BACKGROUND
human enterovirus  <dig>  is a non-enveloped, single-stranded positive-sense rna virus that belongs to the enterovirus species a genogroup in the picornaviridae family.
it began circulating in the netherlands as early as  <dig> and was first described in the usa in  <dig>  <cit> .
ev-a <dig> and coxsackievirus a <dig>  are the two major etiological agents that cause hand, foot, and mouth disease ; periodic large epidemics have occurred in recent decades, and it has become a severe public health problem .
previous studies have shown that ev-a <dig> usually causes hfmd with severe neurological complications, including aseptic meningitis, brainstem encephalitis, poliomyelitis, encephalomyelitis, and even death .
in  <dig>  a large outbreak of hfmd caused by highly neurovirulent ev-a <dig> emerged in malaysia and led to  <dig> deaths among young children  <cit> .
in  <dig>  a large outbreak of enterovirus infection occurred in taiwan that resulted in  <dig> severe cases in children and  <dig> deaths.
of the  <dig> children who died,  <dig>  were under 5 years of age  <cit> .
in  <dig>  the largest recorded outbreak of ev-a71-associated hfmd occurred in mainland china, comprising > <dig>  million cases and including  <dig>  patients who exhibited severe neurological complications and  <dig> deaths  <cit> .
ev-a <dig> has one serotype and can be classified into three genotypes  and many subtypes .
in taiwan, the major subtypes of ev-a <dig> were c <dig> in  <dig>  b <dig> in the  <dig> epidemic, c <dig> in the 2004- <dig> epidemic, c <dig> in the 2006- <dig> epidemic, b <dig> in the 2008- <dig> epidemic, c <dig> in the  <dig> epidemic, and b <dig> in the 2011- <dig> epidemic  <cit> .
the predominant ev-a <dig> genotypes detected in singapore were b <dig> in 1997- <dig>  b <dig> in 2000- <dig>  c <dig> in  <dig>  and b <dig> in 2006- <dig>  in mainland china in 1998- <dig>  all the strains were clustered in the c <dig> subgenotype of ev-a <dig>
most research has been focused on developing vaccines against ev-a <dig> .
given the successful experience in the development of inactivated whole viruses for poliovirus, influenza virus, and rabies virus, inactivated ev-a <dig> whole-virus vaccines have been produced by five manufacturers in mainland china, taiwan, and singapore.
these vaccines have completed phase iii  and phase i , respectively  <cit> .
in mainland china, beijing vigoo biological co., ltd , sinovac biotec co., ltd , and the chinese academy of medical science  have used ev-a <dig> subgenotype c <dig> as a virus seed because it is the prevalent genotype in mainland china; however, vigoo and sinovac chose distinct strains, fy and h <dig>  respectively, which were all isolated from anhui province in south china  <cit> .
thus far, no vaccine has effectively prevented ev-a <dig> infection in hfmd patients is available.
previously, lethal mouse model in ev-a <dig> infection has been a pivotal evaluation role in the development of ev-a <dig> vaccines  <cit> .
however, ev-a <dig> viral isolates from hfmd patients in northeastern china  <cit>  have not been previously studied in a mouse model or for vaccine development.
our group has isolated and identified several circulating ev-a <dig> strains from hospitalized hfmd children in northeastern china who had either severe or mild disease.
we determined that these strains are complex recombinant viruses involving multiple type a human enterovirus   <cit> .
in the present study, we examined and compared the virulence, pathological changes, and progression induced by the circulating ev-a <dig> viruses, including changchun  and fuyang  strains, in a neonatal mouse model.
these strains showed different virulence, and a series of lethal strains could be used as a tool for vaccine evaluation.
furthermore, the ev-a <dig> vaccine candidate cc <dig> strain with the highest virulence also presented a broadly cross-neutralizing capacity and protection to neonatal mice from lethal-dose infect with various ev-a <dig> viruses.
at the same time, the sera of the immunized dams and their pups showed higher neutralization titers against various ev-a <dig> viruses.
the lethal challenge and protection in mouse model from circulating primary ev-a <dig> strains and the select vaccine candidates can be very helpful for vaccine development and evaluation.
methods
cells and viruses
african green monkey kidney epithelial cell line, vero cells  were cultured in modified eagle’s medium   containing 10 % fetal bovine serum   and 3 % l-glutamine at 37 °c with 5 % co <dig>  the cc strains of ev-a <dig> viruses were isolated from throat swabs of hfmd patients in changchun, china in  <dig>  viruses were continuously subcultured to the tenth passage in order to ensure stable titers and genetic features.
fy <dig>  and shzh <dig>  viruses were gifts from dr. qi jin .
brcr  viruses were received from dr. py.
mao .
cv-a16-cc <dig> is preserved in our laboratory and was isolated from a hfmd patient in changchun, china .
viruses were harvested when the cytopathogenic effect  reached 90 %, and the viral titers were determined in vero cells by the microplate cpe method and calculated by the reed–muench method  <cit> .
neonatal mouse infection model
one-day-old specific-pathogen-free  icr neonatal mice  were used to establish the animal model of viral infection.
all welfare and experimental procedures were carried out strictly in accordance with the guide for the care and use of laboratory animals and the related ethical regulations of the first hospital of jilin university.
all efforts were made to minimize animal’s suffering.
the neonatal mice were divided into different groups, randomly and each group contains three litters , and inoculated intracerebrally with ev-a <dig> viruses or mem , respectively.
the survival rates and mean clinical symptoms were monitored daily for 21 days post-infection.
the mean clinical symptoms were scored as follows:  <dig>  healthy;  <dig>  lethargy or weakness;  <dig>  wasting;  <dig>  limb shake;  <dig>  paralysis in hind limb;  <dig>  moribund or dead.
the control mice were healthy throughout the experiments.
the median lethal dose  was calculated as described by the reed–muench method  <cit> .
histopathological and immunohistochemical analysis
three mice with grade  <dig> to  <dig> of clinical disease from each of three experimental groups, ev-a71cc <dig> , brcr , ev-a71cc <dig> , and three mice of grade  <dig> to  <dig> from the control group were subjected to histopathological and ihc analysis at 5 days post-infection.
after the mice were anesthetized, tissues and organs, including lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain, were harvested and immersion-fixed with 10 % formaldehyde solution for 5 days.
then, all the samples were dehydrated via an ethanol gradient, clarified through dimethylbenzene, and embedded in paraffin, and 4-μm sections were obtained for hematoxylin and eosin  staining.
for ihc examination, 4-μm sections of tissue samples were dewaxed and hydrated through an ethanol gradient.
antigens were then restored by microwaving for 15 min at 95 °c in citrate buffer.
used hydrogen peroxide  treatment to inhibit endogenous peroxidase activity of the samples.
ev-a <dig> viral antigens were detected by anti-ev-a <dig> polyclonal antibody of rabbit  and streptavidin-peroxidase anti-rabbit igg kit .
viral loads in newborn infected mouse tissues
injected intracerebrally with ev-a71cc <dig>  or control medium,  <dig> viral challenged mice and  <dig> negative control mice were then used to detecting viral loads.
all samples including blood, lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain were harvested from the experimental group  on days  <dig>   <dig>  and  <dig> post-infection and from the control group  with no challenge on day  <dig>  all the collected tissue samples were weighed individually and stored at -80 °c for further viral detection.
the collected tissue samples were disrupted and homogenized in sterile phosphate buffer with the freeze-thaw/grinding method, then centrifuged.
the clarified supernatants were collected, and viral loads in the tissue supernatant and blood were determined by real-time fluorescence quantitative reverse transcriptase pcr  and the results were expressed in log <dig> copies/ml of blood or log <dig> copies/mg of tissue.
the 95 % confidence interval of the negative control values determined in various organs and tissues were regarded as the reference value for methodological sensitivity .
inactivation, purification, and immunogenicity of ev-a <dig> virus candidate vaccine
the vero cells infected by the seven strains of ev-a <dig> candidate virus were collected and centrifuged at 4500 × g for 30 min; the collected supernatants were inactived with formalin at 4 °c for 72 h. the effect of viral inactivation was tested by cpe, the inactived viruses were concentrated by ultrafiltration, following purified on sepharose  <dig> fast flow gel.
the protein concentration of the final virus elution was measured with a bca kit , and the viral purity was determined by silver stain plus reagent .
the purified, inactivated ev-a <dig> derived viral proteins and negative control antigens prepared in similar fashion were mixed with alum and  <dig>  ml  of the alum adjuvant vaccine was used for each immunization via intraperitoneal injection .
seven groups  of female adult icr mice were immunized i.p.
with a different ev-a <dig> vaccine candidate, cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig>  or fy <dig> two times at 2-week intervals.
serum was collected on the fourth week after immunization, and neutralization titers against the various ev-a <dig> viruses were measured by tcid <dig> assay in vero cells.
serum neutralization test
serum neutralization titers  were determined by the tcid <dig> reduction assay in vero cells.
isopycnic mixing  of the serially diluted sera was carried out with the working concentration  variously circulating ev-a <dig> strain stocks; the mixtures were incubated at 37 °c for 2 h in 96-well plates.
subsequently, the vero cells  were seeded onto 96-well plates  for infection at 35 °c with 5 % co <dig> and cultured for 7 days.
the cpe of the vero cells was observed by light microscopy.
the highest serum dilution that could inhibit cpe in >50 % of the wells were determined as serum neutralization titers.
protective efficacy of vaccine-induced maternal antibody for neonatal mice
eight-week-old female icr mice  were injected i.p.
with inactivated ev-a71cc <dig> vaccine or negative control phosphate-buffered saline  on weeks  <dig> and  <dig>  respectively.
the inactivated vaccine contained 10 μg of ev-a71cc <dig> virus.
the first injection was 1 h after mate.
after delivery, two experiments were carried out to confirm the protective efficacy of the humoral immune response of the ev-a <dig> vaccine candidate.
first, three immunized maternal mice and their pups in the vaccine and negative control groups were euthanized, sera were collected for neutralizing antibodies assays against various ev-a <dig> strains.
second, four groups of pups  were intracerebrally challenged with different lethal doses of ev-a <dig> strains on day  <dig>  challenged mice were monitored for 21 days.
statistical analysis
the clinical scores, viral loads, and antibody titers were analyzed with nonparametric one-way anova tests.
the survival rates were determined by log-rank test.
all statistical analysis were expressed as means ± the standard error of the mean .
p <  <dig>  was considered significant.
RESULTS
establishment of a neonatal mice model using primate ev-a <dig> viruses that produce strain-specific morbidity and mortality
although it has been determined that the dominant strains of ev-a <dig> have maintained a c <dig> genotype since  <dig> in mainland china, there have been no reports of the development and use of a lethal animal model employing primary ev-a <dig> isolates.
in  <dig>  multiple ev-a <dig> viruses  were isolated from infected hfmd patients in northeast china by our group and determined to be recombinant forms of ev-a <dig> viruses  <cit> .
to determine whether these ev-a <dig> viruses are lethal to neonatal mice and could be developed as a tool for vaccine evaluation, we intracerebrally injected multiple circulating ev-a <dig> viruses, i.e., cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig>  fy <dig>  and shzh <dig>  as well as prototype ev-a <dig> brcr , into one-day-old mice .
the clinical scores and survival rates of infected mice were monitored for 21 days.
the results showed that the mice that were infected with cc <dig>  cc <dig>  cc <dig>  and fy <dig> became sick on day  <dig> post-infection and present a gradual aggravation tropism, with a clinical score of grade  <dig> on days  <dig>   <dig>   <dig>   <dig>  respectively; and cc <dig>  cc <dig>  fy <dig> infected mice all were dead by days  <dig>   <dig>   <dig>  respectively, cc <dig> group had 78 % survival rate .
however, cc <dig>  cc <dig>  shzh <dig>  and brcr did not cause mortality of the neonatal mice; the mice only showed some mild lethargy and inactivity in the early days of infection  .
as expected, the mice of negative control group had a grade  <dig> of clinical score and 100 % survival rate.fig.
<dig> distinct pathogenicity of circulating cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig>  and fy <dig> or brcr viruses in neonatal mice.
newborn icr mice were infected with indicated viruse .
negative control mice were inoculated with mem instead of virus.
survival rates and mean clinical scores were continually observed for 21 days after infection.
the results are from three independent experiments, and each litter contained 8- <dig> mice.
the mean clinical symptoms were scored as follows:  <dig>  healthy;  <dig>  lethargy or weakness;  <dig>  wasting;  <dig>  limb shake;  <dig>  paralysis in hind limb;  <dig>  moribund or dead
lethal circulating ev-a <dig> viruses produce dose-dependent morbidity and mortality
since the circulating ev-a <dig> viruses cause lethality in neonatal mice, we looked further into their virulence by measuring the ld <dig> of the viruses.
groups of one-day-old mice divided as described above were injected intracerebrally with 10-fold serial dilutions of cc <dig> , cc <dig> , cc <dig> , or fy <dig> , with mem as the negative control for each group .
the mice that were infected with cc <dig> at titers of  <dig>  ~  <dig>  ccid50/ml started to show symptoms on days  <dig>   <dig>  and  <dig> post-infection, respectively; the symptoms were grade  <dig> and gradually became more severe until they reached grade  <dig> on days  <dig>   <dig>  and  <dig>  respectively .
the three titers of cc <dig>  caused death beginning on days  <dig>   <dig>  and  <dig>  respectively, with a 100 % mortality on days  <dig>   <dig>  and  <dig> post-infection, respectively .
for cc <dig> at  <dig>  ccid50/ml, the virus caused a 45 % mortality in the infected mice, with a reversible mean clinical score of grade  <dig> .
cc <dig> at  <dig>  ccid50/ml did not cause mortality, and it produced only a short period of grade  <dig> symptoms on days 8 ~  <dig> .fig.
<dig> circulating cc <dig>  cc <dig>  cc <dig>  and fy <dig> resulted in dose-related disease and mortality.
one-day-old icr mice  were intracerebrally challenged with 10-fold serial dosages of viruses.
a ev-a71cc <dig> challenged with  <dig>  ccid50/ml to  <dig>  ccid50/ml.
b ev-a71cc <dig> challenged with  <dig>  ccid50/ml to  <dig>  ccid50/ml.
c ev-a71cc <dig> challenged with  <dig>  ccid50/ml to  <dig>  ccid50/ml.
d fy <dig> challenged with  <dig>  ccid50/ml to  <dig>  ccid50/ml.
control animals were given medium instead of virus.
mortality and clinical symptoms were monitored and recorded daily for 21 days after infection.
one representative from three independent tests is shown.
the ld <dig> values are  <dig>  ccid50/ml,  <dig>  ccid50/ml,  <dig>  ccid50/ml, and  <dig>  ccid50/ml, respectively
for cc <dig>  the challenged mice given  <dig>  ccid50/ml,  <dig>  ccid50/ml, or  <dig>  ccid50/ml began to sicken on days  <dig>   <dig>  and  <dig>  respectively, with a 0 %, 80 %, and 90 % survival rate , respectively .
the challenged mice with  <dig>  ccid50/ml began to sicken on day  <dig> , and no deaths occurred during the period of observation .
the mice that were infected with cc <dig> in various titers all became sick on day  <dig> post-infection ; after an increased tropism in clinical grade, only the  <dig>  ccid50/ml group reached grade  <dig> on day  <dig>  whereas the  <dig>  and  <dig>  ccid50/ml groups had mean clinical scores of grade  <dig> during the whole test period .
the challenged mice with  <dig>  ~  <dig>  ccid50/ml began to die on days  <dig>   <dig>  and  <dig>  respectively  .
in the  <dig>  ccid50/ml group, no obvious symptoms or death of the infected mice were observed .
the mice challenged with  <dig>  ccid50/ml,  <dig>  ccid50/ml,  <dig>  ccid50/ml, or  <dig>  ccid50/ml fy <dig> began to sicken on days  <dig>   <dig>   <dig>  and  <dig> , with 100 %, 100 %, 60 %, and 10 % mortality rates by days  <dig>   <dig>   <dig>  and  <dig>  respectively.
the mice that were infected with the lowest dosage of  <dig>  ccid50/ml exhibited only mild symptoms  on days  <dig> to  <dig> and no deaths .
in the aforementioned four experiments, no clinical symptoms or death were observed in the negative control group , indicating that the disease and death of the mice were specifically attributable to the ev-a <dig> viruses.
in conclusion, the symptoms and mortality rate produced by the four pathogenic virus strains increased in a dose-dependent manner.
the median lethal doses  for cc <dig>  cc <dig>  cc <dig>  and fy <dig> were about  <dig>  ccid50/ml,  <dig>  ccid50/ml,  <dig>  ccid50/ml, and  <dig>  ccid50/ml, respectively.
cc <dig> appeared to be the most pathogenic strain .
thus, we have identified multiple circulating ev-a <dig> viruses that can induce strain-specific mortality and disease symptoms in newborn mice.
pathology in the mice post-infected with a lethal dose of circulating cc063
the newborn mice challenged with any lethal dosage of ev-a <dig> virus all exhibited typical clinical symptoms such as wasting and hind-limb paralysis.
to understand the pathological changes caused by cc strains in the neonatal mice, we selected the mice infected with the circulating cc <dig> strain, with strong virulence at the representative endpoint of grade  <dig> clinical score and conducted a series of pathologic analysis of multiple tissues to reveal changes that might be related to the death of the newborn mice.
the results showed that the hind-limb muscle and spinal skeletal muscle fibers appeared severe necrosis, including muscle bundle fracture, muscle fiber swelling, and nuclear dissolution and shrinkage  when compared to those of the non-infected mice .
however, no detectable pathological symptoms were found in the intestine, liver, spleen, kidney, or brain .
in addition, no obvious pathological changes were found in the heart or lungs .
these results demonstrated that circulating ev-a71cc <dig> has a strong tropism cause severe lesions in the muscle tissues.fig.
<dig> pathological analysis of infected newborn mice after intracerebral challenge with a lethal dose  of ev-a71cc <dig>  one-day-old icr mice were intracerebrally inoculated with ev-a71cc <dig>  or medium .
representative images from the hind-limb muscle, spine muscle, cardiac muscle, and lung tissue at 6 days post-infection are shown.
infected mice  exhibited severe necrosis in the hind-limb muscle  and spinal skeletal muscle , but no obvious histological changes in cardiac muscle  or lung tissue  by he staining.
the results for non-infected mice were used as a control .
magnification, 400 ×
to better understand the distribution of viral antigen in selected tissues of cc063-infected newborn mice, we performed ihc staining of the associated tissues of the infected mice.
the results showed widespread expression of viral antigen in the tissues, including the hind-limb muscle , spinal muscle , cardiac muscle , lung , and intestine, liver, brain, spleen, and kidney .
in contrast to the pathological changes in the hind-limb and spinal muscles, which expressed widespread viral antigen, we did not detect high viral antigen expression in the other tissues.
no viral antigen was detected in the non-infected mice , confirmed that the detection was specific.
our results suggested that even cc <dig> could lead to widespread infection, but only induced pathogenesis in the hind-limb and spinal muscles of the mice.fig.
<dig> immunohistochemical  staining results for infected mice after intracerebral challenge with a lethal dose  of ev-a71cc <dig>  one-day-old icr mice were intracerebrally inoculated with ev-a71cc <dig>  or medium .
representative sections from mouse tissues at 6 days post-infection  are shown.
the viral antigen  was detected in the hind limb muscle , spinal skeletal muscle , cardiac muscle , and lung tissue .
in contrast, no viral antigens were detected in the corresponding tissues  of the non-infected mice.
magnification, 400 ×
viral loads in selected tissues of cc063-infected mice
to further characterized the viral replication of cc <dig> viruses in infected mice, we determined viral loads in various organs at various time points post-infection .
viral loads were first detected in the heart , liver , brain , and blood  at 2 days post-infection in the cc063-infected mice.
four days after challenge, the virus was detected in all tested tissues.
the viral loads in various tissues of the infected mice, including heart, liver, spleen, lung, kidney, brain, intestine, spine muscle, hind-limb muscle, and blood reached higher levels of  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg, and  <dig>  copies/ml, respectively, on day  <dig> post-infection.
this result suggested that the virus had spread systemically by day  <dig> post-infection.
by day  <dig>  the viral loads in all the tissues except the intestine had steadily increased, and the virus copy numbers were  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg,  <dig>  copies/mg, and  <dig>  copies/ml, respectively.
in the negative control mice, no viral loads were detected in selected tissues, indicating that our results were specific.
the highest viral loads were found in the spinal muscle, hind-limb muscle, and blood in the later stages .
the detection of viral loads was also consistent with our ihc analysis , suggesting that cc <dig> has a strong tropism for spinal muscle and hind-limb muscle.fig.
<dig> kinetics of viral load levels in various tissues of ev-a71cc063-infected  one-day-old icr mice.
the viral rna copies were detected by qrt-pcr at days  <dig>   <dig>  and  <dig> post-infection, respectively.
the results represent the mean virus loads  ± sd .
rna samples from the mock-infected mice and positive virus culture medium were run simultaneously in each qrt-pcr, and known copies of the dna fragments were used as standards to calculate the copy numbers of the viral rna in the infected tissues
ev-a71cc <dig> vaccine candidate showed strongest immunogenicity and broader cross-neutralization capacity
according to the standards of innovative vaccine development reported by liang et al  <cit> , we compared an analyzed the immunogenicity of these ev-a <dig> strains including the cc strains, shzh <dig>  and fy <dig>  eight groups of seven mice were immunized with various ev-a <dig> viruses with equal inactivated virus antigen  according to the immunization procedure as fig.
6a.
the antibody cross-neutralization titer  was determined by using 28 days serum in cellular cytopathogenic effect method in vitro.
as shown in fig.
6b, cc <dig> vaccine candidate generated highest nt, and the geometrical mean  value was  <dig>  the mean cross-neutralization titer value  of cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig>  and fy <dig> were  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> .
moreover, serum of cc <dig> generated higher nt value against other virus strains except itself than other vaccine candidate .
through the comprehensive analysis of immunogenicity and broad neutralized feature, cc <dig> with the strongest virulence was chosen as an eligible vaccine candidate.fig.
<dig> serum cross-neutralization titers generated by seven ev-a <dig> vaccine candidates.
eight groups  of femal adult icr mice were immunized with ev-a <dig> vaccine candidates including cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig>  fy <dig>  and mem  twice at 2 week intervals: a diagram of the experimental procedure.
b cc <dig> vaccine candidate produced highest mean cross-neutralization titers .
mean cross-neutralization titers of the other ev-a <dig> strains were  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  for cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig> and fy <dig>  separately.
the values for the negative control and cv-a16-cc <dig> sera were all < 8
maternal antibody against the cc <dig> vaccine candidate can broadly protect the neonatal mice from lethal virus challenge
we hypothesized that the animal model developed in the present study may be a useful model for ev-a <dig> vaccine evaluation.
to examine this hypothesis, we immunized female mice  with the ev-a <dig> inactivated vaccine candidate and mated these mice after vaccine injection .
serum samples of three immunized dams and their pups on day  <dig> were collected.
neutralizing antibody  titers against various ev-a <dig> strains were measured.
the ntab titers of female mice immunized against cc <dig>  cc <dig>  cc <dig>  cc <dig>  cc <dig>  shzh <dig>  fy <dig>  or brcr reached an average of  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> , respectively.
the ntab titers of non-immunized mice were not detectable .
the serum ntab titers of pups born to the immunized female mice averaged  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> , respectively.
as expected, no ntab was detected in those pups born to non-immunized mice .fig.
<dig> broadly neutralizing antibody in immunized dams and pups.
a experimental design.
adult female mice were immunized with inactivated ev-a <dig> viral preparation and subsequently examined for serum neutralizing antibody titers as described in section of the methods.
the sera were collected from  <dig> dams and their pups  after delivery as described in section of the methods
next, we determined whether our ev-a <dig> vaccine candidate could protect neonatal mice from challenge with various lethal viruses.
after delivery, we intracerebrally challenged the pups were with lethal a dose of cc <dig> , cc <dig> , cc <dig> , or fy <dig>  on day  <dig>  the neonatal mice born to unimmunized mice  in the four groups started to die an average of 6 days after virus inoculation, and all were dead by day  <dig> post-infection .
in contrast, newborn mice of maternal immunized with vaccine candidate showed a 100 % survival rate and expressed lower clinical scores  in the early stage of the tests .
these results suggested that the animal model developed in this study can be used to evaluate ev-a <dig> vaccine candidates and that our inactivated ev-a <dig> vaccine candidate offers high efficiency and broad-spectrum cross-protection against diverse ev-a <dig> viruses in a neonatal mouse model.fig.
<dig> broadly protection of maternal immunization with the inactivated ev-a71cc <dig> against lethal viruses challenge in neonatal mice.
one-day-old icr mice  born to immunized mice were intracerebrally challenged with a cc <dig> , b cc <dig> , c cc <dig> , or d fy <dig>  and had a 100 % survival rate during the test period of 21 days.
the neonatal mice born to unimmunized mice  in the four groups all had a 100 % mortality rate and suffered increasing grades of clinical score
discussion
animal models plays a critical role in the development and evaluation of potential ev-a <dig> vaccines.
since normal adult mice are insensitive to ev-a <dig> infection, some researchers have suggested using neonatal mice or transgenic mice challenged with single viruses to evaluate the protective efficacy of vaccines  <cit> .
in our study, we have identified multiple lethal circulating ev-a <dig> viruses in a neonatal mouse model and have analyzed the pathogenic features of the lethal and non-lethal strains.
these circulating ev-a <dig> strains were isolated from different regions of china.
early genome analysis demonstrated that these ev-a <dig> strains are circulating recombinant viruses  <cit>  that are distinct from the prototype ev-a <dig> .
interestingly, although the prototype ev-a <dig>  did not cause lethal infection in the neonatal mice, several primary ev-a <dig> isolates readily generated pathogenic infections in these mice.
on the other hand, some primary ev-a <dig> isolates could not cause lethal infection, despite the fact that they are genetically closely related to the pathogenic primary ev-a <dig> viruses.
primary circulating ev-a <dig> viruses cause many symptoms in neonatal mice, including wasting, limb-shake weakness, and hind leg paralysis.
he staining showed severe lesions in the hind limb and spinal skeletal muscles, but not in the cardiac muscles or lung tissues; these findings are very different from those in the neonatal mice infected with lethal cv-a <dig> viruses, which suffered from severe lung damage  <cit> .
we could also detected viral antigen in the hind limb, cardiac, and spinal skeletal muscles.
as expected, a large amount of viral antigen was detected in the brain tissue , a point that has been emphasized in many previous studies  <cit> .
virus loads in these organs were consistent with our results.
however, the expression of viral antigen could also be detected by ihc  in many other tissues of the infected mice that showed tissue-specific pathogenesis.
several viruses, such as cc <dig>  cc <dig>  shzh <dig>  and brcr, were unable to induce neonatal death in the mice .
consistent with the findings, pathological changes and viral loads could not be detected in the organs of these infected mice , suggestnig that the absence of viral replication in the neonatal mice might be the main reason that the virus could not induce animal death.
our mouse model should be useful for future identification of viral determinants of pathogenic infection.
previous studies have demonstrated that 5’utr and vp <dig> may be the major virulence associated regions of the ev-a <dig>  we performed the 5’utr and vp <dig> sequences alignment of the virulent and non-virulent ev-a <dig> viruses .
we found that the non-virulent ev-a <dig> contain mutations that are not detected in the virulent strains.
future studies will be required to determine whether these mutations influence virulence of ev-a <dig> in the mouse model.
the eight ev-a <dig> strains evaluated in our mouse model have been associated with various disease symptoms in human.
only ev-a <dig> brcr and cc <dig> have been associated with severe neurological diseases in people.
interestingly, brcr is pathogenic in people but not virulent in the mouse model.
it is possible that brcr and cc <dig> could use different cellular receptors.
the presence of certain cellular receptor in mouse allowed cc <dig> but not brcr to replicate in the mouse model.
although the lethal mouse model may not be suitable for all ev-a <dig> strains, it is still useful for evaluating immune responses after ev-a <dig> vaccine immunization and protection of these immune responses against viral infection in this sensitive and convenient animal model.fig.
<dig> amino acid sequences alignment of vp <dig> region of virulent  and non-virulent  ev-a <dig> strains
fig.
<dig> nucleotide sequences alignment of 5’utr region of virulent  and non-virulent  ev-a <dig> strains
widespread hfmd has raised serious public health concerns in the asia-pacific region  <cit> .
coxsackievirus  <dig>  and enterovirus  <dig>  can both cause hfmd, major studies are focused on the latter as ev-a <dig> infection often associated with severe complications, including various neurological symptoms.
interestingly, we observed that administration of only the higher dosage of the various ev-a <dig> strains  could induce neonatal mouse death, as compared to circulating cv-a <dig> viruses   <cit> .
the pathogenesis induced by ev-a <dig> and cv-a <dig> needs to be further investigated in the future.
our study demonstrates that primary isolates of circulating ev-a <dig> viruses differ greatly in their ability to elicit broad-range neutralizing antibodies.
using these viruses isolated from diverse areas, we characterized ev-a <dig> vaccine candidates with the highest virulence and determined that these candidates, derived from virus from northeast china, could protect neonatal mice from challenge with diverse lethal strains.
moreover, we found that vaccine candidate cc <dig> had the highest virulence  , the strongest immunogenicity , and the broadest cross-protection .
therefore, using a sensitive mouse model and ev-a <dig> viruses, we have successfully studied the viral pathogenesis and identified a potential vaccine candidate.
CONCLUSIONS
since no effective antiviral agents are available, ev-a <dig> vaccine will play an important role in controlling ev-a71-induced hfmd in children.
the selection of a vaccine candidate strain is the crucial factor, especially for the ev-a <dig> vaccine, because of its high mutation rate and frequent recombination  <cit> .
in general, the virus vaccine candidate strain should have higher virulence, better immunogenicity, broad cross-protection, and genetic stability  <cit> .
for vaccine evaluation, with the exception of the cellular cpe method in vitro, animal model systems can serve directly evaluation to protective efficacy and immunogenicity of candidate vaccines.
indeed, we observed high immunogenicity of the ev-a <dig> vaccine candidate both in vitro and in vivo.
even more important is the fact that the ev-a <dig> candidate vaccine in our study demonstrated broad cross-protection against lethal challenge with multiple ev-a <dig> viruses from the jilin and anhui provinces of china.
our study therefore provides important guidance for future hfmd vaccine development and evaluation which may lead to the discovery of potential vaccine candidates for the prevention and control of hfmd.
junliang chang and jingliang li contributed equally to this work
competing interests
the authors declare that they have no competing interests.
authors’ contributions
xfy, wyz, ww participated in the study design and the preparation of the manuscript, jc,jl,xl,gl,jy participated in the laboratory experimental work, jc and ww participated in the analysis of the data.
all authors read and approved the final manuscript.
we thank chunyan dai, xiuying zhang, qunying mao, zhenglun liang, panyong mao, qi jin and pangyong mao for for critical reagents and deborah mcclellan for editorial assistance.
this work was supported in part by funding from the chinese ministry of science and technology , the chinese ministry of education , the national natural science foundation of china , the key laboratory of molecular virology, jilin province , and the health and family planning commission of jilin province .

 human enterovirus is a non-enveloped, single-stranded positive-sense rna virus that belongs to the enterovirus species a genogroup in the picornaviridae family. it began circulating in the netherlands as early as and was first described in the usa in. ev-a and coxsackievirus a are the two major etiological agents that cause hand, foot, and mouth disease; periodic large epidemics have occurred in recent decades, and it has become a severe public health problem. previous studies have shown that ev-a usually causes hfmd with severe neurological complications, including aseptic meningitis, brainstem encephalitis, poliomyelitis, encephalomyelitis, and even death. in a large outbreak of hfmd caused by highly neurovirulent ev-a emerged in malaysia and led to deaths among young children. in a large outbreak of enterovirus infection occurred in taiwan that resulted in severe cases in children and deaths. of the children who died, were under 5 years of age. in the largest recorded outbreak of ev-a71-associated hfmd occurred in mainland china, comprising > million cases and including patients who exhibited severe neurological complications and deaths. ev-a has one serotype and can be classified into three genotypes and many subtypes. in taiwan, the major subtypes of ev-a were c in b in the epidemic, c in the 2004- epidemic, c in the 2006- epidemic, b in the 2008- epidemic, c in the epidemic, and b in the 2011- epidemic. the predominant ev-a genotypes detected in singapore were b in 1997- b in 2000- c in and b in 2006- in mainland china in 1998- all the strains were clustered in the c subgenotype of ev-a most research has been focused on developing vaccines against ev-a. given the successful experience in the development of inactivated whole viruses for poliovirus, influenza virus, and rabies virus, inactivated ev-a whole-virus vaccines have been produced by five manufacturers in mainland china, taiwan, and singapore. these vaccines have completed phase iii and phase i, respectively. in mainland china, beijing vigoo biological co., ltd, sinovac biotec co., ltd, and the chinese academy of medical science have used ev-a subgenotype c as a virus seed because it is the prevalent genotype in mainland china; however, vigoo and sinovac chose distinct strains, fy and h respectively, which were all isolated from anhui province in south china. thus far, no vaccine has effectively prevented ev-a infection in hfmd patients is available. previously, lethal mouse model in ev-a infection has been a pivotal evaluation role in the development of ev-a vaccines. however, ev-a viral isolates from hfmd patients in northeastern china have not been previously studied in a mouse model or for vaccine development. our group has isolated and identified several circulating ev-a strains from hospitalized hfmd children in northeastern china who had either severe or mild disease. we determined that these strains are complex recombinant viruses involving multiple type a human enterovirus. in the present study, we examined and compared the virulence, pathological changes, and progression induced by the circulating ev-a viruses, including changchun and fuyang strains, in a neonatal mouse model. these strains showed different virulence, and a series of lethal strains could be used as a tool for vaccine evaluation. furthermore, the ev-a vaccine candidate cc strain with the highest virulence also presented a broadly cross-neutralizing capacity and protection to neonatal mice from lethal-dose infect with various ev-a viruses. at the same time, the sera of the immunized dams and their pups showed higher neutralization titers against various ev-a viruses. the lethal challenge and protection in mouse model from circulating primary ev-a strains and the select vaccine candidates can be very helpful for vaccine development and evaluation. methods cells and viruses african green monkey kidney epithelial cell line, vero cells were cultured in modified eagle’s medium containing 10 % fetal bovine serum and 3 % l-glutamine at 37 °c with 5 % co the cc strains of ev-a viruses were isolated from throat swabs of hfmd patients in changchun, china in viruses were continuously subcultured to the tenth passage in order to ensure stable titers and genetic features. fy and shzh viruses were gifts from dr. qi jin. brcr viruses were received from dr. py. mao. cv-a16-cc is preserved in our laboratory and was isolated from a hfmd patient in changchun, china. viruses were harvested when the cytopathogenic effect reached 90 %, and the viral titers were determined in vero cells by the microplate cpe method and calculated by the reed–muench method. neonatal mouse infection model one-day-old specific-pathogen-free icr neonatal mice were used to establish the animal model of viral infection. all welfare and experimental procedures were carried out strictly in accordance with the guide for the care and use of laboratory animals and the related ethical regulations of the first hospital of jilin university. all efforts were made to minimize animal’s suffering. the neonatal mice were divided into different groups, randomly and each group contains three litters, and inoculated intracerebrally with ev-a viruses or mem, respectively. the survival rates and mean clinical symptoms were monitored daily for 21 days post-infection. the mean clinical symptoms were scored as follows: healthy; lethargy or weakness; wasting; limb shake; paralysis in hind limb; moribund or dead. the control mice were healthy throughout the experiments. the median lethal dose was calculated as described by the reed–muench method. histopathological and immunohistochemical analysis three mice with grade to of clinical disease from each of three experimental groups, ev-a71cc, brcr, ev-a71cc, and three mice of grade to from the control group were subjected to histopathological and ihc analysis at 5 days post-infection. after the mice were anesthetized, tissues and organs, including lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain, were harvested and immersion-fixed with 10 % formaldehyde solution for 5 days. then, all the samples were dehydrated via an ethanol gradient, clarified through dimethylbenzene, and embedded in paraffin, and 4-μm sections were obtained for hematoxylin and eosin staining. for ihc examination, 4-μm sections of tissue samples were dewaxed and hydrated through an ethanol gradient. antigens were then restored by microwaving for 15 min at 95 °c in citrate buffer. used hydrogen peroxide treatment to inhibit endogenous peroxidase activity of the samples. ev-a viral antigens were detected by anti-ev-a polyclonal antibody of rabbit and streptavidin-peroxidase anti-rabbit igg kit. viral loads in newborn infected mouse tissues injected intracerebrally with ev-a71cc or control medium, viral challenged mice and negative control mice were then used to detecting viral loads. all samples including blood, lung, intestine, liver, kidney, hind limb muscle, spleen, heart, spinal muscle and brain were harvested from the experimental group on days and post-infection and from the control group with no challenge on day all the collected tissue samples were weighed individually and stored at -80 °c for further viral detection. the collected tissue samples were disrupted and homogenized in sterile phosphate buffer with the freeze-thaw/grinding method, then centrifuged. the clarified supernatants were collected, and viral loads in the tissue supernatant and blood were determined by real-time fluorescence quantitative reverse transcriptase pcr and the results were expressed in log copies/ml of blood or log copies/mg of tissue. the 95 % confidence interval of the negative control values determined in various organs and tissues were regarded as the reference value for methodological sensitivity. inactivation, purification, and immunogenicity of ev-a virus candidate vaccine the vero cells infected by the seven strains of ev-a candidate virus were collected and centrifuged at 4500 × g for 30 min; the collected supernatants were inactived with formalin at 4 °c for 72 h. the effect of viral inactivation was tested by cpe, the inactived viruses were concentrated by ultrafiltration, following purified on sepharose fast flow gel. the protein concentration of the final virus elution was measured with a bca kit, and the viral purity was determined by silver stain plus reagent. the purified, inactivated ev-a derived viral proteins and negative control antigens prepared in similar fashion were mixed with alum and  ml of the alum adjuvant vaccine was used for each immunization via intraperitoneal injection. seven groups of female adult icr mice were immunized i.p. with a different ev-a vaccine candidate, cc cc cc cc cc shzh or fy two times at 2-week intervals. serum was collected on the fourth week after immunization, and neutralization titers against the various ev-a viruses were measured by tcid assay in vero cells. serum neutralization test serum neutralization titers were determined by the tcid reduction assay in vero cells. isopycnic mixing of the serially diluted sera was carried out with the working concentration variously circulating ev-a strain stocks; the mixtures were incubated at 37 °c for 2 h in 96-well plates. subsequently, the vero cells were seeded onto 96-well plates for infection at 35 °c with 5 % co and cultured for 7 days. the cpe of the vero cells was observed by light microscopy. the highest serum dilution that could inhibit cpe in >50 % of the wells were determined as serum neutralization titers. protective efficacy of vaccine-induced maternal antibody for neonatal mice eight-week-old female icr mice were injected i.p. with inactivated ev-a71cc vaccine or negative control phosphate-buffered saline on weeks and respectively. the inactivated vaccine contained 10 μg of ev-a71cc virus. the first injection was 1 h after mate. after delivery, two experiments were carried out to confirm the protective efficacy of the humoral immune response of the ev-a vaccine candidate. first, three immunized maternal mice and their pups in the vaccine and negative control groups were euthanized, sera were collected for neutralizing antibodies assays against various ev-a strains. second, four groups of pups were intracerebrally challenged with different lethal doses of ev-a strains on day challenged mice were monitored for 21 days. statistical analysis the clinical scores, viral loads, and antibody titers were analyzed with nonparametric one-way anova tests. the survival rates were determined by log-rank test. all statistical analysis were expressed as means ± the standard error of the mean. p <  was considered significant.  establishment of a neonatal mice model using primate ev-a viruses that produce strain-specific morbidity and mortality although it has been determined that the dominant strains of ev-a have maintained a c genotype since in mainland china, there have been no reports of the development and use of a lethal animal model employing primary ev-a isolates. in multiple ev-a viruses were isolated from infected hfmd patients in northeast china by our group and determined to be recombinant forms of ev-a viruses. to determine whether these ev-a viruses are lethal to neonatal mice and could be developed as a tool for vaccine evaluation, we intracerebrally injected multiple circulating ev-a viruses, i.e., cc cc cc cc cc fy and shzh as well as prototype ev-a brcr, into one-day-old mice. the clinical scores and survival rates of infected mice were monitored for 21 days. the results showed that the mice that were infected with cc cc cc and fy became sick on day post-infection and present a gradual aggravation tropism, with a clinical score of grade on days respectively; and cc cc fy infected mice all were dead by days respectively, cc group had 78 % survival rate. however, cc cc shzh and brcr did not cause mortality of the neonatal mice; the mice only showed some mild lethargy and inactivity in the early days of infection. as expected, the mice of negative control group had a grade of clinical score and 100 % survival rate.fig. distinct pathogenicity of circulating cc cc cc cc cc shzh and fy or brcr viruses in neonatal mice. newborn icr mice were infected with indicated viruse. negative control mice were inoculated with mem instead of virus. survival rates and mean clinical scores were continually observed for 21 days after infection. the results are from three independent experiments, and each litter contained 8- mice. the mean clinical symptoms were scored as follows: healthy; lethargy or weakness; wasting; limb shake; paralysis in hind limb; moribund or dead lethal circulating ev-a viruses produce dose-dependent morbidity and mortality since the circulating ev-a viruses cause lethality in neonatal mice, we looked further into their virulence by measuring the ld of the viruses. groups of one-day-old mice divided as described above were injected intracerebrally with 10-fold serial dilutions of cc, cc, cc, or fy, with mem as the negative control for each group. the mice that were infected with cc at titers of  ~  ccid50/ml started to show symptoms on days and post-infection, respectively; the symptoms were grade and gradually became more severe until they reached grade on days and respectively. the three titers of cc caused death beginning on days and respectively, with a 100 % mortality on days and post-infection, respectively. for cc at ccid50/ml, the virus caused a 45 % mortality in the infected mice, with a reversible mean clinical score of grade. cc at ccid50/ml did not cause mortality, and it produced only a short period of grade symptoms on days 8 ~ .fig. circulating cc cc cc and fy resulted in dose-related disease and mortality. one-day-old icr mice were intracerebrally challenged with 10-fold serial dosages of viruses. a ev-a71cc challenged with ccid50/ml to ccid50/ml. b ev-a71cc challenged with ccid50/ml to ccid50/ml. c ev-a71cc challenged with ccid50/ml to ccid50/ml. d fy challenged with ccid50/ml to ccid50/ml. control animals were given medium instead of virus. mortality and clinical symptoms were monitored and recorded daily for 21 days after infection. one representative from three independent tests is shown. the ld values are ccid50/ml, ccid50/ml, ccid50/ml, and ccid50/ml, respectively for cc the challenged mice given ccid50/ml, ccid50/ml, or ccid50/ml began to sicken on days and respectively, with a 0 %, 80 %, and 90 % survival rate, respectively. the challenged mice with ccid50/ml began to sicken on day, and no deaths occurred during the period of observation. the mice that were infected with cc in various titers all became sick on day post-infection; after an increased tropism in clinical grade, only the ccid50/ml group reached grade on day whereas the and ccid50/ml groups had mean clinical scores of grade during the whole test period. the challenged mice with  ~  ccid50/ml began to die on days and respectively. in the ccid50/ml group, no obvious symptoms or death of the infected mice were observed. the mice challenged with ccid50/ml, ccid50/ml, ccid50/ml, or ccid50/ml fy began to sicken on days and, with 100 %, 100 %, 60 %, and 10 % mortality rates by days and respectively. the mice that were infected with the lowest dosage of ccid50/ml exhibited only mild symptoms on days to and no deaths. in the aforementioned four experiments, no clinical symptoms or death were observed in the negative control group, indicating that the disease and death of the mice were specifically attributable to the ev-a viruses. in conclusion, the symptoms and mortality rate produced by the four pathogenic virus strains increased in a dose-dependent manner. the median lethal doses for cc cc cc and fy were about ccid50/ml, ccid50/ml, ccid50/ml, and ccid50/ml, respectively. cc appeared to be the most pathogenic strain. thus, we have identified multiple circulating ev-a viruses that can induce strain-specific mortality and disease symptoms in newborn mice. pathology in the mice post-infected with a lethal dose of circulating cc063 the newborn mice challenged with any lethal dosage of ev-a virus all exhibited typical clinical symptoms such as wasting and hind-limb paralysis. to understand the pathological changes caused by cc strains in the neonatal mice, we selected the mice infected with the circulating cc strain, with strong virulence at the representative endpoint of grade clinical score and conducted a series of pathologic analysis of multiple tissues to reveal changes that might be related to the death of the newborn mice. the results showed that the hind-limb muscle and spinal skeletal muscle fibers appeared severe necrosis, including muscle bundle fracture, muscle fiber swelling, and nuclear dissolution and shrinkage when compared to those of the non-infected mice. however, no detectable pathological symptoms were found in the intestine, liver, spleen, kidney, or brain. in addition, no obvious pathological changes were found in the heart or lungs. these results demonstrated that circulating ev-a71cc has a strong tropism cause severe lesions in the muscle tissues.fig. pathological analysis of infected newborn mice after intracerebral challenge with a lethal dose of ev-a71cc one-day-old icr mice were intracerebrally inoculated with ev-a71cc or medium. representative images from the hind-limb muscle, spine muscle, cardiac muscle, and lung tissue at 6 days post-infection are shown. infected mice exhibited severe necrosis in the hind-limb muscle and spinal skeletal muscle, but no obvious histological changes in cardiac muscle or lung tissue by he staining. the results for non-infected mice were used as a control. magnification, 400 × to better understand the distribution of viral antigen in selected tissues of cc063-infected newborn mice, we performed ihc staining of the associated tissues of the infected mice. the results showed widespread expression of viral antigen in the tissues, including the hind-limb muscle, spinal muscle, cardiac muscle, lung, and intestine, liver, brain, spleen, and kidney. in contrast to the pathological changes in the hind-limb and spinal muscles, which expressed widespread viral antigen, we did not detect high viral antigen expression in the other tissues. no viral antigen was detected in the non-infected mice, confirmed that the detection was specific. our results suggested that even cc could lead to widespread infection, but only induced pathogenesis in the hind-limb and spinal muscles of the mice.fig. immunohistochemical staining results for infected mice after intracerebral challenge with a lethal dose of ev-a71cc one-day-old icr mice were intracerebrally inoculated with ev-a71cc or medium. representative sections from mouse tissues at 6 days post-infection are shown. the viral antigen was detected in the hind limb muscle, spinal skeletal muscle, cardiac muscle, and lung tissue. in contrast, no viral antigens were detected in the corresponding tissues of the non-infected mice. magnification, 400 × viral loads in selected tissues of cc063-infected mice to further characterized the viral replication of cc viruses in infected mice, we determined viral loads in various organs at various time points post-infection. viral loads were first detected in the heart, liver, brain, and blood at 2 days post-infection in the cc063-infected mice. four days after challenge, the virus was detected in all tested tissues. the viral loads in various tissues of the infected mice, including heart, liver, spleen, lung, kidney, brain, intestine, spine muscle, hind-limb muscle, and blood reached higher levels of copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, and copies/ml, respectively, on day post-infection. this result suggested that the virus had spread systemically by day post-infection. by day the viral loads in all the tissues except the intestine had steadily increased, and the virus copy numbers were copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, copies/mg, and copies/ml, respectively. in the negative control mice, no viral loads were detected in selected tissues, indicating that our results were specific. the highest viral loads were found in the spinal muscle, hind-limb muscle, and blood in the later stages. the detection of viral loads was also consistent with our ihc analysis, suggesting that cc has a strong tropism for spinal muscle and hind-limb muscle.fig. kinetics of viral load levels in various tissues of ev-a71cc063-infected one-day-old icr mice. the viral rna copies were detected by qrt-pcr at days and post-infection, respectively. the results represent the mean virus loads  ± sd. rna samples from the mock-infected mice and positive virus culture medium were run simultaneously in each qrt-pcr, and known copies of the dna fragments were used as standards to calculate the copy numbers of the viral rna in the infected tissues ev-a71cc vaccine candidate showed strongest immunogenicity and broader cross-neutralization capacity according to the standards of innovative vaccine development reported by liang et al, we compared an analyzed the immunogenicity of these ev-a strains including the cc strains, shzh and fy eight groups of seven mice were immunized with various ev-a viruses with equal inactivated virus antigen according to the immunization procedure as fig. 6a. the antibody cross-neutralization titer was determined by using 28 days serum in cellular cytopathogenic effect method in vitro. as shown in fig. 6b, cc vaccine candidate generated highest nt, and the geometrical mean value was the mean cross-neutralization titer value of cc cc cc cc shzh and fy were and. moreover, serum of cc generated higher nt value against other virus strains except itself than other vaccine candidate. through the comprehensive analysis of immunogenicity and broad neutralized feature, cc with the strongest virulence was chosen as an eligible vaccine candidate.fig. serum cross-neutralization titers generated by seven ev-a vaccine candidates. eight groups of femal adult icr mice were immunized with ev-a vaccine candidates including cc cc cc cc cc shzh fy and mem twice at 2 week intervals: a diagram of the experimental procedure. b cc vaccine candidate produced highest mean cross-neutralization titers. mean cross-neutralization titers of the other ev-a strains were and for cc cc cc cc shzh and fy separately. the values for the negative control and cv-a16-cc sera were all < 8 maternal antibody against the cc vaccine candidate can broadly protect the neonatal mice from lethal virus challenge we hypothesized that the animal model developed in the present study may be a useful model for ev-a vaccine evaluation. to examine this hypothesis, we immunized female mice with the ev-a inactivated vaccine candidate and mated these mice after vaccine injection. serum samples of three immunized dams and their pups on day were collected. neutralizing antibody titers against various ev-a strains were measured. the ntab titers of female mice immunized against cc cc cc cc cc shzh fy or brcr reached an average of and, respectively. the ntab titers of non-immunized mice were not detectable. the serum ntab titers of pups born to the immunized female mice averaged and, respectively. as expected, no ntab was detected in those pups born to non-immunized mice.fig. broadly neutralizing antibody in immunized dams and pups. a experimental design. adult female mice were immunized with inactivated ev-a viral preparation and subsequently examined for serum neutralizing antibody titers as described in section of the methods. the sera were collected from dams and their pups after delivery as described in section of the methods next, we determined whether our ev-a vaccine candidate could protect neonatal mice from challenge with various lethal viruses. after delivery, we intracerebrally challenged the pups were with lethal a dose of cc, cc, cc, or fy on day the neonatal mice born to unimmunized mice in the four groups started to die an average of 6 days after virus inoculation, and all were dead by day post-infection. in contrast, newborn mice of maternal immunized with vaccine candidate showed a 100 % survival rate and expressed lower clinical scores in the early stage of the tests. these results suggested that the animal model developed in this study can be used to evaluate ev-a vaccine candidates and that our inactivated ev-a vaccine candidate offers high efficiency and broad-spectrum cross-protection against diverse ev-a viruses in a neonatal mouse model.fig. broadly protection of maternal immunization with the inactivated ev-a71cc against lethal viruses challenge in neonatal mice. one-day-old icr mice born to immunized mice were intracerebrally challenged with a cc, b cc, c cc, or d fy and had a 100 % survival rate during the test period of 21 days. the neonatal mice born to unimmunized mice in the four groups all had a 100 % mortality rate and suffered increasing grades of clinical score discussion animal models plays a critical role in the development and evaluation of potential ev-a vaccines. since normal adult mice are insensitive to ev-a infection, some researchers have suggested using neonatal mice or transgenic mice challenged with single viruses to evaluate the protective efficacy of vaccines. in our study, we have identified multiple lethal circulating ev-a viruses in a neonatal mouse model and have analyzed the pathogenic features of the lethal and non-lethal strains. these circulating ev-a strains were isolated from different regions of china. early genome analysis demonstrated that these ev-a strains are circulating recombinant viruses that are distinct from the prototype ev-a. interestingly, although the prototype ev-a did not cause lethal infection in the neonatal mice, several primary ev-a isolates readily generated pathogenic infections in these mice. on the other hand, some primary ev-a isolates could not cause lethal infection, despite the fact that they are genetically closely related to the pathogenic primary ev-a viruses. primary circulating ev-a viruses cause many symptoms in neonatal mice, including wasting, limb-shake weakness, and hind leg paralysis. he staining showed severe lesions in the hind limb and spinal skeletal muscles, but not in the cardiac muscles or lung tissues; these findings are very different from those in the neonatal mice infected with lethal cv-a viruses, which suffered from severe lung damage. we could also detected viral antigen in the hind limb, cardiac, and spinal skeletal muscles. as expected, a large amount of viral antigen was detected in the brain tissue, a point that has been emphasized in many previous studies. virus loads in these organs were consistent with our results. however, the expression of viral antigen could also be detected by ihc in many other tissues of the infected mice that showed tissue-specific pathogenesis. several viruses, such as cc cc shzh and brcr, were unable to induce neonatal death in the mice. consistent with the findings, pathological changes and viral loads could not be detected in the organs of these infected mice, suggestnig that the absence of viral replication in the neonatal mice might be the main reason that the virus could not induce animal death. our mouse model should be useful for future identification of viral determinants of pathogenic infection. previous studies have demonstrated that 5’utr and vp may be the major virulence associated regions of the ev-a we performed the 5’utr and vp sequences alignment of the virulent and non-virulent ev-a viruses. we found that the non-virulent ev-a contain mutations that are not detected in the virulent strains. future studies will be required to determine whether these mutations influence virulence of ev-a in the mouse model. the eight ev-a strains evaluated in our mouse model have been associated with various disease symptoms in human. only ev-a brcr and cc have been associated with severe neurological diseases in people. interestingly, brcr is pathogenic in people but not virulent in the mouse model. it is possible that brcr and cc could use different cellular receptors. the presence of certain cellular receptor in mouse allowed cc but not brcr to replicate in the mouse model. although the lethal mouse model may not be suitable for all ev-a strains, it is still useful for evaluating immune responses after ev-a vaccine immunization and protection of these immune responses against viral infection in this sensitive and convenient animal model.fig. amino acid sequences alignment of vp region of virulent and non-virulent ev-a strains fig. nucleotide sequences alignment of 5’utr region of virulent and non-virulent ev-a strains widespread hfmd has raised serious public health concerns in the asia-pacific region. coxsackievirus and enterovirus can both cause hfmd, major studies are focused on the latter as ev-a infection often associated with severe complications, including various neurological symptoms. interestingly, we observed that administration of only the higher dosage of the various ev-a strains could induce neonatal mouse death, as compared to circulating cv-a viruses. the pathogenesis induced by ev-a and cv-a needs to be further investigated in the future. our study demonstrates that primary isolates of circulating ev-a viruses differ greatly in their ability to elicit broad-range neutralizing antibodies. using these viruses isolated from diverse areas, we characterized ev-a vaccine candidates with the highest virulence and determined that these candidates, derived from virus from northeast china, could protect neonatal mice from challenge with diverse lethal strains. moreover, we found that vaccine candidate cc had the highest virulence, the strongest immunogenicity, and the broadest cross-protection. therefore, using a sensitive mouse model and ev-a viruses, we have successfully studied the viral pathogenesis and identified a potential vaccine candidate.  since no effective antiviral agents are available, ev-a vaccine will play an important role in controlling ev-a71-induced hfmd in children. the selection of a vaccine candidate strain is the crucial factor, especially for the ev-a vaccine, because of its high mutation rate and frequent recombination. in general, the virus vaccine candidate strain should have higher virulence, better immunogenicity, broad cross-protection, and genetic stability. for vaccine evaluation, with the exception of the cellular cpe method in vitro, animal model systems can serve directly evaluation to protective efficacy and immunogenicity of candidate vaccines. indeed, we observed high immunogenicity of the ev-a vaccine candidate both in vitro and in vivo. even more important is the fact that the ev-a candidate vaccine in our study demonstrated broad cross-protection against lethal challenge with multiple ev-a viruses from the jilin and anhui provinces of china. our study therefore provides important guidance for future hfmd vaccine development and evaluation which may lead to the discovery of potential vaccine candidates for the prevention and control of hfmd. junliang chang and jingliang li contributed equally to this work competing interests the authors declare that they have no competing interests. authors’ contributions xfy, wyz, ww participated in the study design and the preparation of the manuscript, jc,jl,xl,gl,jy participated in the laboratory experimental work, jc and ww participated in the analysis of the data. all authors read and approved the final manuscript. we thank chunyan dai, xiuying zhang, qunying mao, zhenglun liang, panyong mao, qi jin and pangyong mao for for critical reagents and deborah mcclellan for editorial assistance. this work was supported in part by funding from the chinese ministry of science and technology, the chinese ministry of education, the national natural science foundation of china, the key laboratory of molecular virology, jilin province, and the health and family planning commission of jilin province.
although animal models have been developed using mouse-adapted ev-a strains, mouse models using primary ev-a isolates are scarce. lethal animal models with circulating ev-a infection would contribute to studies of pathogenesis as well as vaccine development and evaluation. thus, our newly established animal model using primary ev-a isolates is helpful for future studies on viral pathogenesis and vaccine and drug development. an inactivated ev-a vaccine candidate offered complete protection from death induced by various circulating ev-a viruses to neonatal mice that were born to immunized female mice. circulating enterovirus -associated hand, foot, and mouth disease is on the rise in the asian-pacific region. in this study, we established a lethal mouse model using primary ev-a isolates from patients infected with serotypes that are currently circulating in humans. we also characterized the dose-dependent virulence and pathologic changes of circulating ev-a in this mouse model. most importantly, we have established this mouse model as a suitable system for ev-a vaccine evaluation. enterovirus 71mouse modelvaccine candidateissue-copyright-statement© the author 2015 the sera of the immunized dams and their pups showed higher neutralization titers against multiple circulating ev-a viruses.
 circulating enterovirus -associated hand, foot, and mouth disease is on the rise in the asian-pacific region. although animal models have been developed using mouse-adapted ev-a strains, mouse models using primary ev-a isolates are scarce. lethal animal models with circulating ev-a infection would contribute to studies of pathogenesis as well as vaccine development and evaluation.  in this study, we established a lethal mouse model using primary ev-a isolates from patients infected with serotypes that are currently circulating in humans. we also characterized the dose-dependent virulence and pathologic changes of circulating ev-a in this mouse model. most importantly, we have established this mouse model as a suitable system for ev-a vaccine evaluation. an inactivated ev-a vaccine candidate offered complete protection from death induced by various circulating ev-a viruses to neonatal mice that were born to immunized female mice. the sera of the immunized dams and their pups showed higher neutralization titers against multiple circulating ev-a viruses.  thus, our newly established animal model using primary ev-a isolates is helpful for future studies on viral pathogenesis and vaccine and drug development. keywords enterovirus 71mouse modelvaccine candidateissue-copyright-statement© the author 2015 