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A mouse muscle-adapted enterovirus 71 strain with increased


Microbes and Infection 13 (2011) 862e870 www.elsevier.com/locate/micinf

Original article

A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice
Wei Wang, Jianying Duo, Jiangning Liu, Chunmei Ma, Lianfeng Zhang, Qiang Wei**, Chuan Qin*
Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, No.5 Panjiayuan Nanli, Chaoyang Dist, Beijing 100021, PR China Received 20 December 2010; accepted 30 April 2011 Available online 12 May 2011

Abstract Enterovirus 71 (EV71) infections can usually cause epidemic hand, foot, and mouth disease (HFMD), and occasionally lead to aseptic meningitis, encephalitis, and polio-like illness. Skeletal muscles have been thought to be crucial for the pathogenesis of EV71-related diseases. However, little is known about the virulence of mouse muscle-adapted EV71. The EV71 0805 were subjected to four passages in the mouse muscle to generate a mouse-adapted EV71 strain of 0805a. In comparison with the parental EV71 0805, the mouse muscle-adapted EV71 0805a displayed stronger cytotoxicity against Rhabdomyosarcoma (RD) cells and more ef?cient replication in RD cells. Furthermore, infection with the EV71 0805a signi?cantly inhibited the gain of body weight, accompanied by increased muscle virus load and multiple tissue distribution in the infected mouse. Histological examinations indicated that infection with the EV71 0805 did not cause obvious pathogenic lesions in mice, while infection with the muscle-adapted 0805a resulted in severe necrotizing myositis in the skeletal and cardio muscles, and intestinitis in mice on day 5 post infection. Further analysis revealed many mutations in different regions of the genome of mouse muscle-adapted virus. Collectively, these data demonstrated the mouse muscle-adapted EV71 0805a with increased virulence in mice. ? 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Enterovirus 71; Mouse-adapted; Myotropism; ICR mouse

1. Introduction Enterovirus 71 (EV71), a member of the genus Enterovirus, family Picornaviridae [1e3], is the major causative agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease. Several outbreaks of HFMD have occurred in Eastern and Southeastern Asian countries and regions, including Singapore [4,5], South Korea [6], Malaysia [7], Japan [8], Vietnam [9], Taiwan [10], and China mainland [11]. Although EV71-mediated HFMD is usually a self-limited disease in most children, infection with EV71 can sometimes cause severe neurological diseases, such as brainstem encephalitis and polio-like paralysis [2,12]. Therefore, EV71 has become one of the most important enteroviruses known to
* Corresponding author. Tel.: ?86 10 67770815; fax: ?86 10 67710812. ** Corresponding author. Tel.: ?86 10 67776049; fax: ?86 10 67761136. E-mail addresses: weiqiang0430@sohu.com (Q. Wei), qinchuan001@ yeah.net (C. Qin).

cause fatalities in children, representing a major public health concern [13,14]. Following EV71 infection, the virus can invade into many organs and tissues in humans and rodents, including the brain, muscle, intestine, lung, and others [15,16], because multiple types of receptors and other factors contribute to the viral tissue tropism [17]. Previous studies have shown that EV71 in human and mouse skeletal muscles is associated with the development of persistent infection [18,19]. Interestingly, the mouse-adapted strain of EV71 predominately infect the skeletal muscles in ICR and NOD/SCID mice [16,20]. While EV71 infection-mediated severe neurological disease is a major lethal factor, the EV71 strains isolated from individual patients of epidemic poliomyelitis-like disease in Bulgaria displayed high tropism for mouse muscle tissues [2]. Furthermore, many EV71 strains, such as B3 and B4, as well as other clinical isolates, can infect mouse muscle, but not the brain [21,22]. Apparently, the muscle acts as a host organ for the initial viral replication, which is crucial for the subsequent

1286-4579/$ - see front matter ? 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2011.04.004

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neuroinvasion [19] and may play an important role in the EV71 infection in humans and mice. However, it is unclear whether mouse muscle-adapted EV71 strain could be generated and how virulent it could be. In addition, the molecular mechanisms underlying the myotropism of EV71 have not been fully understood. In this study, the parental EV71 strain Fuyang-0805 was ?rst passaged in mouse skeletal muscle for four times to generate a mouse muscle-adapted EV71 strain, Fuyang-0805a, with increased myotropism in mice. Subsequently, the phenotype, virulence, and molecular characteristics of the EV71 Fuyang-0805a strain were analyzed for understanding the adaptive evolution of EV71 and the potential mechanisms underlying the myotropism of EV71. 2. Materials and methods 2.1. Cells and viruses Human Rhabdomyosarcoma RD cells line, a major permissive cell line for the isolation and ease culture of clinical strains of EV71 [15,23e26], human colorectal carcinoma Caco-2 cells, African green monkey kidney Vero cells, and human neuroblastom SH-SY5Y cells were maintained in Dulbecco’s Modi?ed Eagle’s Medium (DMEM, Gibco) containing 10e15% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, 100 IU of penicillin, and 100 mg of streptomycin/ ml at 37  C, 5% CO2. The EV71 Fuyang-0805 (GenBank accession number FJ439769, a kind gift from the Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College) [27] was grown in RD cells. For preparation of mouse-adapted strain of EV71, 1-day-old ICR mice (Vital River Laboratory Animal Technology, Beijing, China) were inoculated intraperitoneally (i.p.) with 106 TCID50 of the parental strain of EV71 0805. Five days later, the mice were sacri?ced by aether anesthesia and their limb muscles were dissected out. After homogenization, the muscle lysates were added into the cultured RD cells for the isolation of the virus, which was designed as one passage. The viral strain derived from the fourth passages in mice was used and designated Fuyang-0805a (0805a). To prepare virus stocks, viruses were propagated for one more passage in RD cells to generate a virus stock at 108 TCID50 per ml. The experimental protocol was approved by the Animal Care and Use Committee of the Institute of Laboratory Animal Science of Chinese Academy of Medical Sciences. 2.2. Cell tropism, cytotoxicity and growth kinetics of EV71 To determine the cell tropism and cytotoxicities of EV71 strains, RD, Caco-2, Vero, and SH-SY5Y cells (2 ? 104 cells/ well) were cultured in 96-well plates for 18 h and infected in triplicate with 1 MOI of the strain 0805 or 0805a for varying periods, respectively. The cytotoxicity of these viruses was monitored longitudinally for the cytopathic effect (CPE) and

using a colorimetric assay kit, according to the manufacturers’ instruction (Cell Counting Kit-8; Dojindo, Kumamoto, Japan). Monolayer RD cells at 106 cells/well were cultured in 24well plates and infected in triplicate with 100 TCID50 of the EV71 0805 or 0805a for 1 h at 37  C, respectively. The cells were washed with phosphate buffered saline (PBS) and then cultured in 2% FBS DMEM for varying periods. The cells were harvested and the virus titers were determined by a realtime RT-PCR assay, as described below. 2.3. Virus inoculation and detection in mice ICR mice at one day of age (n ? 120) were fasted for 4 h and inoculated i.p. with 105 TCID50 of EV71 strain 0805 or 0805a (50 ml), respectively. The control mice (n ? 60) were injected with the same volume of RD cell lysate. Their body weights and clinical signs, including ruf?ed fur, hunchbacke, wasting, limb weakness, limb paralysis, moribund and death, were monitored daily up to 9 days after inoculation. In addition, 10 mice per group were sacri?ced on day 0, 1, 3, 5, and 7 post-inoculation, respectively. After perfusion with PBS containing EDTA, their brain, lung, limb muscle, heart, and intestine tissues were immediately dissected out for the extraction of RNA or for histopathological and immunohistochemical examinations, respectively. Total RNA was extracted from different tissues (30 mg) using an RNeasy Mini kit (Qiagen, Hilden, Germany). The RNA was assayed by one-step RT-PCR in a 20-ml reaction mixture (QIAGEN QuantiTect SYBR Green RT-PCR kit) with primers of EV71-S (50 -GCAGCCCAAAAGAACTTCAC-30 ) and EV71-A (50 -ATTTCAGCAGCTTGGAGTGC-30 ) for EV71/BrCr of nucleotides 2372e2598 [11,28], and the conditions consisted of a denaturation step at 95  C for 20 s and 40 cycles of thermal cycling of 95  C for 3 s and 60  C for 30 s. The EV71 virus fragment of nucleotides 2372e2598 was used as real-time PCR standard by adjusting to a concentration gradient of 1 ? 106 copies/ml, 1 ? 105 copies/ml, 1 ? 104 copies/ml, 1 ? 103 copies/ml, and 1 ? 102 copies/ml, and the DNA fragment with known copies was used as standard to calculate the copy number of virus RNA in the infected tissues. Quantitative real-time RT-PCR was performed using the LightCycler system. The limitation of virus detection was 100 copies. 2.4. RT-PCR analysis of EV71 Total RNA was extracted from individual brain, muscle, intestine, and lung samples using a RNeasy Mini kit, according to the manufacturers’ instruction (Qiagen, Hilden, Germany), and reversely transcribed into cDNA using Avian myeloblastosis virus reverse transcriptase (Takara, Dalian, China). The VP1 gene of EV71 was ampli?ed by PCR using 1 ml of cDNA sample, as the template and the primers were same with the real-time PCR. The PCR products were subjected to electrophoresis on 1.5% agarose gels. The mouse GAPDH was used as an internal control.

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2.5. Histopathology The collected tissue samples were ?xed in 10% buffered formalin for 48 h and embedded in paraf?n. The tissue sections at 5 mm were stained with hematoxylin and eosin (HE). Histopathology of mouse skeletal and heart muscles was evaluated for in?ammation, muscle ?ber degeneration and necrosis, while small intestines were evaluated for intestinal villus interstitial edema and epithelial cell vacuolar degeneration by pathologists in a blinded manner. Other tissue samples, including brain, liver, spleen and lung, were also select for histopathology examine. 2.6. Analysis of the viral genome sequences The full-length genome of the EV71 clinical isolates, strain 0805, and the mouse-adapted strain 0805a were characterized by sequencing, as described previously [29,30]. Brie?y, viral RNA was prepared using a high-pure viral RNA puri?cation kit (Qiagen, Hilden, Germany) and reversely transcribed into cDNA using a ReverTra-Plus kit (Invitrogen). The wholegenome of EV71 was ampli?ed by PCR using the speci?c primers [29,30]. The PCR products were puri?ed using the agarose gel DNA puri?cation kit (Takara, Dalian, China), and cloned into a pGEM-T vector (Promega, Madison, WI, USA), followed by transforming into Escherichia coli JM109. A total of 6e8 colonies from each virus library were characterized by DNA sequencing on a 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA). The DNA sequences were analyzed using DNAMAN 6.0 and Bioedit 7.0 [31]. The secondary structure was predicted using the GOR IV program (http://npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl) [32]. 2.7. Statistical analysis Data are expressed as mean ? SD. The difference between groups was analyzed with a ManneWhitney U test using the SPSS software. A p value of <0.05 was considered statistically signi?cant. 3. Results 3.1. Mouse muscle-adapted EV71 0805a with hypervirulence in vitro To examine the virulence and tissue tropism, a wild strain of EV71 0805 was passaged in mouse muscles for four passages to generate the mouse muscle-adapted EV71 strain 0805a, which was detected in the infected RD cells by indirect immuno?uorescent staining with anti-EV71 monoclonal antibody. Next, its cytotoxicity against different types of cells was compared with its parental EV71 0805 (Fig. 1). Both strains of EV71 0805 and 0805a appeared not to have obvious cytotoxicity against nerve cells SY5Y, because the proliferation of cells was similar regardless of infection with, or without, the virus. Furthermore, while infection with EV71 0805 promoted the proliferation of monkey kidney Vero cells, EV71 0805a

infection had no stimulating effect on the proliferation of Vero cells in vitro. More importantly, both EV71 0805 and 0805a displayed strong cytotoxicity against muscle RD and intestinal Caco-2 cells as compared to that of Vero and SY5Y cells, particularly for the EV71 0805a. Evidentially, the proliferation of both types of cells was signi?cantly reduced by 25e95%, as compared with that of un-manipulated cells, and the inhibitory effect of EV71 0805a was more potent than that of EV71 0805 in RD cells. Similar patterns of CPE were observed in different types of cells (data not shown). These data indicated that EV71 0805a had a higher muscle tropism. Characterization of the dynamics of virus replication revealed that while similar numbers of virus copies were detected in ?rst 48 h, the virions of EV71 0805a were signi?cantly greater than that of EV71 0805 at 72 and 96 h post infection and then returned to similar levels of replication in RD cells (Fig. 2). These demonstrated that EV71 0805a replicated faster in RD cells, which was associated with higher cytotoxicity of the virus against RD cells. 3.2. Mouse muscle-adapted EV71 0805a with increased virulence in vivo Next, the in vivo virulence of the EV71 0805 and 0805a was tested. ICR mice at one day of age were infected with EV71 0805 or 0805a, and they were monitored for survival and clinical symptoms up to 21 days post infection. There was no a single mouse that developed neurological symptoms and no a single mouse that was dead throughout the observation period, indicating the low pathogenesis of both strains of viruses in this strain of mice. Analysis of virus invasion revealed that while EV71 0805 was exclusively detected in the muscles, the EV71 0805a was found in the muscles, intestines, lungs, and brain (Fig. 3A), indicating that the mouse muscleadapted EV71 0805a had higher virulence in our experimental system. To further investigate the virulence of virus, ICR mice at one day of age were infected with EV71 0805 or 0805a, respectively. Their body weights were measured longitudinally (Fig. 3B). While mice infected with EV71 0805 grew as much as that of un-manipulated mice, the mice infected with EV71 0805a appeared to grow slowly. Evidentially, the body weights on day 5, 7, and 9 post infections were signi?cantly lighter than that of un-manipulated controls. Characterization of virus antigen in mice indicated that the EV71 0805a was detected in the muscles, heart and intestines, while the EV71 0805 was found only in the muscles of mice on day 5 post infection (data not shown). Speci?cally, analysis of virus in the limb muscle samples indicated that although similar dynamics of virus replication were observed in the EV71 0805 and 0805ainfected mouse muscles, the numbers of the EV71 0805a virions were signi?cantly greater than that of the EV71 0805 detected on day 1, 3, 5, and 7 post infection (Fig. 3C). Further histological examinations indicated that the limb muscles from the EV71 0805a-infected mice displayed massive and widespread necrotizing myositis with some in?ammatory in?ltrates on day 5 post infection (Fig. 4).

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120 100 80 60 40

*

Vero cell

*

*

RD cell
120 100 80 60 40 20

EV71 0805 EV71 0805a

Cell viability (% of control)

20 0 48 72 96

*
48

* *
72 96

0

120 100 80 60 40 20 0 48

SY5Y cell

120 100 80 60 40 20

Caco-2 cell

72

96

0

48

72

96

Hour pos t-infection

Fig. 1. Cytotoxicity of EV71 0805 and 0805a in vitro. Vero, RD, SY5Y, and Caco-2 cells were cultured in 96-well plates for 18 h and infected in triplicate with, or without, 1 MOI of EV71 0805 and 0805a for the indicated periods, respectively. The viabilities of individual types of cells following virus infection were determined using the CCK-8 assay. Data are expressed as mean %?SD of each group of cells from three independent experiments and the cultured cells without virus infection were designated as 100%. *p<0.05 vs. the cells infected with EV71 0805.

Similar pathogenic characteristics were observed in the myocardium from the EV71 0805a-infected mice. Interestingly, villous blunting and crypt hyperplasia were observed as early as at one day post infection (data not shown), and severe vacuolar degeneration of villus cells was detected in the small intestines of the EV71 0805a-infected mice on day 5 post infection (Fig. 4). However, there was no obvious abnormality in the brain, liver, spleen, and lungs of the EV71

0805a-infected mice (data not shown) and no obvious abnormality in any of the tissues tested from the EV71 0805infected mice. 3.3. Comparison of the EV71 0805 genome sequence with mouse-adapted EV71 0805a To understand the mechanisms underlying the hypervirulence of mouse muscle-adapted EV71 0805a, the genomes of EV71 0805 and 0805a were sequenced. These two strains of viruses shared more than 95% of nucleotide identities, but a total of 321 nucleotide mutations were detected in the genome of EV71 strain 0805a, and their distribution varied in different regions (Fig. 5A). Among these mutations, there were 25 non-synonymous mutations, which distributed in different regions of the EV71 0805a genome (Fig. 5B). The frequency of mutations in different regions of the EV71 0805a genome was similar, except for the VP1 region, in which the frequency of mutations was very low (Fig. 5C). The synonymous mutations were mainly located some regions and the highest frequency of synonymous mutations was located in the 3A region (Frequency of mutations per 100bp ? 0.011628, Fig. 5D). Correspondingly, analysis of the deduced amino acid sequences revealed that these nucleotide mutations resulted in 25 amino acid substitution in the EV71 0805a, as compared with that of EV71 0805 (Table 1). The GOR IV program indicated that there was no change in the secondary structure of the VP2, 2A, and 3C proteins, but there were six structural changes in the VP1, 2C, 3A, and 3D proteins.

RD cell
Virus load (log10 copies/ml)
10 8 6 4 2 0 0 50 100 150

*

*
EV71 0805 EV71 0805a

Hour post infection
Fig. 2. The dynamics of EV71 0805 and 0805a replication in RD cells. Monolayer RD cells in 24-well plates were infected in triplicate with 100 TCID50 of EV71 0805 or 0805a at 37  C for 1 h, respectively. The cells were washed with PBS and then cultivated in 2% FBS DMEM for the indicated periods. The virions in the cells were determined by quantitative real-time RTPCR assay. Data are expressed as mean ? SD of virions in each group from three separate experiments. *p < 0.05 vs. EV71 0805.

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Fig. 3. The infected mouse’s weight gain and EV71 virus replication in the mouse infected with EV71 0805 or 0805a. ICR mice at one day of age were fasted for 4 h and inoculated i.p. with, or without, 50 ml of EV71 0805 or 0805a (105 TCID50), respectively. Their body weights were measured at indicated time points. Ten mice from each group were sacri?ced at the indicated time points and their brains, limb muscles, intestines, and lungs samples were collected. The contents of EV71 in the muscles were determined by quantitative real-time RT-PCR. And the presence of EV71 virus was determined by PCR using the speci?c primers from different tissue.(A) Characterization of EV71 virus in different mouse tissues by RT-PCR. Data shown are representative images of each group (n ? 10) on day 5 post-inoculation from three separate experiments. Mk: marker DL2000; P: positive control; N: negative control. B: brain; M: muscle; I: intestine; and L: lung. EV71-Infected RD cell supernatant was as positive control. (B) The changes in mouse body weights. Data are expressed as mean ? SD of body weights (g) of each group of mice (n ? 30e60). (C) Quantitative analysis of EV71 virions in the mouse muscles. Data are expressed as mean ? SD of each group (n ? 6 per time point) of mice from three independent experiments. *p<0.05 vs. EV71 0805 group.

4. Discussion To study the myotropism and virulence of EV71 in the present study, an EV71 strain of 0805 was subjected to four serial passages of mouse muscle adaption to generate a strain of EV71 0805a. In comparison with the parental EV71 0805, we found that the EV71 0805a increased its myotropism and virulence in vitro. First, the EV71 0805a displayed stronger cytotoxicity against RD in vitro. Evidentially, following infection with EV71 0805a, the viability of RD cells was signi?cantly lower than that of cells infected with EV71 0805. Second, the EV71 0805a appeared to replicate faster as the virus load on 72 and 96 h post infection was signi?cantly higher than that of EV71 0805 in RD cells in vitro. Apparently, the mouse muscle-adapted EV71 0805a displayed higher virulence in vitro, providing an excellent model for studying the virulence and pathogenicity of mouse muscle-adapted EV71 in vivo. The parental EV71 strain 0805, a C4 group of virus, was isolated from pharyngeal swab of a child with HFMD in Fuyang city [27], and was a predominant genotype, responsible for recent outbreaks of HFMD in China (GenBank accession number FJ439769). The EV71 0805 has been used in studies of the pathogenesis of EV71 [29,33]. In this study, following i.p inoculation, the EV71 0805 invaded in the muscles of young mice, consistent with a previous report. In contrast, the EV71 0805a not only invaded the muscles, but also spread to other organs, such as the lung, intestine, heart, and liver, even occasionally to the brain of mice. Furthermore, infection of young mice with the EV71 0805a, but not the parental EV71 0805, led to detectable virus antigen in the cytoplasma of muscle cells of mice (data not shown). These

indicated that following infection, the EV71 0805a appeared to effectively replicate and express viral antigens in the muscle cells. Indeed, the virus contents in the muscle of the mice infected with the EV71 0805a were signi?cantly higher than that of the mice infected with EV71 0805, accompanied by signi?cantly reduced body weights in the mice infected with the EV17 0805a, as compared with that in the mice infected with EV71 0805. It is possible that the mouse muscle adaption of EV71 increased the virulence and tropism [16]. Alternatively, the mouse muscle adaption promoted virus replication in the muscle more ef?ciently. Therefore, the increased virus load in the muscle supports the notion that the skeletal muscle is the major site for virus replication [20]. As a result, the increased viruses may effectively invade to other organs outside the muscle. Accordingly, the contents of EV71, particularly for those muscle-adapted EV71 viruses, may determine the pathogenesis of virus infection, at least in mice, and may potentially be used as a prognostic marker of EV71 infection in susceptible individuals. Pathologically, the EV71 is an enterovirus. Previous studies have shown that EV71 can infect the epithelial cells of the intestines, cause pulmonary edema, and invade the central nervous system, particularly for those neurovirulent strains [14,15,22,29]. The parental EV71 0805 was a moderate virulent virus. Evidentially, infection with EV71 0805 had moderate effect on the viability of RD, but not other cells, and did not cause pathogenic lesion in mice. In contrast, the EV71 0805a displayed strong cytotoxicity against RD and Vero cells and obvious pathogenicity in mice. Interestingly, we detected virus in the lung and brain of some mice, but we failed to detect any pathogenic lesion in the lung and brain of mice following infection with the EV71 0805a. The lack of lung and

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Fig. 4. Histological analysis of various tissues in mice. ICR mice at one day of age were inoculated i.p with 2 ? 105 TCID50 of EV71 0805 or 0805a, respectively, and the mice were sacri?ced longitudinally. Their various tissues were dissected out and their tissue sections were subjected to H&E staining, followed by examined histopathologically under a light microscope. Data shown are representative images of each group (n ? 6) of mice on day 5 post-inoculation from ?ve independent experiments (magni?cation 100?). The blue arrows indicate the typical characters of the lesions in different tissues. There was no obvious pathological feature in the brain, lung, spleen, and liver of both groups of mice (data not shown). (For interpretation of the references to color in this ?gure legend, the reader is referred to the web version of this article.)

brain lesions may be due to the lack of strong autoimmune response, which has been thought to cause pulmonary lesions in humans [12,34]. While nuerovirulent EV71 strains usually have unique mutation at position 1994 (Val to Ile) [22], the Val was present at position of 1999 in both EV71 0805 and 0805a, indicating that the mouse muscle adaption did not change their tropism to the brain. These may explain why EV71 0805a was detected in the brains of a few mice, and why there was no pathogenic lesion in the brain following EV71 0805a infection in this strain of mice. More importantly, infection with the EV71 0805a resulted in diffuse necrotizing myositis in the skeletal muscles and myocarditis as well as severe intestinitis, which mimic the features of lesions in humans infected with enterovirus [35,36]. The severe intestinitis was unlikely caused by the route for infection as the epithelial cells of the small intestine are susceptible to virus infection [37]. Indeed, we detected the virus ?rst in the muscle and then in the lung, intestine, and brain following infection, which was similar to a pattern observed in EV71-infected rhesus monkey (data not shown). Apparently, the muscle-adapted EV71 0805a has a high tropism to the muscle, from which the virus spread to

other organs in mice. Therefore, a non-pathogenic EV71 0805, through the muscle adaption, displayed a unique pathogenicity in mice. To identify the molecular mechanisms underlying the virulence of EV71 0805a, we sequenced and compared the genome of various EV71 strains. Although both the gene sequences of strains 0805 and 0805a show above 95.8% identity, there were many mutations in the 0805a that were identi?ed in many genes, including VP1, VP2, 2A, 2C, 3A, 3C, 3D, and others. Interestingly, we found the total numbers of mutations were higher than that of previous reports [15,38], which may be due to the lack of virus selection from the muscle tissue. The high frequency of amino acid substitutions appeared in the 50 UTR, VP1, 2C, 3A, or/and 3D regions of the 0805a genome, which may be associated with increased virulence and unique pathogenicity of EV71 0805a. Indeed, previous studies have shown that the mutations in the VP1, VP2, and 3D regions are correlated with increased virulence of EV71 virus in mice, such as G145E [20,39], E145Q, A170V in the VP1 region [40], K149M in the VP2 region, and V263I in the 3D region [22]. We detected E145Q in the VP1 region, but

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Fig. 5. Mutation analysis of the nucleotide sequences of EV71 strains. The full-length nucleotide sequences of an EV71 clinical isolate 0805 and adapted strain 0805a were ampli?ed by RT-PCR for sequencing. The resulting sequences were analyzed and different types of mutations in individual capsid and non-capsid genes in EV71 0805a were identi?ed. The strain 0805 sequence obtained from Genbank (Accession number FJ439769). Data are expressed as mean number or percentage of mutations in each gene of EV71 0805a. Panel A: The number of synonymous mutations in EV71 0805a, B: The number of non-synonymous mutations in EV71 0805a, C: The frequency of synonymous mutations in EV71 0805a, D: The frequency of non-synonymous mutations in EV71 0805a.

not K149M in the VP2 region of strain 0805a, which may explain the unique pathogenicity of 0805a in mice. The alignment of sequences revealed three amino acid substitutions on the VP1 region, two on VP2, three on 2A, 2C, 3A, 3C, respectively, and nine on the 3D region in strain 0805a.

Table 1 Amino acid substitutions and secondary structure changes predicted in EV71 0805a. Position Secondary structureb Coding Position of Amino acida region amino acids In 0805 In 0805a start end In 0805 In 0805a VP1 22 145 292 37 41 315 47 62 68 16 33 46 68 140 197 261 299 370 H E T V R S A M I N I R H R A E T G Q Q A I K K T L V S V K Y K T G S D / 145 / / / 305 / / 67 / / / / / 194 260 / 372 / 152 / / / H / / / E / / / H / / E / / / / / C E / H

2C

/ / 307 E / / / 72 / / / / / 197 266 / 375 / H / / / / / H H / C

3A

3D

Notably, some of these mutations may change the structure of related proteins, particularly for the VP1, 2C, 3A, and 3D proteins of the 0805a virus. Previous studies have shown that the VP1 gene is a virulence determinant of coxsackievirus B4 and poliovirus chimeras in animal models [41,42]. Wang et al. has found that the E710Q mutation in VP1 of EV71 can result in conformational changes, and thereby affect its binding to host cells. The 2C protein can function as a nucleoside triphosphatase and a director of replication complexes to cell membranes [16]. In addition, the gene for viral RNA-dependent RNA polymerase 3D (3D pol) is crucial for the neurovirulence of polioviruses [43]. Interestingly, only on point mutation at position 1506 in the 3A region was found between the two Singapore strains from fatal and non-fatal cases with HFMD [44]. We speculate that the mutations in the EV71 0805a might be associated with increased virulence and unique pathogenicity in mice. We are interested further investigating the contribution of each mutation in the virulence and pathogenicity of EV71 0805a in mice. In conclusion, our data indicated the mouse muscle-adapted EV71 0805a with increased virulence and unique pathogenicity in mice, which may be attributed to many mutations in different genes of 0805a. Apparently, the EV71 0805a had high tropism to the muscle and can spread to other organs in mice. Therefore, our ?ndings provide new insights into understanding the tropism, virulence, and pathogencity of EV71. Acknowledgments This work was supported by the Ministry of Science and Technology of the People’s Republic of China (contract

The amino acid sequences determined previously of EV71 Fuyang-0805 were deposited in GenBank (Accession number: FJ439769). b /: no change in secondary structure of the amino acids predicted in Fuyang0805a. H: Alpha helix; E: Extended strand; C: Random coil.

a

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number 2009ZX10004-402). We are grateful to Dr. Jianwei Wang of Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College for kindly providing the original stock of EV71 virus. References
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