (Knowles et al., 2011). The P1 region encodes four structural capsid proteins (VP4, VP2, VP3, and VP1), whereas P2 and P3 encode seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). The four capsid proteins form the icosahedral virion structure, with VP1, VP2, and VP3 exposed on the surface and VP4 arranged internally. Among these capsid proteins, VP1 contains major antigenic and neutralization sites (Zhang et al., 2012; Shi et al., 2013) and therefore plays a critical role in the process of virus infection. Phylogenetic analysis grouped all CA16 strains into two genotypes (A and B) (Perera et al., 2007), and the B genotypes can be further divided into the B1 and B2 subgenotypes. Two clusters, B1a and B1b, were recently identified within subgenotype B1 (Zhang et al., 2010a). Both viral and host factors contribute to the pathogenicity of CA16, and virulence supposedly plays a critical role in the pathogenesis of severe neurological diseases caused by enteroviruses. For poliovirus, the major virulence determinants are located in the
J.-F. Han et al. / Virus Research 179 (2014) 212–219
5 -UTR and VP1 genes (De Jesus, 2007). Mutations in the 5 -UTR, VP1 gene, and nonstructural 3D gene of EV71 result in attenuation in mice and cynomologus monkeys (Arita et al., 2005, 2008; Kung et al., 2010). Halim et al. identified a single amino acid residue (L129) in the VP1 protein as the critical determinant of virulence of coxsackievirus B (Halim and Ramsingh, 2000). Overall, CA16 virulence determinants have not been well studied, especially in viruses isolated directly from clinical samples. In this study, two CA16 strains with distinct virulence patterns were isolated from HFMD patients in Guangdong, China in 2009, which provided a unique opportunity to investigate potential virulence determinants by comparing the isolates’ phenotypic and genomic characterizations. 2. Materials and methods 2.1. Clinical samples, cells, and viruses Clinical samples were collected from two HFMD patients in Zhujiang hospital in Guangdong, China in 2009. Both patients were diagnosed with HFMD based on clinical manifestations and laboratory tests. Human rhabdomyosarcoma (RD) cells (ATCC, CCL-136TM ) and African green monkey kidney Vero cells (ATCC, CCL-81TM ) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a 5% CO2 atmosphere. The two CA16 viruses (GD09/24 and GD09/119) were isolated in Vero cells and passaged once before in vitro and in vivo characterization.
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antibody assay (IFA) was performed. RD cells were incubated with CA16 antisera for 1 h at 37 ◦ C. The cells were then incubated with fluorescein isothiocyanate (FITC)-labeled anti-mouse IgG (ZSBIO) at a dilution of 1:300 in Evans Blue for another 30 min. Finally, the cells were rinsed again and visualized under a fluorescent microscope (Olympus) after being dried at room temperature.
2.5. Virus titration Virus titers were determined using a microtitration assay and are represented as 50% tissue culture infectious doses (TCID50 values) in tissue culture or plaque-forming units (PFUs) in plaque assays. Briefly, serially diluted virus samples were used to infect RD cells in 96-well plates, and quadruplicate samples were used for each dilution. The 96-well plates were incubated for 3 days at 37 ◦ C, and the TCID50 values were measured by determining the cytopathic effects. The TCID50 values were calculated using the Reed–Muench method (Reed and Muench, 1938). The plaque-forming assay in RD cells was performed as previously described (Han et al., 2011). Briefly, approximately 4 × 105 cells were incubated overnight and were infected with serially diluted virus suspensions. After adsorption for 1 h, the virus suspension was replaced with DMEM containing 2% FBS and low melting point agarose. The medium was removed 72 h post-infection, and the cells were fixed in 10% formaldehyde and subsequently stained with 1% crystal violet solution. The titer of the virus was expressed as PFU/ml.
2.2. RT-PCR and DNA sequencing To sequence the CA16 isolates, total RNA from CA16-infected RD cells was extracted using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. cDNA was produced by reverse transcription using a PrimeScript RT-PCR Kit (Takara) with an oligo (dT) primer. The overlapping DNA fragments were produced by polymerase chain reaction (PCR) with LA Taq DNA polymerase (Takara). All of the primers used in this study are listed in the supplemental Table. The cycling parameters were denaturation for 3 min at 94 ◦ C, followed by 30 cycles of 30 s at 94 ◦ C, 30 s at 55 ◦ C, and 1 min at 72 ◦ C, and a final extension for 7 min at 72 ◦ C. The PCR products were sequenced and assembled using SeqMan software. The UTRs were determined using a 5 /3 -Rapid Amplification of cDNA Ends (RACE) Kit (Roche). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres. 2013.10.020. 2.3. Genome sequence analysis Homology analysis of the CA16 strains was performed using ClustalX and MegAlign software. A phylogenetic tree was generated using the Molecular Evolutionary Genetics Analysis (MEGA) program via the maximum likelihood and the neighbor-joining methods. The EV71 prototype strain BrCr was set as the outgroup. A total of 26 CA16 strains were collected from GenBank and included in the analysis. Homologous recombination was analyzed using Simplot software. RNA secondary structure prediction was performed using RNAfold, and the structures were drawn with RNAStructure. 2.4. IFA To identify the CA16-specific antigen protein, human RD cells were infected with the CA16 isolates, and an indirect fluorescent
2.6. One-step growth curve One-step growth curves of the CA16 isolates were performed as previously described (Ye et al., 2012). Briefly, RD cell monolayers in 6-well dishes were washed with DMEM and inoculated at an MOI (multiplicity of infection) of 1. After adsorption for 1 h, the cells were thoroughly washed to remove the unbound virus and were cultured at 37 ◦ C for 24, 48, and 72 h. The supernatants were collected, and the virus titers were determined as described above.
2.7. Animal experiments One-day-old BALB/c mice were obtained from the Beijing Laboratory Animal Center. All animal experiments were approved by and performed according to the guidelines of the Animal Experiment Committee of State Key Laboratory of Pathogen and Biosecurity. Groups of mice (n = 5) were inoculated with 10-fold serial viral dilutions via intracranial (i.c.) or intraperitoneal (i.p.) injection. All animals were then monitored for clinical symptoms, paralysis, and death for up to 14 days postinoculation. The survival curves were drawn using GraphPad software, and the 50% lethal doses (LD50 values) for each virus strain were calculated using the Reed–Muench method (Reed and Muench, 1938). For the dead mice, the intact brain was dissected out and rinsed with PBS three times before further assay.
2.8. Nucleotide sequence accession numbers The complete genomic sequences of the CA16/GD09/24 and CA16/GD09/119 isolates described in this study were deposited into GenBank under the accession numbers KC117317 and KC117318, respectively.
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Percent survival
100 80 60 40
GD09/119 GD09/24
20 0
0
2
4
6
8
10
12
14
Days post-inoculation Fig. 2. Mice survival curves of the CA16 strains via intracranial (i.c.) injection. Oneday-old BALB/c mice were intracranially administered 100 PFU virus, and mortality was monitored for 14 days. The survival curves were created using GraphPad software (version 5.0).
Fig. 1. In vitro plaque phenotypes and propagation characteristics of the CA16 isolates. (a) Plaque morphology of the CA16 isolates. A standard plaque-forming assay was performed in RD cells as described elsewhere. (b) One-step growth curve analysis of the CA16 strains in RD cells. Virus supernatants were collected after 24, 48, and 72 h, and the virus titers are expressed as TCID50 /ml.
3. Results
significantly higher compared to that of GD09/24. All of the mice died within 5 days of infection with GD09/119, while 60% of the mice challenged with an equal dose of GD09/24 survived. Virus recovery and genome sequencing of the brains of dead mice confirmed the CA16 infection (data not shown). Importantly, mice receiving i.p. inoculation with varying doses of the CA16 isolates also developed the typical central nervous system (CNS) clinical manifestations, similar to the i.c. infection. The mortality and average survival times of the mice infected with GD09/119 and GD09/24 are shown in Table 1. According to the LD50 of each virus, GD09/119 was approximately 100-fold more virulent than GD09/24. Furthermore, CA16-specific genes could be detected by RT-PCR in the brain and skeletal muscles of dead mice (data not shown), and CA16 was isolated from the brains of dead mice. Collectively, these results demonstrated that although the two clinical CA16 isolates presented similar growth characteristics in vitro, GD09/119 was more virulent than GD09/24 in mice.
3.1. In vitro characterization of the CA16 clinical isolates Two CA16 strains (GD09/24 and GD09/119) were isolated using Vero cells from anal swab samples of HFMD patients. IFA with human convalescent phase sera (1:100 dilution) from confirmed patients with CA16 infections revealed that the CA16-specific viral proteins were expressed in both GD09/24- and GD09/119-infected cells (data not shown). Then, both strains were inoculated into CA16-susceptive RD cells, and the typical cytopathic effects, such as cell rounding, aggregation, and fall-off, appeared 24 h post infection. The plaque-forming assay revealed that both isolates yielded similar small plaque morphologies (Fig. 1a). Furthermore, one-step growth curve analysis showed similar replication kinetics for both isolates in RD cells (Fig. 1b). Both viruses peaked at 72 h postinfection on RD cells, and the highest titer was approximately 6 log TCID50 /ml. 3.2. Virulence comparison of the CA16 isolates in mice The suckling mouse model of CA16 infection was recently described by Mao et al. (Mao et al., 2012). To further characterize the two CA16 clinical isolates, both viruses were inoculated into groups of one-day-old BALB/c mice via i.p. or i.c. routes. Following i.c. infection, both GD09/24 and GD09/119 were both virulent to suckling mice, resulting in the typical neurological symptoms, including significant weight loss, hind limb paralysis, moribund demeanor, and death. As shown in Fig. 2, the mortality of GD09/119 was
3.3. Phylogenetic and homologous recombination analyses Furthermore, to compare the genomic characteristics of these newly isolated CA16 strains, the complete genome sequences of GD09/119 and GD09/24 were determined and deposited in GenBank under the accession numbers KC117317 and KC117318, respectively. The genomic RNAs of both strains were 7409 bp in length, and the 5 -UTR was 745 bp, while the 3 -UTR was 82 bp. BLAST analysis indicated that the two isolates were most homologous with the CA16 strain SZ/HK08-7 (GQ279371), which was isolated in Shenzhen city, Guangdong, China in 2008, with nucleotide identities of 95.6% and 96.5% to GD09/119 and GD09/24, respectively. The nucleotide sequence identities among the different CA16 strains recorded in GenBank (a total of 28 strains) ranged from 78.8% to 96.5%. Maximal amino acid identity (>99.7%) of VP1 was noted among the isolates from the Guangdong region (including shzh05-1, GZ08, and SZ/HK08-7). Amino acid sequence homology analysis revealed 95.9% identity in the 3D proteins and 96.5% identity in the non-structural proteins among the isolates. Importantly, phylogenetic analysis was performed based on both the complete VP1 gene (Fig. 3a) and the complete genome (Fig. 3b) by either the neighbor joining method or the maximum likelihood method using the MEGA program. The results indicated that all CA16 strains circulating in China belonged to the B genotype; the two newly isolated strains, GD09/119 and GD09/24, were both within the B1b genotype (Fig. 3a and b).
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Table 1 Mice challenged with CA16 strains via i.p. route. Strain
Virus dose (PFU)
LD50 (PFU/ml)
AST (d)
GD09/24
104 103 102 10 1
5/5 (100) 2/5 (40) 1/5 (20) 0/5 (0) 0/5 (0)
Death/total mice (mortality, %)
1.48 × 103
6.2 11.6 12.6 14.0 14.0
GD09/119
104 103 102 10 1
5/5 (100) 5/5 (100) 5/5 (100) 2/5 (40) 0/5 (0)
14.8
6.6 7.4 9.0 12.6 14.0
AST, average survival time.
Homologous recombination events occur frequently within enterovirus species (Simmonds and Welch, 2006; Han et al., 2012). Thus, homologous recombination analysis of the two CA16 isolates with other human enterovirus species was performed using SimPlot software. Similarity scanning analysis using GD09/119 as a query sequence showed that most of the 5 half of the genome (nt 750–3600) had high similarity (77.4%) to the CA16 prototype strain G-10 and that the 3 half (nt 3600–7400) of the genome showed high similarity (83.5%) to the EV71 strain BrCr (Fig. 4a). Additionally, bootscanning results revealed that GD09/119 was most closely related to CA16 strain G-10 in the P1 region and to EV71 strain BrCr in the P2 and P3 regions (Fig. 4b). Genetic algorithms for the detection of recombination indicated that the putative breakpoints were
located within the 2A domain at approximately nt 3620. A similar recombination pattern was observed when using GD09/24 as a reference sequence (data not shown). The above analysis demonstrated that the CA16 strains were possibly progeny of intertypic and intratypic recombination events involving the EV71 and CA16 prototypes. 3.4. Genome comparison and virulence determinant analysis To reveal the potential relationship between the genome sequence and pathogenicity phenotypes of these newly isolated CA16 isolates, the complete genome sequences of GD09/119 and GD09/24 were compared in detail. Sequence homology analysis
Fig. 3. Phylogenetic analysis of selected human CA16 strains from different origins. The neighbor-joining tree was generated using MEGA 5.0 software, and prototype EV71 strains were used as the outgroup. (a) Phylogenetic tree based on complete VP1 sequences. (b) Phylogenetic tree based on complete genome sequences. Both GD09/24 and GD09/119 belonged to the B1b subtype.
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J.-F. Han et al. / Virus Research 179 (2014) 212–219 Table 2 Sequence comparison of 5 UTR between CA16 strains. Nucleotide position
Nucleotide change
56 63 71 91 94 100 104 123 153 158 199 232 265 270 275 419 513 517 659 669 671 686 703 719 726
GD09/24
GD09/119
C G T C C T C C A C C T T A A T A C C C G T C C C
T A C T T C T T G T T C C G G C G T T T A C T T T
Table 3 Amino acids sequence comparison between CA16 strains.
Fig. 4. Recombination analysis of CA16. (a) Similarity scanning analysis of CA16 with representative enterovirus strains on the basis of the full-length genome sequences. (b) Bootscanning analysis indicated recombination with EV71 within the nonstructural regions; bootscanning analysis was performed with a window size of 200 nt and a step of 20 nt. The breakpoint was at approximately nt 3620 in the 2A region.
revealed that the complete genome nucleotide identity between the new CA16 isolates was 95.7% and that the amino acid homology was 99.2%. The 3 -UTRs were highly conserved between the two strains, but there were 25 nucleotides that were different in the 5 -UTRs, as shown in Table 2. However, RNA secondary structure predictions using RNAfold software indicated little topological difference between the strains (data not shown). All of the amino acid differences within the open reading frames (ORFs) between the two strains are shown in Table 3. Regarding VP1, the nucleotide identity between GD09/119 and GD09/24 was 95.2%, and the amino acid identity was 99.7%. There were 43 nucleotide changes in the VP1 gene between the newly isolated CA16 strains, but only one amino acid substitution at position 220 (methionine for GD09/24 and leucine for GD09/119) was identified. Sequence alignment with other CA16 strains revealed that VP1L220 was highly conserved among the genotype B strains, except for GD09/24; meanwhile, all genotype A CA16 strains (G-10 and FY18) contained an alanine residue at VP1-220 (Fig. 5). According to the crystal structure of EV71, L220 is located on the GH loop, near the pocket factor-binding site of the VP1 protein. A total of 86 nucleotide changes were present in the VP4, VP2, and VP3 sequences, but all were silent mutations. There were only single amino acid substitutions in 2B (T/M), 3A (S/N), and 3C (N/T), and there were four amino acid substitutions in 2A. Additionally, a total of nine amino acid substitutions were identified within the 3D gene (Table 3). Interestingly, GD09/24 was consistent with the
Coding protein
Nucleotide position
VP4 VP2 VP3 VP1 2A
– – – 220 75 82 129 132 15 – 11 – 55 39 92 169 172 259 323 342 346 451
2B 2C 3A 3B 3C 3D
Amino acid change GD09/24
GD09/119
M T I N M T
L S V S V M
S
N
N K V R K P H L K Y
T E T K R S N S R F
genotype A strain FY18 and strain G-10 at the 3D positions 342(L) and 451(Y), while an amino acid substitution at K39E resulted in a change in polarity. The functional roles of these amino acids remain unknown. 4. Discussion In this study, two CA16 strains were isolated from HFMD patients in Guangdong, China. In vitro characterization confirmed their similar growth properties; however, in vivo tests revealed that these viruses had distinct virulence patterns in mice. These two CA16 strains were both from HFMD patients with mild clinical manifestations, and whether there is any relationship between their virulence phenotypes and disease severity remains unknown.
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Fig. 5. Sequence alignments of selected enterovirus VP1 genes. MegAlign software was used to align different sequences using the Clustal W method. Amino acid 220 of the VP1 gene of the GD09/24 strain was distinct from the other strains, with the exception of strains in subgenotype A.
Previous investigations have indicated no significant differences among the EV71 isolates from fatal and non-fatal cases (Shih et al., 2000; Singh et al., 2002), which is in agreement with our findings in this study. Meanwhile, the in vitro characteristics may be not associated with enterovirus virulence in vivo, which suggests the importance of in vivo phenotypic differences in pathogenesis. In mainland China, CA16 has been recognized as the major causative agent of HFMD during the last century (Zhang et al., 2010a), and CA16 subgenotype B1 predominantly circulates in mainland China (Li et al., 2005; Zong et al., 2011). Our results indicated that the newly isolated CA16 strains belong to genotype B1b and that these strains are closely related to other CA16 strains isolated in mainland China (Fig. 4). Both genotypes B1a and B1b have been identified in Guangdong, China. These results demonstrated that the CA16 strains have been endemic in the Guangdong region for a long time. Recombination among the different enteroviruses has been well documented (Zhang et al., 2010a, 2010b; Hu et al., 2011; Zhao et al., 2011; Chen et al., 2010). Phylogenetic and recombination analysis of the CA16 strains circulating in China revealed that the current CA16 strains were progeny of homologous recombination within enterovirus subgroup A, especially with EV71 (Zhao et al., 2011). Our study revealed that recombination between CA16 and EV71 may be a frequent phenomenon among HFMD pathogens and may be a possible reason for the variation in virulence.
Virulence investigation usually involves the comparison of viral genome sequences using different virulent phenotypes or origins (Oberste, 2008). Complete genome comparison revealed that the virulence-associated sites of the enteroviruses were present in both the UTRs and the polyprotein-coding regions. Li et al. performed comparative genomic analysis of EV71 strains from severe cases and mild cases; their analysis indicated the critical roles of G/Q/R710 and E729 in VP1, K930 in protease 2A, and three nucleotide changes in the 5 -UTR (Li et al., 2011). Previously, Arita et al. suggested that a single mutation in the VP1 (G145E) gene of EV71 was essential for the mouse-adapted phenotype in NOD/SCID mice (Arita et al., 2008). Zaini et al. recently demonstrated that a single mutation in the VP1 (Q145E) gene could generate a virulent phenotype in fiveday-old BALB/c mice (Zaini and McMinn, 2012). Another amino acid residue in VP1 (K244E) was recently identified as a critical determinant of mouse adaptation and virulence through comparison with full-length genomes sequenced directly from different tissues and blood specimens (Cordey et al., 2012). Currently, little is known about the virulent determinants of CA16, and the crystal structure of the CA16 virion remains unknown. Recently, a comparison study of the CA16 strains isolated from Guangxi, China in 2010 suggested that nucleotide variations in the UTR regions and amino acid substitutions within the VP1 gene may be responsible for differences in virulence (Yang et al., 2012). In our study, L220 in the VP1
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gene was highly conserved among most CA16 genotype B strains, except for GD09/24, while the CA16 genotype A strains (G-10 and FY18) contained A220 at the same position (Fig. 5). In particular, residue 220 of the VP1 protein is located within the neutralization epitope (Shi et al., 2013), and passive transfer of antisera against this epitope conferred significant protection against either EV71 or CA16 challenge in mice (Foo et al., 2007; Tian et al., 2012). Recently, the mouse-adapted virulent CA16 strain MAV was developed (Cai et al., 2013); at residue 220, the MAV strain (L) was identical to GD09/119 (L) but different from GD09/24 (M). The crystal structure of EV71 indicated that L220 of VP1 is located in the GH loop and is exposed on the surface of the mature virus, near the pocket factor-binding site (Wang et al., 2012). The functional importance of this amino acid merits further investigation. The enterovirus 3D protein acts as a viral RNA-dependent RNA polymerase (RdRp) and plays a major role in viral RNA synthesis and uridylylation. In our study, a total of nine amino acid substitutions in the 3D protein were identified between the two CA16 strains; however, none of these substitutions were associated with RNA synthesis and uridylylation. The association of these differences with virulence in mice remains unknown. Previous studies suggested that the low fidelity of the 3D polymerase is essential for the high rate of mutation, which aids in the survival of the virus population in the presence of selective pressure (Pfeiffer and Kirkegaard, 2005). Mutations in the 3D region of poliovirus resulted in temperature susceptibility and viral attenuation in mice (Diamond and Kirkegaard, 1994). Comparison of the amino acid sequences of the 3D polymerases of EV71 clinical isolates revealed that the I251T substitution resulted in a strong temperature-sensitive phenotype, which might contribute to virulence attenuation (Kung, 2010). Both biochemical and reverse genetic studies are needed to clarify the critical roles of these amino acid substitutions in the CA16 strains (Liu et al., 2011). In conclusion, two Chinese CA16 strains from HFMD patients in Guangdong, China were isolated and characterized in vitro and in vivo. Phylogenetic and homologous recombinant analyses indicated that the CA16 isolates circulating in mainland China belonged to the genotype B1b and are likely the progeny of CA16 G-10. Combined with whole genome sequences and virulence in mice, potential associations revealed possible virulence-related candidate sites in the two strains. These findings provide insight into the evolutionary relationships among the CA16 isolates in China and provide useful templates for virulence variation research. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81000721 and 31270195) and the Natural Science Foundation of Beijing (Nos. 7112108 and 7122129). CFQ was supported by the Beijing Nova Program of Science and Technology (No. 2010B041). References Arita, M., Ami, Y., Wakita, T., Shimizu, H., 2008. Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model. J. Virol. 82 (4), 1787–1797. Arita, M., Shimizu, H., Nagata, N., Ami, Y., Suzaki, Y., Sata, T., Iwasaki, T., Miyamura, T., 2005. Temperature-sensitive mutants of enterovirus 71 show attenuation in cynomolgus monkeys. J. Gen. Virol. 86 (Pt 5), 1391–1401. Cai, Y., Liu, Q., Huang, X., Li, D., Ku, Z., Zhang, Y., Huang, Z., 2013. Active immunization with a Coxsackievirus A16 experimental inactivated vaccine induces neutralizing antibodies and protects mice against lethal infection. Vaccine 31 (18), 2215–2221. Chan, Y.F., Wee, K.L., Chiam, C.W., Khor, C.S., Chan, S.Y., Amalina, W., Sam, M.Z.I.C., 2012. Comparative genetic analysis of VP4 VP1 and 3D gene regions of enterovirus 71 and coxsackievirus A16 circulating in Malaysia between 1997–2008. Trop. Biomed. 29 (3), 451–466.
Chang, L.Y., Lin, T.Y., Huang, Y.C., Tsao, K.C., Shih, S.R., Kuo, M.L., Ning, H.C., Chung, P.W., Kang, C.M., 1999. Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998. Pediatr. Infect. Dis. J. 18 (12), 1092–1096. Chen, X., Zhang, Q., Li, J., Cao, W., Zhang, J.X., Zhang, L., Zhang, W., Shao, Z.J., Yan, Y., 2010. Analysis of recombination and natural selection in human enterovirus 71. Virology 398 (2), 251–261. Cordey, S., Petty, T.J., Schibler, M., Martinez, Y., Gerlach, D., van Belle, S., Turin, L., Zdobnov, E., Kaiser, L., Tapparel, C., 2012. Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection. PLoS Pathog. 8 (7), e1002826. De Jesus, N.H., 2007. Epidemics to eradication: the modern history of poliomyelitis. Virol. J. 4, 70. Diamond, S.E., Kirkegaard, K., 1994. Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperaturesensitive mutants defective in RNA synthesis. J. Virol. 68 (2), 863–876. Foo, D.G., Alonso, S., Chow, V.T., Poh, C.L., 2007. Passive protection against lethal enterovirus 71 infection in newborn mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect. 9 (11), 1299–1306. Goto, K., Sanefuji, M., Kusuhara, K., Nishimura, Y., Shimizu, H., Kira, R., Torisu, H., Hara, T., 2009. Rhombencephalitis and coxsackievirus A16. Emerg. Infect. Dis. 15, 1689–1691. Halim, S., Ramsingh, A.I., 2000. A point mutation in VP1 of coxsackievirus B4 alters antigenicity. Virology 269 (1), 86–94. Han, J.F., Cao, R.Y., Deng, Y.Q., Tian, X., Jiang, T., Qin, E.D., Qin, C.F., 2011. Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo. Virol. J. 8, 106. Han, J.F., Jiang, T., Fan, X.L., Yang, L.M., Yu, M., Cao, R.Y., Wang, J.Z., Qin, E.D., Qin, C.F., 2012. Recombination of human coxsackievirus B5 in hand, foot, and mouth disease patients, China. Emerg. Infect. Dis. 18 (2), 351–353. Hu, Y.F., Yang, F., Du, J., Dong, J., Zhang, T., Wu, Z.Q., Xue, Y., Jin, Q., 2011. Complete genome analysis of coxsackievirus A2, A4 A5, and A10 strains isolated from hand, foot, and mouth disease patients in China revealing frequent recombination of human enterovirus A. J. Clin. Microbiol. 49 (7), 2426–2434. Knowles, N.J., et al., 2011. Picornaviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J. (Eds.), Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, CA, pp. 855–880. Kung, Y.H., Huang, S.W., Kuo, P.H., Kiang, D., Ho, M.S., Liu, C.C., Yu, C.K., Su, I.J., Wang, J.R., 2010. Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase. Virology 396 (1), 1–9. Li, L., He, Y., Yang, H., Zhu, J., Xu, X., Dong, J., Zhu, Y., Jin, Q., 2005. Genetic characteristics of human enterovirus 71 and coxsackievirus A16 circulating from 1999 to 2004 in Shenzhen, People’s Republic of China. J. Clin. Microbiol. 43, 3835–3839. Li, R., Zou, Q., Chen, L., Zhang, H., Wang, Y., 2011. Molecular analysis of virulent determinants of enterovirus 71. PLoS One 6 (10), e26237. Liu, C.C., Tseng, H.W., Wang, S.M., Wang, J.R., Su, I.J., 2000. An outbreak of enterovirus 71 infection in Taiwan 1998 epidemiologic and clinical manifestations. J. Clin. Virol. 17 (1), 23–30. Liu, F., Liu, Q., Cai, Y., Leng, Q., Huang, Z., 2011. Construction and characterization of an infectious clone of coxsackievirus A16. Virol. J. 8, 534. Mao, Q., Wang, Y., Gao, R., Shao, J., Yao, X., Lang, S., Wang, C., Mao, P., Liang, Z., Wang, J., 2012. A neonatal mouse model of coxsackievirus a16 for vaccine evaluation. J. Virol. 86 (22), 11967–11976. Oberste, M.S., 2008. Comparative genomics of the coxsackie B viruses and related enteroviruses. Curr. Top. Microbiol. Immunol. 323, 33–47. Perera, D., Yusof, M.A., Podin, Y., Ooi, M.H., Thao, N.T., Wong, K.K., Zaki, A., Chua, K.B., Malik, Y.A., Tu, P.V., Tien, N.T., Puthavathana, P., McMinn, P.C., Cardosa, M.J., 2007. Molecular phylogeny of modern coxsackievirus A16. Arch. Virol. 152 (6), 1201–1208. Pfeiffer, J.K., Kirkegaard, K., 2005. Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog. 1 (2), e11. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493–497. Shi, J., Huang, X., Liu, Q., Huang, Z., 2013. Identification of conserved neutralizing linear epitopes within the VP1 protein of coxsackievirus A16. Vaccine 31 (17), 2130–2136. Shih, S.R., Ho, M.S., Lin, K.H., Wu, S.L., Chen, Y.T., Wu, C.N., Lin, T.Y., Chang, L.Y., Tsao, K.C., Ning, H.C., Chang, P.Y., Jung, S.M., Hsueh, C., Chang, K.S., 2000. Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res. 68 (2), 127–136. Simmonds, P., Welch, J., 2006. Frequency and dynamics of recombination within different species of human enteroviruses. J. Virol. 80 (1), 483–493. Singh, S., Poh, C.L., Chow, V.T., 2002. Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in Singapore (2000). Microbiol. Immunol. 46 (11), 801–808. Tian, X., Su, X., Li, X., Li, H., Li, T., Zhou, Z., Zhong, T., Zhou, R., 2012. Protection against enterovirus 71 with neutralizing epitope incorporation within adenovirus type 3 hexon. PLoS One 7 (7), e41381. Wang, C.Y., Li, Lu.F., Wu, M.H., Lee, C.Y., Huang, L.M., 2004. Fatal coxsackievirus A16 infection. Pediatr. Infect. Dis. J. 23 (3), 275–276. Wang, X., Peng, W., Ren, J., Hu, Z., Xu, J., Lou, Z., Li, X., Yin, W., Shen, X., Porta, C., Walter, T.S., Evans, G., Axford, D., Owen, R., Rowlands, D.J., Wang, J., Stuart, D.I., Fry, E.E., Rao, Z., 2012. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19 (4), 424–429.
J.-F. Han et al. / Virus Research 179 (2014) 212–219 Yan, X.F., Gao, S., Xia, J.F., Ye, R., Yu, H., Long, J.E., 2012. Epidemic characteristics of hand, foot, and mouth disease in Shanghai from 2009 to 2010: enterovirus 71 subgenotype C4 as the primary causative agent and a high incidence of mixed infections with coxsackievirus A16. Scand. J. Infect. Dis. 44 (4), 297–305. Yang, E.X., Zhao, H., Zhang, Y., et al., 2012. A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B). Sci. China Life Sci. 55, 336–342. Ye, Q., Li, X.F., Zhao, H., Li, S.H., Deng, Y.Q., Cao, R.Y., Song, K.Y., Wang, H.J., Hua, R.H., Yu, Y.X., Zhou, X., Qin, E.D., Qin, C.F., 2012. A single nucleotide mutation in NS2A of Japanese encephalitis-live vaccine virus (SA14-14-2) ablates NS1’ formation and contributes to attenuation. J. Gen. Virol. 93 (Pt 9), 1959–1964. Zaini, Z., McMinn, P., 2012. A single mutation in capsid protein VP1 (Q145E) of a genogroup C4 strain of human enterovirus 71 generates a mouse-virulent phenotype. J. Gen. Virol. 93 (Pt 9), 1935–1940. Zhang, J., Dong, M., Jiang, B., Dai, X., Meng, J., 2012. Antigenic characteristics of the complete and truncated capsid protein VP1 of enterovirus 71. Virus Res. 167 (2), 337–342.
219
Zhang, Y., Wang, D., Yan, D., Zhu, S., Liu, J., Wang, H., Zhao, S., Yu, D., Nan, L., An, J., Chen, L., An, H., Xu, A., Xu, W., 2010a. Molecular evidence of persistent epidemic and evolution of subgenotype B1 coxsackievirus A16-associated hand, foot, and mouth disease in China. J. Clin. Microbiol. 48 (2), 619–622. Zhang, Y., Zhu, Z., Yang, W., Ren, J., Tan, X., Wang, Y., Mao, N., Xu, S., Zhu, S., Cui, A., Zhang, Y., Yan, D., Li, Q., Dong, X., Zhang, J., Zhao, Y., Wan, J., Feng, Z., Sun, J., Wang, S., Li, D., Xu, W., 2010b. An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol. J. 7, 94. Zhao, K., Han, X., Wang, G., Hu, W., Zhang, W., Yu, X.F., 2011. Circulating coxsackievirus a16 identified as recombinant type a human enterovirus, China. Emerg. Infect. Dis. 17 (8), 1537–1540. Zong, W., He, Y., Yu, S., Yang, H., Xian, H., Liao, Y., Hu, G., 2011. Molecular phylogeny of Coxsackievirus A16 in Shenzhen China, from 2005 to 2009. J. Clin. Microbiol. 49 (4), 1659–1661.
