Original article
A mouse muscle-adapted enterovirus 71strain 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.5Panjiayuan Nanli,Chaoyang Dist,Beijing 100021,PR China
Received 20December 2010;accepted 30April 2011
Available online 12May 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 v
irulence of mouse muscle-adapted EV71.The EV710805were subjected to four passages in the mouse muscle to generate a mouse-adapted EV71strain of 0805a.In comparison with the parental EV710805,the mouse muscle-adapted EV710805a displayed stronger cytotoxicity against Rhabdomyosarcoma (RD)cells and more efficient replication in RD cells.Furthermore,infection with the EV710805a significantly 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 EV710805did 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 5post 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 EV710805a with increased virulence in mice.Ó2011Institut 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 [1e 3],is the major cau
sative 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 EV71can sometimes cause severe neurological diseases,such as brainstem encephalitis and polio-like paralysis [2,12].Therefore,EV71has become one of the most important enteroviruses known to
cause fatalities in children,representing a major public health concern [13,14].
Following EV71infection,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 EV71in human and mouse skeletal muscles is associated with the development of persistent infection [18,19].Interestingly,the mouse-adapted strain of EV71predominately infect the skel-etal muscles in ICR and NOD/SCID mice [16,20].While EV71infection-mediated severe neurological disease is a major lethal factor,the EV71strains isolated from individual patients of epidemic poliomyelitis-like disease in Bulgaria displayed high tropism for mouse muscle tissues [2].Furthermore,many EV71strains,such as B3and B4,as well as other clinical isolates,can infect mouse muscle,but not the br
ain [21,22].Apparently,the muscle acts as a host organ for the initial viral replication,which is crucial for the subsequent
*Corresponding author.Tel.:þ861067770815;fax:þ861067710812.**Corresponding author.Tel.:þ861067776049;fax:þ861067761136.E-mail addresses:weiqiang0430@sohu (Q.Wei),qinchuan001@yeah (C.
Qin).Microbes and Infection 13(2011)862e
870
www.elsevier/locate/micinf
1286-4579/$-see front matter Ó2011Institut Pasteur.Published by Elsevier Masson SAS.All rights reserved.doi:10.1016/j.micinf.2011.04.004
neuroinvasion[19]and may play an important role in the EV71infection in humans and mice.However,it is unclear whether mouse muscle-adapted EV71strain could be gener-ated and how virulent it could b
e.In addition,the molecular mechanisms underlying the myotropism of EV71have not been fully understood.
In this study,the parental EV71strain Fuyang-0805was first passaged in mouse skeletal muscle for four times to generate a mouse muscle-adapted EV71strain,Fuyang-0805a, with increased myotropism in mice.Subsequently,the phenotype,virulence,and molecular characteristics of the EV71Fuyang-0805a strain were analyzed for understanding the adaptive evolution of EV71and 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 clin-ical strains of EV71[15,23e26],human colorectal carcinoma Caco-2cells,African green monkey kidney Vero cells,and human neuroblastom SH-SY5Y cells were maintained in Dulbecco’s Modified Eagle’s Medium(DMEM,Gibco) containing10e15%fetal bovine serum(FBS,Gibco),2mM L-glutamine,100IU of penicillin,and100m g of streptomycin/ ml at37 C,5%CO2.The EV71Fuyang-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,Bei-jing,China)were inoculated intraperitoneally(i.p.)with106 TCID50of the parental strain of EV710805.Five days later, the mice were sacrificed 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 at108TCID50per 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(2Â104cells/ well)were cultured in96-well plates for18h and infected in triplicate with1MOI of the strain0805or0805a for varying periods,respectively.The cytotoxicity of these viruses was monitored lon
gitudinally 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 at106cells/well were cultured in24-well plates and infected in triplicate with100TCID50of the EV710805or0805a for1h at37 C,respectively.The cells were washed with phosphate buffered saline(PBS)and then cultured in2%FBS DMEM for varying periods.The cells were harvested and the virus titers were determined by a real-time RT-PCR assay,as described below.
2.3.Virus inoculation and detection in mice
ICR mice at one day of age(n¼120)were fasted for4h and inoculated i.p.with105TCID50of EV71strain0805or 0805a(50m l),respectively.The control mice(n¼60)were injected with the same volume of RD cell lysate.Their body weights and clinical signs,including ruffled fur,hunchbacke, wasting,limb weakness,limb paralysis,moribund and death, were monitored daily up to9days after inoculation.In addi-tion,10mice per group were sacrificed on day0,1,3,5,and7 post-inoculation,respectively.After perfusion with PBS con-taining EDTA,their brain,lung,limb muscle,heart,and intestine tissues were immediately dissected out for the extraction of RNA or for histopathological and immunohis-tochemical examinations,respectively.
Total RNA was extracted from different tissues(30mg) using an RNeasy Mini kit(Qiagen,Hilden,Germany).The RNA was assayed by one-step RT-PCR in a20-m l 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 nucleotides2372e2598[11,28],and the conditions consisted of a denaturation step at95 C for20s and40cycles of thermal cycling of95 C for3s and60 C for 30s.The EV71virus fragment of nucleotides2372e2598was used as real-time PCR standard by adjusting to a concentration gradient of1Â106copies/m l,1Â105copies/m l,1Â104 copies/m l,1Â103copies/m l,and1Â102copies/m l,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 100copies.
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 VP1gene of EV71was amplified by PCR using 1m l of cDNA sample,as the template and the primers were same wi
th the real-time PCR.The PCR products were subjected to electrophoresis on1.5%agarose gels.The mouse GAPDH was used as an internal control.
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W.Wang et al./Microbes and Infection13(2011)862e870
2.5.Histopathology
The collected tissue samples werefixed in10%buffered formalin for48h and embedded in paraffin.The tissue sections at5m m were stained with hematoxylin and eosin (HE).Histopathology of mouse skeletal and heart muscles was evaluated for inflammation,musclefiber degeneration and necrosis,while small intestines were evaluated for intestinal villus interstitial edema and epithelial cell vacuolar degener-ation 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 EV71clinical isolates,strain 0805,and the mouse-adapted strain0805a were characterized by sequencing,as described previously[29,30].Briefly,viral RNA was prepared using
a high-pure viral RNA purification kit(Qiagen,Hilden,Germany)and reversely transcribed into cDNA using a ReverTra-Plus kit(Invitrogen).The whole-genome of EV71was amplified by PCR using the specific primers[29,30].The PCR products were purified using the agarose gel DNA purification kit(Takara,Dalian,China),and cloned into a pGEM-T vector(Promega,Madison,WI,USA), followed by transforming into Escherichia coli JM109.A total of6e8colonies from each virus library were characterized by DNA sequencing on a3730DNA analyzer(Applied Bio-systems,Foster City,CA,USA).The DNA sequences were analyzed using DNAMAN 6.0and Bioedit7.0[31].The secondary structure was predicted using the GOR IV program (npsa-pbil.ibcp.fr/cgi-bin/secpred_gor4.pl)[32].
2.7.Statistical analysis
Data are expressed as meanÆSD.The difference between groups was analyzed with a Mann e Whitney U test using the SPSS software.A p value of<0.05was considered statisti-cally significant.
3.Results
3.1.Mouse muscle-adapted EV710805a with hypervirulence in vitro
To examine the virulence and tissue tropism,a wild strain of EV710805was passaged in mouse muscles for four passages to generate the mouse muscle-adapted EV71strain 0805a,which was detected in the infected RD cells by indirect immunofluorescent staining with anti-EV71monoclonal anti-body.Next,its cytotoxicity against different types of cells was compared with its parental EV710805(Fig.1).Both strains of EV710805and0805a appeared not to have obvious cyto-toxicity against nerve cells SY5Y,because the proliferation of cells was similar regardless of infection with,or without,the virus.Furthermore,while infection with EV710805promoted the proliferation of monkey kidney Vero cells,EV710805a infection had no stimulating effect on the proliferation of Vero cells in vitro.More importantly,both EV710805and0805a displayed strong cytotoxicity against muscle RD and intestinal Caco-2cells as compared to that of Vero and SY5Y cells, particularly for the EV710805a.Evidentially,the proliferation of both types of cells was significantly reduced by25e95%,as compared with that of un-manipulated cells,and the inhibitory effect of EV710805a was more potent than that of EV710805 in RD cells.Similar patterns of CPE were observed in different types of cells(data not shown).These data indicated that EV710805a had a higher muscle tropism.
Characterization of the dynamics of virus replication revealed that while similar numbers of virus copies were detected infirst48h,the virions of EV710805a were significantly greater than that of EV710
805at72and96h post infection and then returned to similar levels of replication in RD cells(Fig.2).These demonstrated that EV710805a replicated faster in RD cells,which was associated with higher cytotoxicity of the virus against RD cells.
3.2.Mouse muscle-adapted EV710805a with increased virulence in vivo
Next,the in vivo virulence of the EV710805and0805a was tested.ICR mice at one day of age were infected with EV710805or0805a,and they were monitored for survival and clinical symptoms up to21days 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 EV710805was exclusively detected in the muscles,the EV710805a was found in the muscles,intestines, lungs,and brain(Fig.3A),indicating that the mouse muscle-adapted EV710805a had higher virulence in our experimental system.
To further investigate the virulence of virus,ICR mice at one day of age were infected with EV710805or0805a, respectively.Their body weights were measured longitudinally (Fig.3B).While mice infected with EV710805grew as much as that of un-manipulated mice,the mice infected with EV71 080
5a appeared to grow slowly.Evidentially,the body weights on day5,7,and9post infections were significantly lighter than that of un-manipulated controls.Characterization of virus antigen in mice indicated that the EV710805a was detected in the muscles,heart and intestines,while the EV710805was found only in the muscles of mice on day5post infection(data not shown).Specifically,analysis of virus in the limb muscle samples indicated that although similar dynamics of virus replication were observed in the EV710805and0805a-infected mouse muscles,the numbers of the EV710805a virions were significantly greater than that of the EV710805 detected on day1,3,5,and7post infection(Fig.3C).
Further histological examinations indicated that the limb muscles from the EV710805a-infected mice displayed massive and widespread necrotizing myositis with some inflammatory infiltrates on day5post infection(Fig.4).
864W.Wang et al./Microbes and Infection13(2011)862e870
Similar pathogenic characteristics were observed in the myocardium from the EV710805a-infected mice.Interest-ingly,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 in
testines of the EV710805a-infected mice on day 5post 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 EV710805-infected mice.
3.3.Comparison of the EV710805genome sequence with mouse-adapted EV710805a
To understand the mechanisms underlying the hyper-virulence of mouse muscle-adapted EV710805a,the genomes of EV710805and 0805a were sequenced.These two strains of viruses shared more than 95%of nucleotide identities,but a total of 321nucleotide mutations were detected in the genome of EV71strain 0805a,and their distribution varied in different regions (Fig.5A).Among these mutations,there were 25non-synonymous mutations,which distributed in different regions of the EV710805a genome (Fig.5B).The frequency of mutations in different regions of the EV710805a genome was similar,except for the VP1region,in which the frequency of mutations was very low (Fig.5C).The synony-mous mutations were mainly located some regions and the highest frequency of synonymous mutations was located in the 3A region (Frequency of mutations per 100bp ¼0.011628,Fig.5D).Correspondingly,analysis of the deduced amino acid sequences revealed that thes
e nucleotide mutations resulted in 25amino acid substitution in the EV710805a,as compared with that of EV710805(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.
2040
6080100120RD cell
20
40608010012048
7296
20406080100120487296
20
40608010012048
72
96
48
72
96
Hour pos t-infection
C e l l v i a b i l i t y (% o f c o n t r o l )
Fig.1.Cytotoxicity of EV710805and 0805a inreaction member
vitro .Vero,RD,SY5Y ,and Caco-2cells were cultured in 96-well plates for 18h and infected in triplicate with,or without,1MOI of EV710805and 0805a for the indicated periods,respectively.The viabilities of individual types of cells following virus infection were determined using the CCK-8assay.Data are expressed as mean %ÆSD of each group of cells from three independent experiments and the cultured cells without virus infection were designated as 100%.*p ＜0.05vs.the cells infected with EV710805.
RD cell
Hour post infection
V i r u s l o a d (l o g 10 c o p i e s /m l )
Fig.2.The dynamics of EV710805and 0805a replication in RD cells.Monolayer RD cells in 24-well plates were infected in triplicate with 100TCID 50of EV710805or 0805a at 37 C for 1h,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 RT-PCR assay.Data are expressed as mean ÆSD of virions in each group from three separate experiments.*p ＜0.05vs.EV710805.
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4.Discussion
To study the myotropism and virulence of EV71in the present study,an EV71strain of 0805was subjected to four serial passages of mouse muscle adaption to generate a strain of EV710805a.In comparison with the parental EV710805,we found that the EV710805a increased its myotropism and virulence in vitro .First,the EV710805a displayed stronger cytotoxicity against RD in vitro .Evidentially,following infection with EV710805a,the viability of RD cells was significantly lower than that of cells infected with EV710805.Second,the EV710805a appeared to replicate faster as the virus load on 72and 96h post infection was significantly higher than that of EV710805in RD cells in vitro.Apparently,the mouse muscle-adapted EV710805a displayed higher virulence in vitro ,providing an excellent model for studying the virulence and pathogenicity of mouse muscle-adapted EV71in vivo .
The parental EV71strain 0805,a C4group of virus,was isolated from pharyngeal swab of a child with HFMD in Fuyang city [27],and was a predominant genotype,respon-sible for recent outbreaks of HFMD in China (GenBank accession number FJ439769).The EV710805has been used in studies of th
e pathogenesis of EV71[29,33].In this study,following i.p inoculation,the EV710805invaded in the muscles of young mice,consistent with a previous report.In contrast,the EV710805a 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 EV710805a,but not the parental EV710805,led to detectable virus antigen in the cytoplasma of muscle cells of mice (data not shown).These indicated that following infection,the EV710805a 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 EV710805a were significantly higher than that of the mice infected with EV710805,accompanied by significantly reduced body weights in the mice infected with the EV170805a,as compared with that in the mice infected with EV710805.It is possible that the mouse muscle adaption of EV71increased the virulence and tropism [16].Alterna-tively,the mouse muscle adaption promoted virus replication in the muscle more efficiently.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 EV71viruses,may determine the pathogenesis of virus infection,at least in mice,and may potentially be used as a prognostic marker of EV71infection in susceptible individuals.
Pathologically,the EV71is an enterovirus.Previous studies have shown that EV71can 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 EV710805was a moderate viru-lent virus.Evidentially,infection with EV710805had moderate effect on the viability of RD,but not other cells,and did not cause pathogenic lesion in mice.In contrast,the EV710805a 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 EV710805a.The lack of lung
and
Fig.3.The infected mouse’s weight gain and EV71virus replication in the mouse infected with EV710805or 0805a.ICR mice at one day of age were fasted for 4h and inoculated i.p.with,or without,50m l of EV710805or 0805a (105TCID 50),respectively.Their body weights were measured at indicated time points.Ten mice from each group were sacrificed at the indicated time points and their brains,limb muscles,intestines,and lungs samples were collected.The contents of EV71in the muscles were determined by quantitative real-time RT-PCR.And the presence of EV71virus was determined by PCR using the specific primers from different tissue.(A)Characterization of EV71virus in different mouse tissues by RT-PCR.Data shown are representative images of each group (n ¼10)on day 5post-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 ÆSD of body weights (g)of each group of mice (n ¼30e 60).(C)Quantitative analysis of EV71virions in the mouse muscles.Data are expressed as mean ÆSD of each group (n ¼6per time point)of mice from three independent experiments.*p ＜0.05vs.EV710805group.
866W.Wang et al./Microbes and Infection 13(2011)862e 870
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