Circulating HFMD-Associated Coxsackievirus A16 Is Genetically and Phenotypically Distinct from the Prototype CV-A16

Human enteroviruses (HEV) have been linked to hand, foot, and mouth disease (HFMD) in the Pacific and Southeast Asia for decades. Many cases of HFMD have been attributed to coxsackievirus A16 (CV-A16, CA16), based on only partial viral genome determination. Viral phenotypes are also poorly defined. Herein, we have genetically and phenotypically characterized multiple circulating CV-A16 viruses from HFMD patients and determined multiple full-length sequences of these circulating viruses. We discovered that the circulating CV-A16 viruses from HFMD patients are genetically distinct from the proto-type CV-A16 G10. We have also isolated circulating CV-A16 viruses from hospitalized HFMD patients and compared their virological differences. Interestingly, circulating CV-A16 viruses are more pathogenic in a neonatal mouse model than is CV-A16 G10. Thus, we have found circulating recombinant forms of CV-A16 (CRF CV-A16) that are related to, but different from, the prototype CV-A16 G10 that have distinct biological phenotypes.

Human enteroviruses (HEV) belong to the picornaviridae family, which can be further divided into 12 subgroups on the basis of molecular typing: Enterovirus A, Enterovirus B, Enterovirus C,Enterovirus D, Enterovirus E, Enterovirus F, Enterovirus G, Enterovirus H, Enterovirus J,Rhinovirus A, Rhinovirus B and Rhinovirus C. CV-A16, along with enterovirus 71 (EV71), CV-A2 and CV-A4, are members of the Enterovirus A subgroup. The first and prototype CV-A16 strain, CV-A16 G10, was isolated in South Africa almost 60 years ago [9,10] and subsequently sequenced in 1994 [11]. CV-A16 viruses contain a single-stranded positive-sense RNA genome of approximately 7.4kb and only a single open reading frame followed by a poly A tract. The CV-A16 RNA genome can be divided into three main regions: a) two non-coding regions, the 59-UTR and the 39UTR; b) a structural region, P1 (VP1,VP2,VP3,VP4); and c) the non-structural regions P2 (2A,2B,2C) and P3 (3A,3B,3C,3D) [9]. Infection with CV-A16 occurs mainly through Fecal -oral, respiratory and direct personal contact routes. It can also be spread to newborn infants as a result of intrauterine exposure to the infected pregnant mother [1,4,12,13]. In addition to the classic hand, foot and mouth ulcerations, infection with this virus can also cause myocarditis, intractable shock and poliomyelitis-like paralysis [14,15].
Based on partial viral sequencing and/or serologic characterization, a significant proportion of the viruses from HFMD patients have been found to be related to the prototype CV-A16 G10 and have thus been classified as CV-A16 strains. Most reports of CV-A16 genetic characterization are based only on the VP1 or VP4 region [3,[16][17][18][19][20][21]. Based on the limited full-length CV-A16 viral sequences available from HFMD patients that were diagnosed after 2008, it has been determined that circulating CV-A16 strains are quite unrelated to the prototype CV-A16 G10 in most parts of the viral genome [22]. To define the full extent of the genetic changes that might exist within the circulating CV-A16 and to study the functional implications of recombination events within nonstructural regions of the genome (especially within viral accessary protein-coding sequences), full-length genome characterization of circulating CV-A16 is urgently needed to facilitate the development of drugs and/or vaccines to prevent and treat this disease.
In the present study, we have sequenced the full-length genome of eight new CV-A16 viruses from HFMD patients. Surprisingly, we have found that prevalent CV-A16 viruses are recombinants with circulating HEV-A viruses in which only one-third of the viral genome is related to CV-A16 G10. Furthermore, significant differences in pathogenicity were observed between the newly identified circulating recombinant CV-A16 viruses and the prototype CV-A16 G10 in a neonatal mouse model.

Ethics Statement and Sample Selection
This study has been approved by the Ethics Committee of the First Hospital of Jilin University. Written informed consent was obtained from the parents of all the children involved in our study. Eight throat swabs were collected from pediatric patients with clinical diagnoses of CV-A16-related HFMD at the First Hospital of Jilin University in 2010. All specimens were positive for CV-A16 VP1-based characterization assays [9] using a Coxackievirus A16 PCR Kit (DAAN Gene Co., Ltd. of Sun Yat-Sen University).

Viral RNA Extraction, Reverse Transcription-PCR, and PCR Amplification and Sequencing
The enterovirus sequencing strategy used has been previously described [23]. Extraction of total RNA from throat swabs was performed using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Viral cDNA was generated in a 20-ml reaction volume for 1.5 h at 42uC using Oligo-dT primers and Super-Script II reverse transcriptase (Invitrogen) according to the provided instructions. PCR reactions were performed in 50-ml reaction volumes containing 5 ml of cDNA, 2 U of recombinant Taq DNA polymerase (Takara, Japan), 200 pmol specific forward and reverse primers, 0.4 mM concentrations of deoxynucleoside triphosphates, 20 mM Tris-HCl (pH 8.4), 20 mM KCl, and 1.5 mM MgCl 2 . The cycling conditions consisted of 4 min at 95uC, followed by 35 cycles of 94uC for 30 s to 45 s, 50uC to 55uC for 30 s to 45 s, and 72uC for 1 to 2 min, and finally 72uC for 8 min. The PCR products were examined by agarose gel electrophoresis. The primers used for CV-A16 detection are listed in Table S1. The primers were numbered according to CV-A16 strain SHZH00-1. All amplicons were bidirectionally sequenced. Cycle sequencing was performed with the Big Dye Terminator cycle sequencing kit (Applied Biosystems) and an ABI3730 automated DNA sequencer (Applied Biosystems).

Bootscanning and Phylogenetic Analysis
Bootscanning and nucleotide similarity tests were carried out with the Simplot 3.5.1 program [24]. The window and step sizes were determined based on the intent of the analysis and the length of the sequences. The neighbor-joining method and the Kimura 2parameter model were selected for all bootscanning. To confirm the bootscanning results, genetic algorithm recombination detection (GARD) [25] analysis was also used to detect possible recombination breakpoints within the China CV-A16 sequences. Alignment was achieved with the MEGA4 program [26], using the ClustalW method. The length of nucleotides used for analysis varied, depending on the purpose of the particular analysis, and is clearly indicated in the Results section. All phylogenetic analyses were also performed with the MEGA4 program, using the neighbor-joining method and the Kimura 2-parameter as the model, unless otherwise specified. The phylogeny of each tree was determined for 1,000 replicates with random seeds. To determine potential recombination events, we use both RDP4 [27] and Simplot version 3.5.1 to analyze the recombination. RDP4 was screened with the default settings (standard Bonferroni correction, P values #0.05 were considered significant), For the Simplot, a window size of 500 and step size of 20 were used throughout.

Isolation of Viruses from HFMD Patients and Viral Characterization
Vero cells were used to isolate the Changchun024, Chang-chun045, and Changchun090 strains of the CV-A16 virus from the throat swabs of patients with HFMD in 2010. The viral samples were diluted in DMEM medium and filter-sterilized using 0.22-mm filters (Millipore, Bedlford, MA); 300 ml of each filtered sample was inoculated into T25 flasks containing approximately 50% confluent Vero cells. Cultures were monitored daily for evidence of cytopathic effect. Culture supernatants from infected Vero cells showing cytopathic effect were collected, aliquoted, viral-titered, and stored at 280uC. Viral titer was determined according to the Reed-Muench method [28]. CV-A16-G10 prototype virus were obtained from the ATCC. Titered viruses were also evaluated for replication in mouse L929 (ATCC #CCL-1), Vero (ATCC, cat. #CCL-81), and human SK-N-SH cells (ATCC, #HTB-11).

CV-A16-infected Neonatal Mouse Model and Pathological Analysis
One-day-old specific pathogen-free (SPF)-level imprinting control region (ICR) neonatal mice (weighing 1.8-2.0 g, provided by the Experimental Animal Center, College of Basic Medicine, Jilin University) were randomly divided into several groups of three litters per group and 8-10 neonatal mice per litter. The neonatal mice were injected intracerebrally with different viral strains or DMEM medium. Body weight, activity, limb paralysis, morbidity, and death were noted for 21 days post-injection [29,30]. Protocols involving animals used in this study were approved by the Insititute of Virology and AIDS Research Subcommittee of Research Animal Care.
For immunohistochemical analysis, tissue samples were embedded in optimal cutting temperature (OCT) compound and frozen in liquid nitrogen. The frozen tissue samples were then cut into 4mm sections, placed on poly-L-lysine-coated glass slides, and fixed in 3.7% paraformaldehyde. The endogenous peroxidase activity of the tissues was inhibited by treatment with hydrogen peroxide (2.5%). CV-A16 antigen was captured by rabbit anti-CV-A16 polyclonal antibody (made in our laboratory) and detected by a Streptavidin-Peroxidase anti-rabbit IgG Kit (Maixin, Fujian), followed by color development with diaminobenzidine for detection of the antigen-antibody reactions.
To detect the viral loads of challenged mice, all tissues (heart, liver, spleen, lung, kidney, brain, intestine, spine skeletal muscle, and hind-limb muscle) were individually weighed and homogenized in sterile phosphate-buffered saline, disrupted by freezethawing, and centrifuged. RNA was extracted from the samples using TRIzol (Invitrogen) and viral load was determined by realtime PCR as previously described [31,32].

Accession Numbers
The sequences of the eight CV-A16 stains have been entered into the GenBank database under accession numbers KF055238-KF055245.

Clinical Features of HFMD Patients Infected with CV-A16 from Changchun, China
Samples from all eight patients with HFMD were collected from the First Clinical Hospital of Jilin University in 2010. Their clinical features are listed in Table 1. All of the patients were children, ranging in age from 2 months to 4 years old. Most of them presented with typical symptoms of HFMD, such as skin rash, herpangina, myoclonic jerks, fever, and vomiting. Some of the patients had complications: one patient (changchun024) had viral meningitis, and another (changchun097) had bronchial pneumonia. Interestingly, one of the eight cases (changchun090) was a 2month-old child whose mother also had HFMD after delivery.

Circulating CV-A16 Sequences from Recent HFMD Patients do not Cluster with the Proto-type CV-A16 G10
To characterize circulating CV-A16 related to HFMD, fulllength viral sequences were obtained from these eight HFMD patients. Phylogenetic analysis indicated that these full-length CV-A16 sequences from Changchun are not closely related to the prototype CV-A16 G10. A neighbor-joining phylogenetic tree was constructed for the eight sequences, along with 19 reference sequences for HEV-A, using EV68 and poliovirus 1 as outliers (Fig. 1A). This analysis confirmed that all the new CV-A16 sequences indeed belonged to HEV-A, which includes CV-A16-G10 (Fig. 1A). However, clustering of these new CV-A16 sequences (Fig. 1A, blue line) was observed with EV71A (BrCr), EV71B (EV71/9/97/SHA89), and EV71C (S10862-SAR-98) (Fig. 1A, grey line), but not with CV-A16 G10 (Fig. 1A, red line). All the circulating CV-A16 (or CV-A16) strains reported since 1998 share the same recombination pattern and form a close cluster in phylogenetic trees (Fig. S1). The only distinct CV-A16 that is available is the prototype G-10 strain.

Further Analysis of CV-A16 Sequences from China
Since the new HFMD viral sequences contained the CV-A16 VP1 region (data not shown) but clustered within the HEV-A group but not with CV-A16-G10, we examined the viral sequences for evidence of recombination. Bootscanning was performed with the Simplot program to investigate the possibility of recombination within the changchun024 sequence. Various HEV-A sequences were used as reference sequences. indicated that the 59UTR of the changchun024 sequence was closely related to CA4 (Fig. 1B). The P1 region was more closely related to CV-A16-G10. However, the 39 half of the chang-chun024 sequence had some similarity to EV71A but not CV-A16-G10 (Fig. 1B). Such patterns were also observed for changchun029 (Fig. 1C) and the other viruses (data not shown). We therefore carefully examined the 59-UTR. Phylogenetic analysis of the fragment 24-718bp in the 59UTR showed a small cluster of circulating CV-A16 viruses with CA4 and CA14, with a bootstrap value of 74% (Fig. 2B). On the other hand, the P1 region of circulating CV-A16 viruses clearly clustered with CV-A16 G10 ( Fig. 2A). A more detailed bootscanning of the 59-UTR of changchun024 and changchun029 with a smaller window of 200bp showed a dominant CA4 sequence, but not CA14, in the 59-UTR of changchun024 and changchun029 viruses (Fig. 2C). Similar results were observed for the other circulating Changchun recombinant CV-A16 viruses (data not shown).

Detailed Analysis of the P2 and P3 Regions
The bootscanning results indicated that the P2 and P3 regions of circulating CV-A16 viruses showed no significant similarity to CV-A16 G10 (Fig. 1B,1C). To help pinpoint the possible origin of the P2 and P3 regions of the circulating CV-A16 viruses, the 39 sequences were further examined. A more detailed phylogenetic analysis of the P2 and P3 (Fig. 3A) regions demonstrated clustering of circulating CV-A16 viruses with a group of HEV-A viruses, including EV71A, CV-A2, CV-A3, CV-A6, CV-A10, and CV-A12, but not CV-A16 G-10. Bootscanning analysis further confirmed the possible relationship of the P2 and P3 regions of changchun024 to EV71A (Fig. 3B), CV-A2 (Fig. 3C), CV-A12 (Fig. 3D) and CV-A3, CV-A6, CV-A10 (Fig. S2). These results indicated a possible recombination in the P2 and P3 regions between CV-A16 and a virus that is related to EV71A, CV-A2, CV-A3, CV-A6, CV-A10, or CV-A12. Similar results were also observed for other circulating Changchun recombinant CV-A16 viruses (data not shown).
In order to reconfirm our conclusion, we used a series of algorithms in the RDP program, including RDP,GENECONV, BootScan, MaxChi, Chimaera, SiScan, PhylPro, LARD, and 3Seq, to detect the recombination events. The results confirmed those obtained with Bootscan in the Simplot program: when the sequence of changchun024 was analyzed in the absence of reference sequences CV-A2, CV-A3, CV-A6, CV-A10, and CV-A12, seven algorithms detected significant recombination with EV71A at the P2-P3 region; when the sequence of changchun024 was analyzed in the absence of reference sequences EV71A, CV-A3, CV-A6, CV-A10, and CV-A12, recombination was detected in two regions: five algorithms detected recombination with CV-A2 in the P2 region and four algorithms detected recombination with CV-A2 in the P3 region. This was consistent with the bootscan results. Of particular interest was the recombination with CV-A2 detected in the 3D region, which has not been previously reported. In a similar fashion, when the sequence of chang-chun024 was analyzed in the absence of reference sequences EV71A, CV-A2, CV-A3, CV-A6 and CV-A10, recombination with CV-A12 was detected using six algorithms. All of the recombination events had high P-values (Table 2).

Biological Characterization of CRF CV-A16 from HFMD Patients
Since circulating recombinant CV-A16 viruses differ from CV-A16 G10 genetically, we were interested in whether these viruses also differed in their biological properties. We evaluated viral replication in multiple cell lines. Like CV-A16 G10, CV-A16 changchun045 and CV-A16 changchun090 replicated and caused a cytopathic effect in African green monkey kidney Vero cells, a cell line sensitive to most enterovirus infections (Fig. 4A, 4B). The CPE started at 48 h after infection and peaked at 96 h after infection (Fig. 4B). A previous report has indicated that CV-A16 G10 cannot infect the mouse fibroblast cell line L929 [33]. Our data indicated that CV-A16 changchun045 and CV-A16 changchun090 also did not replicate or cause a cytopathic effect in L929 cells (Fig. 4B). When similar viral titers of various CV-A16 viruses were used to infect human neuroblastoma SK-N-SH cells, we found that that all the Changchun-circulating CV-A16 strains and CV-A16 G10 could induce an obvious cytopathic effect by 96 h post-infection (Fig. 4B). Thus, our results indicated that all the Changchun recombinant CV-A16 and CV-A16-G10 strains were unable to infect L929 cells, but they had the same ability to infect and induce cell death in human neuroblastoma cells.

Different Pathogenetic Outcomes in Neonatal Mice for the Prototype CV-A16-G10 and Circulating Recombinant forms of CV-A16
As described above, the Changchun circulating CV-A16 strains are recombinant and differ from the prototype CV-A16 G10 in most regions of the viral genome. Although viral replication in certain cell lines appeared to be unaltered by the recombination, whether these recombinants have different pathogenetic potential still needed to be evaluated. To address this issue, we established a mouse model of lethal CV-A16 infection in order to compare the pathogenesis of the prototype CV-A16 G10 and the Changchuncirculating CV-A16 strains. We found that CV-A16 chang-chun024, which was isolated from a HFMD patient with viral meningitis, was highly pathogenic in newborn mice. To evaluate any possible differences in viral pathogenesis, we established a grading system for evaluating the clinical condition of the mice: no abnormalities (grade 0), lethargy and inactivity (grade 1), wasting (grade 2), limb shaking and weakness (grade 3), hind-limb paralysis (grade 4), and moribund or dead (grade 5) [29]. The negative control, DMEM medium, and CV-A16 changchun024 in DMEM medium were injected intracerebrally into one-day-old neonatal mice. The negative control group showed no disease symptoms and had a 100% survival rate. The mice that were infected with CV-A16 changchun024 became sick on day 5 post-infection, with a mean clinical score of grade 4, and all were dead by day 10  (Fig. 5B). Similarly, all mice that were challenged with chang-chun045 and changchun090 developed clinical symptoms with a clinical score up to 4, and all were dead by day 8 (Fig. 5B). In contrast, the clinical symptoms of G10-infected mice were much less severe than those of mice infected with the circulating CV-A16 viruses (Fig. 5B). The majority of the mice infected with CV-A16 G10 survived after infection up to day 21 (Fig. 5A). Our results indicated that in this lethal-challenge mouse model, recombinant  Table 2. Summary of Changchun024 recombination events detected by RDP4.
circulating CV-A16 viruses were more pathogenic than the prototype CV-A16 G10. Consistent with these findings, the viral loads of different organs and blood from the CV-A16-infected newborn mice showed that dramatically higher viral loads were present in Changchun024-infected newborn mice compared to the organs of mice infected with the prototype CV-A16 G10 viruses (Fig. 6A). In particular, our data clearly indicated that circulating recombinant form CV-A16 had significantly higher replication activity in the spine muscle and hind limb muscle. Further immunohistochemical analysis also supported our results in the hind limb muscle of challenged mice (Fig. 6B). In general, CV-A16 Changchun024 had higher virus loads than CV-A16-G10 in all tested samples of organs and blood. In particular, the CV-A16 Changchun024 loads in the spine muscle and hind limb muscle from infected mice were much higher (.10 5 copies/mg tissue) than the loads in these muscles from CV-A16 G10-challenged mice, which contained no significant detection of CV-A16 G10. These findings are consistent with Changchun024 being much more virulent in this model system.

Discussion
Coxsackievirus A16, like other enteroviruses, shows a high mutation rate during viral replication due to the deficiency of proofreading activity. In the current study, we have determined eight full-length sequences of CV-A16 viruses from recent HFMD patients. Our study revealed significant differences in the genomic structures between the prototype CV-A16 G10 and the currently circulating CV-A16 viruses. Further analysis of several CRF CV-A16 viruses isolated from hospitalized patients revealed similar viral phenotypes, which were distinct from that of CV-A16 G10.
We first demonstrated that circulating CV-A16 viruses from recent HFMD patients in China are actually complex recombinant viruses involving multiple type A HEV, including CA4, CV-A16, and possibly EV71, CV-A2, CV-A3, CV-A6,CV-A10 or CV-A12. The 59UTR region of these viruses had the highest similarity to CV-A4. Most of the structural protein (P1) region resembled that of the prototype CV-A16 G10 strain. However, the non-structural protein domains (P2 and P3) showed similarity to multiple type A HEV viruses but not CV-A16 G10.
The detailed characterization of eight new CV-A16-related fulllength viral sequences as well as three earlier CV-A16-related sequences from China confirm the previous report [22] that the 59UTR region of the viral genome has the highest similarity to CA4. Interestingly, CA4 has been detected in HFMD patients in Zhejiang province (central China) [22] and in Gansu province (northwest China) [7]. Although the P1 regions of the currently circulating recombinant CV-A16 viruses are mostly related to the prototype CV-A16-G10, they still differ from CV-A16 G10 by almost 20% at the amino acid level. On the other hand, the P1 regions of all the circulating CV-A16 viruses have divergences from each other of less than 10%.
The non-structural protein domains (P2 and P3) of circulating CV-A16 viruses are most divergent from the same regions in CV-A16 G10. The origin of these P2 and P3 regions is also not clear. Recent reports have suggested that circulating CV-A16 strains are recombinant viruses that have recombined with different HEV-A type viruses. However, these reports are not in total agreement concerning the possible origin of the P2 and P3 regions in these circulating recombinant CV-A16 viruses. Chan Yoke-Fun et al. and Cyril C.Y.Yip et al. claimed that the P2 and P3 regions of circulating CV-A16 strains in China were mainly from EV71A [34,35]. On the other hand, Ke Zhao et al. found that only a 1.5kb fragment (4,406-5,585 bp) of these nonstructural domains was similar to EV71A [34,35]. In the present study, we have observed a similar pattern for the newly identified circulating recombinant CV-A16 viruses (Fig. 2). Based on phylogenetic analysis, the P2 and P3 regions of the circulating recombinant CV-A16 viruses are actually more closely related to a group of HEV type A viruses, including EV71A, CV-A2, CV-A3, CV-A6, CV-A10, and CV-A12 ( Fig. 2A). In this case, bootscanning results can be significantly influenced by which reference viral sequence is used ( Fig. 2B; Fig. S1). These findings emphasize the importance of using a more complete set of HEV reference sequences for bootscanning analysis. Since the exact functions of the nonstructural region (P2 and P3) in viral replication and pathogenesis are still not well defined, future studies should be encouraged to explore how and why so many enteroviruses share a similar nonstructural region.
Despite the fact that only a third of the viral genome of the circulating CV-A16 viruses shares similarity with CV-A16 G10, these viruses had a similar replication capacity in multiple mammalian cell lines. All the viruses replicated well in human neuroblastoma SK-N-SH cells and African green monkey kidney Vero cells, and none could efficiently replicate in the mouse fibroblast cell line L929. Thus far, two cellular receptors have been identified for CV-A16 and EV71 viruses: human scavenger receptor B2 (SCARB2) and P-selectin glycoprotein ligand-1(PSGL-1) [33,36,37]. Unlike PSGL-1, which is mainly expressed in lymphoid cells, SCARB2 is widely expressed in both EV71 and CV-A16 target cells. However, mouse SCARB2 (unlike human SCARB2) could not support efficient EV71 or CV-A16 infection in L929 cells. It is likely that the cellular tropism of the circulating CV-A16 viruses is similar to that of the prototype CV-A16 G10. . Disease and mortality rate differences in newborn mice caused by different strains of CV-A16. Differences in virulence between the prototype CV-A16-G10 and circulating recombinant forms of CV-A16 in neonatal mice were compared. One-day-old ICR mice were intracerebrally inoculated with 10 3.5 TCID50/ml G10, Changchun024, Changchun045, Changchun090 viruses in DMEM medium. The negative control mice were given medium instead of the virus suspension. The survival rates (A) and clinical scores (B) were monitored and recorded daily after infection for 21 days. The clinical score was graded as follows: 0: no abnormalities; 1: lethargy and inactivity; 2: wasting; 3: limb shake weakness; 4: hind-limb paralysis; 5: moribund or dead. Each group contained six to ten mice. doi:10.1371/journal.pone.0094746.g005 Surprisingly, circulating CV-A16 viruses were more pathogenic than the prototype CV-A16 G10 in neonatal mice despite similar replication patterns in tissue culture. Circulating CV-A16 viruses induced more disease-related symptoms in neonatal mice than did CV-A16 G10. Furthermore, whereas circulating CV-A16 viruses produced a 100% death rate in infected mice, the majority of the CV-A16-G10-infected mice survived. To our knowledge, this is the first example of a positive or negative effect of a CV-A16 recombinant on viral pathogenesis in an animal model. It is well known that many enterovirus recombinants have gained activity related to viral replication or spread in animal models, and, while the implications of these findings for human disease have yet to be determined, our results raise the possibility of evolution to increased virulence. The 59UTR and the nonstructural P2/P3 regions showed the most difference between CV-A16 G10 and the circulating recombinant CV-A16 viruses. The contribution of these regions to viral pathogenesis should be explored in future experiments. In addition, it is still not clear if the significantly higher viral loads in the spine muscle and hind limb muscle of CRF CV-A16-infected newborn mice are due to specific receptor expression or more suitable viral replication environments; similar results were also detected in our CV-A16 vaccine candidate studies [32]. It is equally interesting to note that multiple CV-A16 virus strains could cause a lethal infection in the neonatal mice, despite the fact that mouse PSGL-1 and SCARB2 are not expected to mediate efficient replication of CV-A16 viruses in these mice. Our findings raises the question of whether additional cellular receptors contribute to CV-A16 infection in our neonatal mouse model.
To date, there is still no effective vaccine or drug against CV-A16 infection. The significant differences among CV-A16 viruses in cellular infection systems and animal models may be important factors for vaccine development and evaluation. The establishment of animal models of lethal CV-A16 infection that could distinguish between different CV-A16 viral strains would also be critical for subsequent studies of the pathogenesis of CV-A16. Further elucidation of the mechanisms that govern the differences in these different model systems may also shed light on our understanding of CV-A16 enterovirus recombination and viral pathogenesis. One-day-old mice were intracerebrally inoculated with Changchun 024 or prototype G10 viruses. Virus loads were assessed by real time quantitative PCR in samples of the heart, liver, spleen, lung, kidney, brain, intestine, spine muscle, hind limb muscle and blood from the infected newborn mice. Samples were collected at the indicated time; (B) Immunohistochemical analysis of hind limb muscle from Changchun024 or CV-A16-G10 challenged newborn mice. doi:10.1371/journal.pone.0094746.g006