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Association of viral replication capacity with the pathogenicity of enterovirus 71
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Le-le Sun a , Jia-Kun Wang b , Xiao-qing Cui c , Shu-Bin Hao d , Jing Li a , Li Zhao a , Xiao-jing Yuan d , Hong-ling Wen a,∗ , Xue-jie Yu a , Zhi-Yu Wang a a
Department of Virology, School of Public Health, Shandong University, Jinan, Shandong 250012, People’s Republic of China Laiwu City Center for Disease Control and Prevention, Shandong Province 271100, People’s Republic of China c Shanghai City Center for Disease Control and Prevention, Shanghai 200336, People’s Republic of China d Shandong Medical Equipment Quality Supervision and Inspection Center, Jinan, Shandong 250101, People’s Republic of China
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Article history: Received 11 December 2013 Received in revised form 28 March 2014 Accepted 22 April 2014 Available online xxx
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Keywords: Replication Pathogenicity EV71
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1. Introduction
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Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease, which is associated with fatal neurological disease. The mechanism of EV71 pathogenesis remains obscure. We compared the replication capacity of the severe and mild enterovirus 71 isolates. The replication kinetics of EV71 in RD cells and ICR mice was determined by qRT-PCR. The lung, muscular, brain, intestine tissues were used for histopathological and immunohistochemical assays. The growth curves of EV71 strains in RD cells showed that the severe EV71 strains (SDLY107 and SDLY52) replicated faster and generated more viral RNA than the mild EV71 strains (SDLY11 and SDLY1). The mice infected by the severe EV71 strains (SDLY107) showed more severe clinical symptoms, pathological changes and higher viral load than the mice infected by the mild EV71 strains (SDLY11). These results suggest that there was a difference in replication capacity between the severe and mild EV71 strains, which was possibly associated with EV71 pathogenesis. © 2014 Published by Elsevier B.V.
Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus, belonging to family Picornaviridae, genus Enterovirus, species A. EV71 infection usually causes hand-foot-mouth disease (HFMD) in children under 5 years old. HFMD is usually a mild disease in children, but in some cases HFMD may be a severe neurogenic disease, complicated with aseptic meningitis, encephalitis, acute flaccid paralysis and fatal neurogenic pulmonary edema (McMinn, 2002). Since EV71 was first identified from a California patient in 1969, outbreaks of EV71 infection have been reported around the world (Schmidt et al., 1974; Chan et al., 2000), especially in the AsiaPacific region, and EV71 infections have contributed to millions of HFMD cases and hundreds of deaths in children since 1997 (Chan et al., 2003; Yang et al., 2009). However, to date no specific drugs or vaccines are commercially available for EV71 prevention and treatment and the pathogenic mechanism of EV71 remains obscure. Immature immunity and proinflammatory cytokines are thought to associate with EV71 pathogenesis (Huang et al., 1999;
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∗ Corresponding author. Tel.: +86 15098708509. E-mail address: [email protected] (H.-l. Wen).
Wang et al., 2012). However, it is not clear why some EV71 strains caused fatal disease and some EV71 strains only caused mild disease in humans. Comparative genomic analysis showed that the virulence of EV71 is probably determined by two positions (Gly/Gln/Arg710) and Glu729) on the DE and EF loop of VP1, one position (Lys930) on the surface of protease 2A, and four positions (C158, G272, U488 and A700/U700) in the 5 -NTR region (Li et al., 2011; Yeh et al., 2011; Reed and Muench, 1938). An EV71 replicon system showed that temperature-sensitive phenotype of EV71 was governed by the threonine at position 251 of virus 3D polymerase (Kung et al., 2010). However, how the molecular mechanisms of these virulent determinants of EV71 caused different clinical outcomes and whether these virulent determinants of EV71 associated with replication capacity of different EV71 isolates are not clear. The objective of this study was to investigate whether viral replication capacity was different between the severe EV71 strains (SDLY 107 isolated from a death case, and SDLY 52 isolated from a severe case with nerve complications) and the mild EV71 strains (SDLY 11 and SDLY 1 both isolated from HFMD patients without neurological complications) in RD cells, meanwhile SDLY 107 and SDLY 11 were chosen as the representative isolates for further study in ICR mice, and finally attempted to determine the pathogenesis of EV71.
http://dx.doi.org/10.1016/j.virusres.2014.04.014 0168-1702/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Sun, L.-l., et al., Association of viral replication capacity with the pathogenicity of enterovirus 71. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.014
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2. Materials and methods
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2.1. Cells and viruses
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RD (rhabdomyosarcoma) cell was cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% heatinactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a 5% CO2 humidified atmosphere. The severe EV71 strains (SDLY 107 and SDLY 52) and the mild EV71 strains (SDLY 11 and SDLY 1) were isolated from patients with neurological complications and from patients without neurological complications, respectively, from Linyi City, Shandong Province, China in 2011 (Wen et al., 2011; Si et al., 2012). RD cell monolayers at 80–90% confluence were infected with EV71 isolates and then incubated at 37 ◦ C with 5% CO2 in air until cytopathic effects (CPE) were visible. The virus infected cells were freezed and thawed for three times and then centrifuged at 8000 × g for 5 min. The supernatant was harvested and the TCID50 for each virus was measured by the Reed and Muench formula (Reed and Muench, 1938).
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2.2. Growth curves of EV71 Strains in RD Cells
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RD (rhabdomyosarcoma) cell was cultured in 24-well plate. In each well, seed 5 × 104 RD cells in 1 mL of growth medium 24 h before being infected with EV71 strains at multiplicity of infection (MOI) = 100. Then the infecting RD cells were cultured in the medium that contained 2% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, and 100 g/ml streptomycin in a 5% CO2 humidified atmosphere at 37 ◦ C and 39.5 ◦ C, respectively. During incubation, the viruses were harvested at the following time points: 0, 12, 24, 36, 48, 60, 72, 84 and 96 h post infection and stored at −80 ◦ C. Total viral RNAs were extracted using a RNA extraction kit (E.Z.N.A.® Viral RNA Kit, OMEGA, Guangzhou, China) following the manufacturer’s instructions. The extracted RNAs were quantified by real-time quantitative PCR (qRT-PCR).
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2.3. Animal experiments
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All work with mice followed National Institutes of Health guidelines and was approved by the Animal Care Committee of Shandong University (Approved protocol no. 20130305). The ICR mice were obtained from Beijing HFK Bio-Technology. Co. For mice experiments, five female ICR mice (1 day or 2 weeks) per group were intraperitoneally inoculated with SDLY 107 or SDLY 11 (50 l of EV71 at 2000TCID50 for 1-day mouse, and 150 l of EV71 at 2000TCID50 for 2-weeks mouse). Control group was inoculated with saline. All mice were monitored daily for clinical symptoms after inoculation. Groups of one-day-old ICR suckling mice were weighted at day 0 d, 2 d, 4 d, 6 d, 8 d and 10 d. The infected mice were dissected and the lung, brain, intestine and muscle tissue was collected at day 3 d, 4 d, 5 d, 6 d, and 7 d for histopathological and immunohistochemical analysis, and the muscle tissue were collected every day for 2 weeks to measure the viral RNA copy number. Total RNA was extracted from the muscle tissues with Total RNA Kit II (OMEGA, Guangzhou, China) according to the manufacturer’s instructions. The extracted RNAs were quantified by qRT-PCR. 2.4. Histopathological analysis Lung, brain, intestine and muscle tissues were fixed in 4% (v/v) paraformaldehyde solution. Twenty-four hours after fixation, all tissues were embedded in paraffin, serial sections were cut for 5 mm latitudinally and 4 m longitudinally, and mounted on glass slides for pathological and immunohistochemical examination. The
sections were stained with hematoxylin-eosin (HE) to assess general histopathology.
2.5. Immunohistochemical analysis The paraffin sections were baked at 60 ◦ C overnight, deparaffinized with dimethylbenzene, and dehydrated with ethanol. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigens were retrieved by heat treatment using the microwave in 0.01 M sodium citrate buffer. The slides were sequentially incubated with 5% bovin serum albumin (BSA) for 20 min, 1:1000 rat antibodies to EV71 (ABCAM, Shanghai, China) overnight at 4 ◦ C, goat anti-rat antibody conjugated with horseradish peroxidase (ZSGB-BIO, ZF-0312) for 30 min at 37 ◦ C. Immunoreactivity signals were detected using a 3,3-diaminobenzidine (DAB, OriGene, Beijing, China) substrate, and the cells were counterstained with Mayer’s hematoxylin. After a six graded ethanol series and a two graded dimethylbenzene series, the stained slides were observed using a light microscope.
2.6. Real-time quantitative PCR (qRT-PCR) The extracted RNAs were reverse transcribed (RT) using ReverTra Ace qPCR RT Kit (TOYOBO, Shanghai, China) into cDNA. Real-time fluorescent quantitative polymerase chain reaction was conducted with SYBR Green I RT-PCR Master Kit (Roche, Shanghai, China) using Roche’s LightCycler 480. The primer sequences were as follows: VP1 (226 bp) forward: 5 -GCAGCCCAAAAGAACTTCAC3 , reverse: 5 -ATTTCAGCAGCTTGGAGTGC-3 . In the RT step, a 10 l reaction volume contained the following components: 2 l RNA sample, 0.5 l primer mix, 0.5 l RT Enzyme Mix, 2 l 5× RT buffer, 5 l Nuclease-free Water. The reaction was performed at 37 ◦ C for 15 min, 98 ◦ C for 5 min. The RNA sample was treated with 65 ◦ C for 5 min before RT step. In the Real-time PCR step, a 20 l reaction volume contained the following components: 10 l 2× SYBR Green I RT-PCR Master Mix, 0.4 l primer (25 M), and 2 l cDNA, 7.2 l nuclease-free water. The PCR protocol was as follows: denaturation at 94 ◦ C for 10 min; 45 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and elongation at 72 ◦ C for 1 min. Signals were detected by the CT value.
2.7. The establishment of qRT-PCR standard curve EV71 VP1 was amplified as described above. The amplified products were separated by electrophoresis on 1.5% agarose gels (voltage: 100 V), stained with ethidium bromide and photographed using an ultraviolet imaging system (ProteinSimple). We used Gel Extraction Kit (OMEGA, Guangzhou, China) to collect the VP1, and then cloned VP1 into PMD19T Vector followed by PMD19T-VP1 transformation into DH5␣ and plasmid extraction using Plasmid Miniprep Kit II (OMEGA, Guangzhou, China). All protocols were following the manufacturer’s instructions. The PMD19T-VP1 nucleic acid concentration was measured by spectrophotometer (Thermo, NANODROP 200C), and calculated the PMD19T-VP1 concentration of copies according to the equation (6.02 × 1023 ) × (DNA g/l × 10−9 )/(DNA length × 660). The PMD 19T-VP1 was diluted to eight folds series, 108 , 107 , 106 , 105 , 104 , 103 , 102 , 101 copies/l. The CT value was detected by qRT-PCR. The standard curve was established by using CT value as the longitudinal axis, logarithm of the VP1 concentration of copies as the horizontal axis.
Please cite this article in press as: Sun, L.-l., et al., Association of viral replication capacity with the pathogenicity of enterovirus 71. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.014
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Fig. 1. Growth Curves of EV71 Strains in RD Cells at different time points after EV71 infection at multiplicity of infection (MOI) = 100. (A) The severe (SDLY107, SDLY52) and mild (SDLY1, SDLY11) EV71 isolates were grown in RD cells at 39.5 ◦ C. (B) The severe (SDLY107, SDLY52) and mild (SDLY1, SDLY11) EV71 isolates were grown in RD cells at 37 ◦ C. At each time point, data are means ± SD of three samples; each carried out in triplicate.
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3. Result 3.1. Growth Curves of EV71 Strains in RD Cells determined by qPCR The severe EV71 strains (SDLY107 and SDLY52) and the mild strains (SDLY1 and SDLY11) were harvested on RD cell every 12 h post infection and the viral RNA was quantified to determine
whether the replication of EV71 severe and mild strains were different. The growth curves of both severe and mild strains in RD cells infected at multiplicity of infection (MOI) = 100 is shown in Fig. 1. Strain SDLY107 and SDLY52 generated approximately 10-fold of viral copies/l of strain SDLY11 between 36 and 96 h after infection at 39.5 ◦ C (Fig. 1A). However, the growth curves of SDLY107 and SDLY52 were slightly higher than SDLY11 and SDLY1 after infection at 37 ◦ C (Fig. 1B). These results demonstrate that the replication
Fig. 2. Growth Curves of EV71 Strains in RD Cells at different time points after EV71 infection at multiplicity of infection (MOI) = 100. (A) The fatal strain SDLY 107 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (B) The severe strain SDLY 52 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (C) The mild strain SDLY 11 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (D) The mild strain SDLY 1 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively; at each time point, data are means ± SD of three samples; each carried out in triplicate. The curves plotted in this figure are the same ones than in Fig. 1.
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Fig. 3. Difference of viral load between 39.5 ◦ C and 37 ◦ C at different time points after EV71 infection at multiplicity of infection (MOI) = 100. The difference of viral load between 39.5 ◦ C and 37 ◦ C showed that the growth of both severe and mild EV71 strains was inhibited, especially in mild EV71 strains. Data are the mean ± SD of three independent experiments; each carried out in triplicate.
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capacity of severe EV71 strains were higher than mild EV71 strains at high temperature (39.5 ◦ C) rather than at optimal temperature (37 ◦ C). In addition, outside the ranges of optimal temperature (37 ◦ C vs 39.5 ◦ C), the growth of both severe and mild EV71 strains was inhibited, especially in mild EV71 strains (Figs. 2A–D and 3). All together these data suggest that the different replication capacity of both severe and mild EV71 strains was only achieved at high temperatures, and this finding may partly interpret that the durations of fever were significantly longer in severe HFMD children than in mild HFMD children. 3.2. The symptoms of mice after EV71 infection The 1-day-old ICR mice injected with SDLY107 developed symptoms, including piloerection, arched back, limb paralysis at day 3–4 and recovered at day 7–8 post infection, while the mice infected with SDLY 11 showed symptoms at day 5–6 and recovered at day 8–9 post infection. The 2-weeks-old ICR mice injected with SDLY 107 and SDLY11 only showed weight loss, and there was no significant alteration in the control group (normal saline). The growth curve of mice body weight showed that 1-day-old ICR mice injected with SDLY107 (group A) and injected with SDLY11 (group B) had a more slowly growth trend of weight than control group (group C). The weight of mice in group A was a slightly heavier than mice in group B (Fig. 4).
The pathology of lung, brain, intestine, and muscle in EV71 infected mice was evaluated by H&E staining. The tissues of 1-dayold mice infected with SDLY 107 and SDLY 11 exhibited distinct pathological changes at day 4 post infection, while 1-day-old mice normal control had no obvious pathological changes. The 1-dayold mice infected with SDLY 107 had more obvious pathological changes than the 1-day-old mice infected with SDLY 11 (Fig. 5). The structure of small intestinal mucosal was destroyed, accompanying with the intestinal villus falling off, vacuolar degenerating in the intestinal villus epithelial cell and inflammatory cell infiltrating (Fig. 5G–I). The muscle tissues had an accumulation of inflammatory cells (Fig. 5J–L). The lung tissues showed thickening of alveolar septa, hyperemia of blood capillaries, mononuclear and lymphocyte inflammatory infiltration, and exudation of edema fluid were also found in the alveolar cavities (Fig. 5A–C). The necrosis foci were seen in brain tissues, in which some cells dissolved and formed liquefactive necrosis, in addition, the number of microglia increased (Fig. 5D–F). 3.4. Immunohistochemical findings Immunohistochemical staining showed strong reaction with EV71 specific antigen in lung and muscle tissues of the 1-day-old mice infected with SDLY 107 and SDLY11 4 days post infection (Fig. 6). The results suggested that EV71 are mainly distributed in the muscle and lung tissue. 3.5. EV71 replication kinetics in ICR mice determined by qPCR We performed qPCR assays to detect the replication kinetics of EV71 in ICR mice. Based on the immunohistochemical results that EV71 are mainly distributed in the muscle and lung tissues, we collected the hindlimb muscle for the quantitative detection of the EV71 using qPCR assays. The EV71 replication curves showed that the 1-day-old mice infected with SDLY 107 and the 1-day-old mice infected with SDLY11 had the same viral replication tendency. However, there were two peaks of growth in 1-day-old mice infected by SDLY107 (the highest copies of viral RNA were 2.07 × 104 copies/mg and 6.02 × 104 copies/mg at day 1 and 5 post infection, respectively), while there was only one peak of growth in the 1-day-old mice infected with SDLY11 (the highest copies of viral RNA were 3.27 × 104 copies/mg), in addition, the growth peak of SDLY107 strain appeared earlier than SDLY11 strain (Fig. 7). In accordance with the viral replication curves of the 1-day-old mice, the viral load peak of 2 weeks old mice infected with SDLY 107 appears earlier than 2 weeks old mice infected with SDLY 11 (Fig. 8), and the highest copies of viral RNA were 2.57 × 104 copies/mg and 3.47 × 103 copies/mg, respectively. 4. Discussion
Fig. 4. Growth curve of weight of the 1d old mice infected by EV71. Groups of 1 d old suckling mice (n = 5 per group) were inoculated intraperitoneally (i.p.) with SDLY107 (group A) or SDLY11 (group B), respectively. The growth curve of mice body weight at different time points after EV71 infection was observed. The control group (group C) was injected with saline. At each time point, data are means ± SD of five samples; each carried out in triplicate.
EV71 is a major cause of hand-foot-and-mouth disease and is associated with fatal neurological disease (McMinn, 2002). While many factors contribute to the disease process, different virulent strains of EV71 can lead to diverse clinical outcomes (Yan et al., 2013; Wen et al., 2013; Li-mei et al., 2011). It is not clear why different EV71 strains caused different clinical outcomes in humans. In this study, we focused on the molecular mechanism of replication of EV71 genome, and demonstrated that there was a difference in replication capacity between the severe EV71 strain and the mild EV71 strain in RD cells and ICR mice. As EV71 is a member of the Picornaviridae family, an internal ribosome entry site (IRES) has been used to facilitate the expression of proteins in a single transcript, and 3D polymerase plays a role in
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Fig. 5. Representative histology of various tissues of ICR mice infected by EV71. Tissues of mock control (A, D, G and J), ICR mice infected by SDLY107 (C, F, I and L), ICR mice infected by SDLY11 (B, E, H and K). Typical lesions were indicated as the arrow: thickening of alveolar septa of lung tissues (B and C); liquefactive necrosis of brain tissues (E and F); intestinal villus loss of intestinal tissues (H and I); inflammatory cell infiltration of muscle tissues (L). Magnification: A, B, C, G, H, I, J, K, L:200×, D, E, F:100×.
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the replication of EV71, therefore, variations in these regions may affect the process of viral replication. A number of studies showed that four positions (C158, G272, U488 and A700/U700) in the 5 -NTR region (Li et al., 2011; Yeh et al., 2011) and one position (thr251) in 3D polymerase (Kung et al., 2010) were variant between the severe and mild EV71 strains. Another studies also demonstrated variation in the complete genomes of EV71 strains associated with different clinical phenotypes, especially in the 5 -UTR and 3D regions,
which were related to the translation and replication of EV71 (Yeh et al., 2011; Wen et al., 2013). A hypothesis was that these variation sites governing the range of different clinical symptoms resulted in the difference of EV71 replication capacity. In support of this idea, we found that the severe EV71 strains grew faster than the mild EV71 strains at 39.5 ◦ C in RD cells, while it was not obvious at 37 ◦ C. Our results also showed that the replication of both the severe EV71 strains and mild EV71 strains were suppressed at high
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Fig. 6. Representative images of immunostaining for lung and muscle tissues of EV71 infected mice. Target protein positive staining is brownish-yellow or snuff. The positive protein was seen both on the muscle tissues of mice infected with SDLY107 (B)/SDLY11 (C) and lung tissues of mice infected with SDLY107 (E)/SDLY11 (F). The control group (A and D) was parallel with the experimental group. Magnification: 400×.
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temperatures (39.5 ◦ C), and the suppression was more obvious in mild strains. Some studies showed that a high fever was existed in EV71 patients, and patients with CNS involvement had longer durations of fever than patients without CNS involvement (Ru et al., 2010; Liu et al., 2008; Li et al., 2002), suggesting that the temperature might play a role in the replication of EV71, so we conducted this experiment at 39.5 ◦ C to simulate in vivo temperature environments. Based on these findings, it was reasonable to assume that the severe EV71 strains replicate faster than the mild EV71 strains at high temperatures and are more likely to cause severe disease. Recently, the suitable animal model for finding clues on EV71 pathogenesis in humans was not so satisfactory. Previous studies have used monkeys and neonatal mice as models for this disease. In our studies, given the ethical and economical reasons, we used mice as models. In mice experiments, viral replication curves confirmed that the viral load peak of SDLY 107 appears earlier than
Fig. 7. EV71 replication kinetics in the 1 day old mice at different time points after EV71 infection. The 1 day old mice were infected with SDLY107 (A) isolated from death case and SDLY11 (B) isolated from mild case at a 2000CCID50 for 50 l, and the amounts of virus RNA were quantified by qRT-PCR assay. The control group (C) was parallel with the experimental group. At each time point, data are means ± SD of three samples; each carried out in triplicate.
SDLY 11 in ICR mice, suggesting that the difference of EV71 replication capacity existed in ICR mice, which was consistent with the results in RD cells. Together, the difference of EV71 replication may be involved in EV71 pathogenesis. However, mice that are more than a few weeks old lose their susceptibility to EV71 which was not observed in viral replication kinetics of 2-weeks old mice model (Fig. 8), although the 2-weeks old mice showed no clinical symptoms except for weight loss. The reasons for this difference remain to be clarified. Furthermore, the viral replication sites in the mouse (mainly in muscle tissues) and clinical outcomes of mice EV71 infection were different from humans. Thus, an appropriate small animal model should be developed for the study of this disease. In several laboratories, transgenic mouse model was used for the study of EV71 pathogenesis, and the pathological features in transgenic mouse were similar to those of EV71 infection in humans (Fujii et al., 2013). Transgenic mouse would be a promising animal model for the study of EV71 infection.
Fig. 8. EV71 replication kinetics in two weeks old mice at different time points after EV71 infection. Two weeks old mice were infected with SDLY107 (A) isolated from death case and SDLY11 (B) isolated from mild case at a 2000CCID50 for 150 l, and the amounts of virus RNA were quantified by qRT-PCR assay. The control group (C) was parallel with the experimental group. At each time point, data are means ± SD of three samples; each carried out in triplicate.
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Kung et al. reported that the encephalitis strain EV71 was temperature-resistant (Tr) at 40 ◦ C in Vero cells and easier to replicate in HTB-14 (astrocytoma) than the herpangina isolate EV71 (Kung et al., 2007). Fukuhara M et al. studied the replication dynamics of three EV71 strains (major symptom was HFMD) in RD cells using mathematical modeling and also found that viral productivity was different (Fukuhara et al., 2013). In our study, we further confirmed that the severe EV71 strains replicate faster than the mild EV71 strains in RD cells at 39.5 ◦ C, and the difference in replication was partly caused by the unsuitable temperature, in addition, for the first time we demonstrated that the difference of viral replication existed in the ICR mice. Therefore, it is reasonable to predict that during EV71 infection, high replication EV71 isolates may lead to severe disease. In summary, our study demonstrated that there was a difference in replication capacity between the severe and mild enterovirus 71 isolates in mice and in vitro, which is possibly associated with EV71 pathogenesis. The molecular mechanism of virulence determinant governing the viral replication needs to be further studied. Our results would provide a new insight into the pathophysiology of EV71-related HFMD. Acknowledgements We are grateful to the Shandong Medical Equipment Quality Supervision and Inspection Center and Shandong Linyi City People’s Hospital for supplying medical equipment and clinical samples. References Chan, L.G., Parashar, U.D., Lye, M.S., et al., 2000. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. Clin. Infect. Dis. 31 (3), 678–683. Chan, K.P., Goh, K.T., Chong, C.Y., et al., 2003. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg. Infect. Dis. 9 (1), 78. Fujii, K., Nagata, N., Sato, Y., et al., 2013. Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc. Natl. Acad. Sci. U. S. A. 110 (36), 14753–14758. Fukuhara, M., Iwami, S., Sato, K., et al., 2013. Quantification of the dynamics of enterovirus 71 infection by experimental-mathematical investigation. J. Virol. 87 (1), 701–705.
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Huang, C.C., Liu, C.C., Chang, Y.C., et al., 1999. Neurologic complications in children with enterovirus 71 infection. N. Engl. J. Med. 341 (13), 936–942. Kung, C.M., King, C.C., Lee, C.N., et al., 2007. Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan. J. Med. Virol. 79 (1), 60–68. Kung, Y.H., Huang, S.W., Kuo, P.H., et al., 2010. Introduction of a strong temperaturesensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase. Virology 396 (1), 1–9. Li, C.C., Yang, M.Y., Chen, R.F., et al., 2002. Clinical manifestations and laboratory assessment in an enterovirus 71 outbreak in southern Taiwan. Scand. J. Infect. Dis. 34 (2), 104–109. Li, R., Zou, Q., Chen, L., et al., 2011. Molecular analysis of virulent determinants of enterovirus 71. PLoS ONE 6 (10), e26237. Li-mei, S., Huan-ying, Z., Hui-zhen, Z., et al., 2011. An enterovirus 71 epidemic in Guangdong Province of China, 2008: epidemiological, clinical, and virogenic manifestations. Jpn. J. Infect. Dis. 64 (1), 13–18. Liu, Y.X., Xie, J.J., He, Y.X., et al., 2008. Study of the clinical and laboratory features of hand-foot-mouth disease]. Zhonghua shi yan he lin chuang bing du xue za zhi 22 (6), 475. McMinn, P.C., 2002. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol. Rev. 26 (1), 91–107. Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Epidemiol. 27 (3), 493–497. Ru, W.P., Kang, K., You, A.G., et al., 2010. Analysis on clinical and epidemiological characteristics of severe cases infected by EV71. Zhonghua shi yan he lin chuang bing du xue za zhi 24 (6), 448. Schmidt, N.J., Lennette, E.H., Ho, H.H., 1974. An apparently new enterovirus isolated from patients with disease of the central nervous system. J. Infect. Dis. 129 (3), 304–309. Si, L., Gao, F., Wen, H., et al., 2012. Genetic characteristics of enterovirus 71 strains isolated in Linyi, Shandong province, 2009–2010. Chin. J. Public Health 28 (11), 1446–1448. Wang, S.M., Lei, H.Y., Liu, C.C., 2012. Cytokine immunopathogenesis of enterovirus 71 brain stem encephalitis. Clin. Dev. Immunol. 2012. Wen, H., Hao, S., Gao, F., et al., 2011. Sequence analysis for length genomes of human enterovirus 71 strains isolated in Linyi, Shandong Province. J. Microbiol. Immunol. 31 (7), 603–608. Wen, H., Si, L., Yuan, X., et al., 2013. Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes. Virol. J. 10 (1), 115. Yan, Y., Guo, J., Zhou, J.Z., et al., 2013. Analysis on molecular epidemiological features of enterovirus type 71 in Guizhou Province, 2008–2011. Bing du xue bao bian ji wei yuan hui 29 (2), 176–179. Yang, F., Ren, L., Xiong, Z., et al., 2009. Enterovirus 71 outbreak in the People’s Republic of China in 2008. J. Clin. Microbiol. 47 (7), 2351–2352. Yeh, M.T., Wang, S.W., Yu, C.K., et al., 2011. A single nucleotide in stem loop II of 5 -untranslated region contributes to virulence of enterovirus 71 in mice. PLoS ONE 6 (11), e27082.
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Contents lists available at ScienceDirect
Virus Research journal homepage: www.elsevier.com/locate/virusres
Association of viral replication capacity with the pathogenicity of enterovirus 71
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Le-le Sun a , Jia-Kun Wang b , Xiao-qing Cui c , Shu-Bin Hao d , Jing Li a , Li Zhao a , Xiao-jing Yuan d , Hong-ling Wen a,∗ , Xue-jie Yu a , Zhi-Yu Wang a a
Department of Virology, School of Public Health, Shandong University, Jinan, Shandong 250012, People’s Republic of China Laiwu City Center for Disease Control and Prevention, Shandong Province 271100, People’s Republic of China c Shanghai City Center for Disease Control and Prevention, Shanghai 200336, People’s Republic of China d Shandong Medical Equipment Quality Supervision and Inspection Center, Jinan, Shandong 250101, People’s Republic of China
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Article history: Received 11 December 2013 Received in revised form 28 March 2014 Accepted 22 April 2014 Available online xxx
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Keywords: Replication Pathogenicity EV71
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1. Introduction
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Enterovirus 71 (EV71) is a major cause of hand-foot-and-mouth disease, which is associated with fatal neurological disease. The mechanism of EV71 pathogenesis remains obscure. We compared the replication capacity of the severe and mild enterovirus 71 isolates. The replication kinetics of EV71 in RD cells and ICR mice was determined by qRT-PCR. The lung, muscular, brain, intestine tissues were used for histopathological and immunohistochemical assays. The growth curves of EV71 strains in RD cells showed that the severe EV71 strains (SDLY107 and SDLY52) replicated faster and generated more viral RNA than the mild EV71 strains (SDLY11 and SDLY1). The mice infected by the severe EV71 strains (SDLY107) showed more severe clinical symptoms, pathological changes and higher viral load than the mice infected by the mild EV71 strains (SDLY11). These results suggest that there was a difference in replication capacity between the severe and mild EV71 strains, which was possibly associated with EV71 pathogenesis. © 2014 Published by Elsevier B.V.
Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus, belonging to family Picornaviridae, genus Enterovirus, species A. EV71 infection usually causes hand-foot-mouth disease (HFMD) in children under 5 years old. HFMD is usually a mild disease in children, but in some cases HFMD may be a severe neurogenic disease, complicated with aseptic meningitis, encephalitis, acute flaccid paralysis and fatal neurogenic pulmonary edema (McMinn, 2002). Since EV71 was first identified from a California patient in 1969, outbreaks of EV71 infection have been reported around the world (Schmidt et al., 1974; Chan et al., 2000), especially in the AsiaPacific region, and EV71 infections have contributed to millions of HFMD cases and hundreds of deaths in children since 1997 (Chan et al., 2003; Yang et al., 2009). However, to date no specific drugs or vaccines are commercially available for EV71 prevention and treatment and the pathogenic mechanism of EV71 remains obscure. Immature immunity and proinflammatory cytokines are thought to associate with EV71 pathogenesis (Huang et al., 1999;
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∗ Corresponding author. Tel.: +86 15098708509. E-mail address: [email protected] (H.-l. Wen).
Wang et al., 2012). However, it is not clear why some EV71 strains caused fatal disease and some EV71 strains only caused mild disease in humans. Comparative genomic analysis showed that the virulence of EV71 is probably determined by two positions (Gly/Gln/Arg710) and Glu729) on the DE and EF loop of VP1, one position (Lys930) on the surface of protease 2A, and four positions (C158, G272, U488 and A700/U700) in the 5 -NTR region (Li et al., 2011; Yeh et al., 2011; Reed and Muench, 1938). An EV71 replicon system showed that temperature-sensitive phenotype of EV71 was governed by the threonine at position 251 of virus 3D polymerase (Kung et al., 2010). However, how the molecular mechanisms of these virulent determinants of EV71 caused different clinical outcomes and whether these virulent determinants of EV71 associated with replication capacity of different EV71 isolates are not clear. The objective of this study was to investigate whether viral replication capacity was different between the severe EV71 strains (SDLY 107 isolated from a death case, and SDLY 52 isolated from a severe case with nerve complications) and the mild EV71 strains (SDLY 11 and SDLY 1 both isolated from HFMD patients without neurological complications) in RD cells, meanwhile SDLY 107 and SDLY 11 were chosen as the representative isolates for further study in ICR mice, and finally attempted to determine the pathogenesis of EV71.
http://dx.doi.org/10.1016/j.virusres.2014.04.014 0168-1702/© 2014 Published by Elsevier B.V.
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2. Materials and methods
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RD (rhabdomyosarcoma) cell was cultured in Dulbecco’s modified Eagle’s medium (HyClone) supplemented with 10% heatinactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, and 100 g/ml streptomycin at 37 ◦ C in a 5% CO2 humidified atmosphere. The severe EV71 strains (SDLY 107 and SDLY 52) and the mild EV71 strains (SDLY 11 and SDLY 1) were isolated from patients with neurological complications and from patients without neurological complications, respectively, from Linyi City, Shandong Province, China in 2011 (Wen et al., 2011; Si et al., 2012). RD cell monolayers at 80–90% confluence were infected with EV71 isolates and then incubated at 37 ◦ C with 5% CO2 in air until cytopathic effects (CPE) were visible. The virus infected cells were freezed and thawed for three times and then centrifuged at 8000 × g for 5 min. The supernatant was harvested and the TCID50 for each virus was measured by the Reed and Muench formula (Reed and Muench, 1938).
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2.2. Growth curves of EV71 Strains in RD Cells
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RD (rhabdomyosarcoma) cell was cultured in 24-well plate. In each well, seed 5 × 104 RD cells in 1 mL of growth medium 24 h before being infected with EV71 strains at multiplicity of infection (MOI) = 100. Then the infecting RD cells were cultured in the medium that contained 2% heat-inactivated fetal bovine serum (FBS) (HyClone), 100 U/ml penicillin, and 100 g/ml streptomycin in a 5% CO2 humidified atmosphere at 37 ◦ C and 39.5 ◦ C, respectively. During incubation, the viruses were harvested at the following time points: 0, 12, 24, 36, 48, 60, 72, 84 and 96 h post infection and stored at −80 ◦ C. Total viral RNAs were extracted using a RNA extraction kit (E.Z.N.A.® Viral RNA Kit, OMEGA, Guangzhou, China) following the manufacturer’s instructions. The extracted RNAs were quantified by real-time quantitative PCR (qRT-PCR).
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All work with mice followed National Institutes of Health guidelines and was approved by the Animal Care Committee of Shandong University (Approved protocol no. 20130305). The ICR mice were obtained from Beijing HFK Bio-Technology. Co. For mice experiments, five female ICR mice (1 day or 2 weeks) per group were intraperitoneally inoculated with SDLY 107 or SDLY 11 (50 l of EV71 at 2000TCID50 for 1-day mouse, and 150 l of EV71 at 2000TCID50 for 2-weeks mouse). Control group was inoculated with saline. All mice were monitored daily for clinical symptoms after inoculation. Groups of one-day-old ICR suckling mice were weighted at day 0 d, 2 d, 4 d, 6 d, 8 d and 10 d. The infected mice were dissected and the lung, brain, intestine and muscle tissue was collected at day 3 d, 4 d, 5 d, 6 d, and 7 d for histopathological and immunohistochemical analysis, and the muscle tissue were collected every day for 2 weeks to measure the viral RNA copy number. Total RNA was extracted from the muscle tissues with Total RNA Kit II (OMEGA, Guangzhou, China) according to the manufacturer’s instructions. The extracted RNAs were quantified by qRT-PCR. 2.4. Histopathological analysis Lung, brain, intestine and muscle tissues were fixed in 4% (v/v) paraformaldehyde solution. Twenty-four hours after fixation, all tissues were embedded in paraffin, serial sections were cut for 5 mm latitudinally and 4 m longitudinally, and mounted on glass slides for pathological and immunohistochemical examination. The
sections were stained with hematoxylin-eosin (HE) to assess general histopathology.
2.5. Immunohistochemical analysis The paraffin sections were baked at 60 ◦ C overnight, deparaffinized with dimethylbenzene, and dehydrated with ethanol. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigens were retrieved by heat treatment using the microwave in 0.01 M sodium citrate buffer. The slides were sequentially incubated with 5% bovin serum albumin (BSA) for 20 min, 1:1000 rat antibodies to EV71 (ABCAM, Shanghai, China) overnight at 4 ◦ C, goat anti-rat antibody conjugated with horseradish peroxidase (ZSGB-BIO, ZF-0312) for 30 min at 37 ◦ C. Immunoreactivity signals were detected using a 3,3-diaminobenzidine (DAB, OriGene, Beijing, China) substrate, and the cells were counterstained with Mayer’s hematoxylin. After a six graded ethanol series and a two graded dimethylbenzene series, the stained slides were observed using a light microscope.
2.6. Real-time quantitative PCR (qRT-PCR) The extracted RNAs were reverse transcribed (RT) using ReverTra Ace qPCR RT Kit (TOYOBO, Shanghai, China) into cDNA. Real-time fluorescent quantitative polymerase chain reaction was conducted with SYBR Green I RT-PCR Master Kit (Roche, Shanghai, China) using Roche’s LightCycler 480. The primer sequences were as follows: VP1 (226 bp) forward: 5 -GCAGCCCAAAAGAACTTCAC3 , reverse: 5 -ATTTCAGCAGCTTGGAGTGC-3 . In the RT step, a 10 l reaction volume contained the following components: 2 l RNA sample, 0.5 l primer mix, 0.5 l RT Enzyme Mix, 2 l 5× RT buffer, 5 l Nuclease-free Water. The reaction was performed at 37 ◦ C for 15 min, 98 ◦ C for 5 min. The RNA sample was treated with 65 ◦ C for 5 min before RT step. In the Real-time PCR step, a 20 l reaction volume contained the following components: 10 l 2× SYBR Green I RT-PCR Master Mix, 0.4 l primer (25 M), and 2 l cDNA, 7.2 l nuclease-free water. The PCR protocol was as follows: denaturation at 94 ◦ C for 10 min; 45 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and elongation at 72 ◦ C for 1 min. Signals were detected by the CT value.
2.7. The establishment of qRT-PCR standard curve EV71 VP1 was amplified as described above. The amplified products were separated by electrophoresis on 1.5% agarose gels (voltage: 100 V), stained with ethidium bromide and photographed using an ultraviolet imaging system (ProteinSimple). We used Gel Extraction Kit (OMEGA, Guangzhou, China) to collect the VP1, and then cloned VP1 into PMD19T Vector followed by PMD19T-VP1 transformation into DH5␣ and plasmid extraction using Plasmid Miniprep Kit II (OMEGA, Guangzhou, China). All protocols were following the manufacturer’s instructions. The PMD19T-VP1 nucleic acid concentration was measured by spectrophotometer (Thermo, NANODROP 200C), and calculated the PMD19T-VP1 concentration of copies according to the equation (6.02 × 1023 ) × (DNA g/l × 10−9 )/(DNA length × 660). The PMD 19T-VP1 was diluted to eight folds series, 108 , 107 , 106 , 105 , 104 , 103 , 102 , 101 copies/l. The CT value was detected by qRT-PCR. The standard curve was established by using CT value as the longitudinal axis, logarithm of the VP1 concentration of copies as the horizontal axis.
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Fig. 1. Growth Curves of EV71 Strains in RD Cells at different time points after EV71 infection at multiplicity of infection (MOI) = 100. (A) The severe (SDLY107, SDLY52) and mild (SDLY1, SDLY11) EV71 isolates were grown in RD cells at 39.5 ◦ C. (B) The severe (SDLY107, SDLY52) and mild (SDLY1, SDLY11) EV71 isolates were grown in RD cells at 37 ◦ C. At each time point, data are means ± SD of three samples; each carried out in triplicate.
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3. Result 3.1. Growth Curves of EV71 Strains in RD Cells determined by qPCR The severe EV71 strains (SDLY107 and SDLY52) and the mild strains (SDLY1 and SDLY11) were harvested on RD cell every 12 h post infection and the viral RNA was quantified to determine
whether the replication of EV71 severe and mild strains were different. The growth curves of both severe and mild strains in RD cells infected at multiplicity of infection (MOI) = 100 is shown in Fig. 1. Strain SDLY107 and SDLY52 generated approximately 10-fold of viral copies/l of strain SDLY11 between 36 and 96 h after infection at 39.5 ◦ C (Fig. 1A). However, the growth curves of SDLY107 and SDLY52 were slightly higher than SDLY11 and SDLY1 after infection at 37 ◦ C (Fig. 1B). These results demonstrate that the replication
Fig. 2. Growth Curves of EV71 Strains in RD Cells at different time points after EV71 infection at multiplicity of infection (MOI) = 100. (A) The fatal strain SDLY 107 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (B) The severe strain SDLY 52 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (C) The mild strain SDLY 11 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively. (D) The mild strain SDLY 1 were grown in RD cells at 39.5 ◦ C and 37 ◦ C, respectively; at each time point, data are means ± SD of three samples; each carried out in triplicate. The curves plotted in this figure are the same ones than in Fig. 1.
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Fig. 3. Difference of viral load between 39.5 ◦ C and 37 ◦ C at different time points after EV71 infection at multiplicity of infection (MOI) = 100. The difference of viral load between 39.5 ◦ C and 37 ◦ C showed that the growth of both severe and mild EV71 strains was inhibited, especially in mild EV71 strains. Data are the mean ± SD of three independent experiments; each carried out in triplicate.
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capacity of severe EV71 strains were higher than mild EV71 strains at high temperature (39.5 ◦ C) rather than at optimal temperature (37 ◦ C). In addition, outside the ranges of optimal temperature (37 ◦ C vs 39.5 ◦ C), the growth of both severe and mild EV71 strains was inhibited, especially in mild EV71 strains (Figs. 2A–D and 3). All together these data suggest that the different replication capacity of both severe and mild EV71 strains was only achieved at high temperatures, and this finding may partly interpret that the durations of fever were significantly longer in severe HFMD children than in mild HFMD children. 3.2. The symptoms of mice after EV71 infection The 1-day-old ICR mice injected with SDLY107 developed symptoms, including piloerection, arched back, limb paralysis at day 3–4 and recovered at day 7–8 post infection, while the mice infected with SDLY 11 showed symptoms at day 5–6 and recovered at day 8–9 post infection. The 2-weeks-old ICR mice injected with SDLY 107 and SDLY11 only showed weight loss, and there was no significant alteration in the control group (normal saline). The growth curve of mice body weight showed that 1-day-old ICR mice injected with SDLY107 (group A) and injected with SDLY11 (group B) had a more slowly growth trend of weight than control group (group C). The weight of mice in group A was a slightly heavier than mice in group B (Fig. 4).
The pathology of lung, brain, intestine, and muscle in EV71 infected mice was evaluated by H&E staining. The tissues of 1-dayold mice infected with SDLY 107 and SDLY 11 exhibited distinct pathological changes at day 4 post infection, while 1-day-old mice normal control had no obvious pathological changes. The 1-dayold mice infected with SDLY 107 had more obvious pathological changes than the 1-day-old mice infected with SDLY 11 (Fig. 5). The structure of small intestinal mucosal was destroyed, accompanying with the intestinal villus falling off, vacuolar degenerating in the intestinal villus epithelial cell and inflammatory cell infiltrating (Fig. 5G–I). The muscle tissues had an accumulation of inflammatory cells (Fig. 5J–L). The lung tissues showed thickening of alveolar septa, hyperemia of blood capillaries, mononuclear and lymphocyte inflammatory infiltration, and exudation of edema fluid were also found in the alveolar cavities (Fig. 5A–C). The necrosis foci were seen in brain tissues, in which some cells dissolved and formed liquefactive necrosis, in addition, the number of microglia increased (Fig. 5D–F). 3.4. Immunohistochemical findings Immunohistochemical staining showed strong reaction with EV71 specific antigen in lung and muscle tissues of the 1-day-old mice infected with SDLY 107 and SDLY11 4 days post infection (Fig. 6). The results suggested that EV71 are mainly distributed in the muscle and lung tissue. 3.5. EV71 replication kinetics in ICR mice determined by qPCR We performed qPCR assays to detect the replication kinetics of EV71 in ICR mice. Based on the immunohistochemical results that EV71 are mainly distributed in the muscle and lung tissues, we collected the hindlimb muscle for the quantitative detection of the EV71 using qPCR assays. The EV71 replication curves showed that the 1-day-old mice infected with SDLY 107 and the 1-day-old mice infected with SDLY11 had the same viral replication tendency. However, there were two peaks of growth in 1-day-old mice infected by SDLY107 (the highest copies of viral RNA were 2.07 × 104 copies/mg and 6.02 × 104 copies/mg at day 1 and 5 post infection, respectively), while there was only one peak of growth in the 1-day-old mice infected with SDLY11 (the highest copies of viral RNA were 3.27 × 104 copies/mg), in addition, the growth peak of SDLY107 strain appeared earlier than SDLY11 strain (Fig. 7). In accordance with the viral replication curves of the 1-day-old mice, the viral load peak of 2 weeks old mice infected with SDLY 107 appears earlier than 2 weeks old mice infected with SDLY 11 (Fig. 8), and the highest copies of viral RNA were 2.57 × 104 copies/mg and 3.47 × 103 copies/mg, respectively. 4. Discussion
Fig. 4. Growth curve of weight of the 1d old mice infected by EV71. Groups of 1 d old suckling mice (n = 5 per group) were inoculated intraperitoneally (i.p.) with SDLY107 (group A) or SDLY11 (group B), respectively. The growth curve of mice body weight at different time points after EV71 infection was observed. The control group (group C) was injected with saline. At each time point, data are means ± SD of five samples; each carried out in triplicate.
EV71 is a major cause of hand-foot-and-mouth disease and is associated with fatal neurological disease (McMinn, 2002). While many factors contribute to the disease process, different virulent strains of EV71 can lead to diverse clinical outcomes (Yan et al., 2013; Wen et al., 2013; Li-mei et al., 2011). It is not clear why different EV71 strains caused different clinical outcomes in humans. In this study, we focused on the molecular mechanism of replication of EV71 genome, and demonstrated that there was a difference in replication capacity between the severe EV71 strain and the mild EV71 strain in RD cells and ICR mice. As EV71 is a member of the Picornaviridae family, an internal ribosome entry site (IRES) has been used to facilitate the expression of proteins in a single transcript, and 3D polymerase plays a role in
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Fig. 5. Representative histology of various tissues of ICR mice infected by EV71. Tissues of mock control (A, D, G and J), ICR mice infected by SDLY107 (C, F, I and L), ICR mice infected by SDLY11 (B, E, H and K). Typical lesions were indicated as the arrow: thickening of alveolar septa of lung tissues (B and C); liquefactive necrosis of brain tissues (E and F); intestinal villus loss of intestinal tissues (H and I); inflammatory cell infiltration of muscle tissues (L). Magnification: A, B, C, G, H, I, J, K, L:200×, D, E, F:100×.
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the replication of EV71, therefore, variations in these regions may affect the process of viral replication. A number of studies showed that four positions (C158, G272, U488 and A700/U700) in the 5 -NTR region (Li et al., 2011; Yeh et al., 2011) and one position (thr251) in 3D polymerase (Kung et al., 2010) were variant between the severe and mild EV71 strains. Another studies also demonstrated variation in the complete genomes of EV71 strains associated with different clinical phenotypes, especially in the 5 -UTR and 3D regions,
which were related to the translation and replication of EV71 (Yeh et al., 2011; Wen et al., 2013). A hypothesis was that these variation sites governing the range of different clinical symptoms resulted in the difference of EV71 replication capacity. In support of this idea, we found that the severe EV71 strains grew faster than the mild EV71 strains at 39.5 ◦ C in RD cells, while it was not obvious at 37 ◦ C. Our results also showed that the replication of both the severe EV71 strains and mild EV71 strains were suppressed at high
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Fig. 6. Representative images of immunostaining for lung and muscle tissues of EV71 infected mice. Target protein positive staining is brownish-yellow or snuff. The positive protein was seen both on the muscle tissues of mice infected with SDLY107 (B)/SDLY11 (C) and lung tissues of mice infected with SDLY107 (E)/SDLY11 (F). The control group (A and D) was parallel with the experimental group. Magnification: 400×.
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temperatures (39.5 ◦ C), and the suppression was more obvious in mild strains. Some studies showed that a high fever was existed in EV71 patients, and patients with CNS involvement had longer durations of fever than patients without CNS involvement (Ru et al., 2010; Liu et al., 2008; Li et al., 2002), suggesting that the temperature might play a role in the replication of EV71, so we conducted this experiment at 39.5 ◦ C to simulate in vivo temperature environments. Based on these findings, it was reasonable to assume that the severe EV71 strains replicate faster than the mild EV71 strains at high temperatures and are more likely to cause severe disease. Recently, the suitable animal model for finding clues on EV71 pathogenesis in humans was not so satisfactory. Previous studies have used monkeys and neonatal mice as models for this disease. In our studies, given the ethical and economical reasons, we used mice as models. In mice experiments, viral replication curves confirmed that the viral load peak of SDLY 107 appears earlier than
Fig. 7. EV71 replication kinetics in the 1 day old mice at different time points after EV71 infection. The 1 day old mice were infected with SDLY107 (A) isolated from death case and SDLY11 (B) isolated from mild case at a 2000CCID50 for 50 l, and the amounts of virus RNA were quantified by qRT-PCR assay. The control group (C) was parallel with the experimental group. At each time point, data are means ± SD of three samples; each carried out in triplicate.
SDLY 11 in ICR mice, suggesting that the difference of EV71 replication capacity existed in ICR mice, which was consistent with the results in RD cells. Together, the difference of EV71 replication may be involved in EV71 pathogenesis. However, mice that are more than a few weeks old lose their susceptibility to EV71 which was not observed in viral replication kinetics of 2-weeks old mice model (Fig. 8), although the 2-weeks old mice showed no clinical symptoms except for weight loss. The reasons for this difference remain to be clarified. Furthermore, the viral replication sites in the mouse (mainly in muscle tissues) and clinical outcomes of mice EV71 infection were different from humans. Thus, an appropriate small animal model should be developed for the study of this disease. In several laboratories, transgenic mouse model was used for the study of EV71 pathogenesis, and the pathological features in transgenic mouse were similar to those of EV71 infection in humans (Fujii et al., 2013). Transgenic mouse would be a promising animal model for the study of EV71 infection.
Fig. 8. EV71 replication kinetics in two weeks old mice at different time points after EV71 infection. Two weeks old mice were infected with SDLY107 (A) isolated from death case and SDLY11 (B) isolated from mild case at a 2000CCID50 for 150 l, and the amounts of virus RNA were quantified by qRT-PCR assay. The control group (C) was parallel with the experimental group. At each time point, data are means ± SD of three samples; each carried out in triplicate.
Please cite this article in press as: Sun, L.-l., et al., Association of viral replication capacity with the pathogenicity of enterovirus 71. Virus Res. (2014), http://dx.doi.org/10.1016/j.virusres.2014.04.014
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Kung et al. reported that the encephalitis strain EV71 was temperature-resistant (Tr) at 40 ◦ C in Vero cells and easier to replicate in HTB-14 (astrocytoma) than the herpangina isolate EV71 (Kung et al., 2007). Fukuhara M et al. studied the replication dynamics of three EV71 strains (major symptom was HFMD) in RD cells using mathematical modeling and also found that viral productivity was different (Fukuhara et al., 2013). In our study, we further confirmed that the severe EV71 strains replicate faster than the mild EV71 strains in RD cells at 39.5 ◦ C, and the difference in replication was partly caused by the unsuitable temperature, in addition, for the first time we demonstrated that the difference of viral replication existed in the ICR mice. Therefore, it is reasonable to predict that during EV71 infection, high replication EV71 isolates may lead to severe disease. In summary, our study demonstrated that there was a difference in replication capacity between the severe and mild enterovirus 71 isolates in mice and in vitro, which is possibly associated with EV71 pathogenesis. The molecular mechanism of virulence determinant governing the viral replication needs to be further studied. Our results would provide a new insight into the pathophysiology of EV71-related HFMD. Acknowledgements We are grateful to the Shandong Medical Equipment Quality Supervision and Inspection Center and Shandong Linyi City People’s Hospital for supplying medical equipment and clinical samples. References Chan, L.G., Parashar, U.D., Lye, M.S., et al., 2000. Deaths of children during an outbreak of hand, foot, and mouth disease in Sarawak, Malaysia: clinical and pathological characteristics of the disease. Clin. Infect. Dis. 31 (3), 678–683. Chan, K.P., Goh, K.T., Chong, C.Y., et al., 2003. Epidemic hand, foot and mouth disease caused by human enterovirus 71, Singapore. Emerg. Infect. Dis. 9 (1), 78. Fujii, K., Nagata, N., Sato, Y., et al., 2013. Transgenic mouse model for the study of enterovirus 71 neuropathogenesis. Proc. Natl. Acad. Sci. U. S. A. 110 (36), 14753–14758. Fukuhara, M., Iwami, S., Sato, K., et al., 2013. Quantification of the dynamics of enterovirus 71 infection by experimental-mathematical investigation. J. Virol. 87 (1), 701–705.
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