Study On The Pathogenesis Of Enterovirus 71

1 BackgroundHand,foot and mouth disease(HFMD)is one of the common infectious disease caused by enterovirus.Coxsackie virus A16(CoxA16)and enterovirus 71(EV71)are two main pathogens.HFMD mainly causes fever and rash and ulcer in the parts of hand,foot and mouth in children under 5 years old.Some serious patients may develop fatal complications such as myocarditis,pulmonary edema and aseptic meningitis.EV71 infection is mostly related to the serious condition.Serious HFMD and death related to EV71 infection were often reported every time the HFMD outbreak occurred,which posted a great threat to the public health.Serious EV71 infection mainly presented neurological diseases manifesting aseptic meningitis,encephalitis or poliomyelitis-like acute flaccid paralysis which resulted in the pulmonary edema,pulmonary hemorrhage and myocarditis,leading to the the high rate of disability and lethality.Epidemics of HFMD occurred in China from 2007 to 2008.Our team reported an 8-month-old female infant with the fatal enterovius 71 infection and had a study in the aspects of clinical,etiology,and pathology etc.Results showed that EV71 can cause disseminated infections involving multiple organs.Pulmonary edema and hemorrhage are the main cause of death.Pathological observation of central nervous system showed that the brain tissue presented obvious inflammation while the inflammation of the cerebellum and the brain stem was not obvious.Immunochemical detection showed that EV71 antigen was positive in the brain glial cells,the cerebellar granule cells and the brain stem neurons,indicating that multiple neuron cells of the brain tissues are the main target objects of EV71.Many studies indicated that the pathogenesis of EV71 was regulated by many factors,but more researches were still needed to clarify the pathogenesis of EV71 infection,which was significant to the control and treatment of EV71 infection.2 Objective(1)To study the target organs for enterovirus 71(EV71)in EV71 infected BALB/c mice and study the potential pathogenic mechanism of EV71.(3)To analyze the potential pathogenic mechanism of EV71 in Vero cells.3 MethodsThe EV71 used in the study was a clinical isolate derived from a fatal case of enterovirus 71 infection in 2008,Guangzhou.Studying methods were as follows:(1)The isolated virus was titrated in the Vero cells.The end point titer was calculated in tissue culture infective dose 50(TCID50)per mL as described by Reed and Muench.(2)An EV71 clinical isolate was inoculated to Vero cells and the EV71 VP1 antigen was detected by immunofluorescence.(3)Ultrastructures of the EV71 infected Vero cells were observed by electron transmission microscopy.(4)5-day-old BALB/c suckling mice were infected with an EV71 strain(888/GZ/CHN/2008).Clinical presentation was noted.Tissues of the infected mice were processed for histopathological examination and the distribution of EV71 antigen was detected by immunochemical staining.(5)Injury of the skeletal muscle tissues was studied by in situ hybridization and ultrastructural observation.(6)Applying the stereological principles and methods,viral particles and mitochondria in EV71 infected Vero cells were studied by quantitative methods.4 Results(1)TCID50 of the EV71 strain used in this experiment was 10-5.81/0.1mL.(2)The EV71 virus infected Vero cells became round,appeared crimple and progressed to death.The EV71 particles were detected in the cytoplasm by immunofluorescence.The ultrastructure showed that large numbers of viral particles existed in the cytoplasm with agglomerate distribution and lattice arrangement.Most of the mitochondria presented swelling,vacuole and degeneration.(3)5-day-old BALB/c suckling mice were infected with an EV71 strain.Results of the histopathological study showed obvious myofibril fracture and myocyte disruption.The result of immunohistochemistry and in situ hybridization proved that a large amount of EV71 existed in the skeletal muscle tissues.Except four EV71 infected mice,the brain tissues showed mild inflammatory cells infiltration,but no positive EV71 antigen was detected in these tissues.Different from the obviously damaged skeletal muscle,in the EV71 infected mice the myocardia were intact.Pulmonary edema and hemorrhage were seen,other tissues and organs were normal.(4)Ultrastructural observation of skeletal muscle tissues of the EV71 infected mice and the EV71 infected Vero cells showed that large numbers of mitochondria were vacuolated.In some vacuolated mitochondria of the EV71 Vero cells,high electron density granules were seen.Quantitative analysis of the granules in the mitochondria were consistent with those outside the mitochondria.5 Conclusions(1)The EV71 clinical isolate used in this study presented evident myotropism.Skeletal muscles,including the limbs,the intercostal spaces and even along the spine,are main target organs for EV71 in the infected suckling mice(2)EV71 proliferated in the cytoplasm of the infected Vero cells and invaded the mitochondria,leading to the direct injury of the mitochondrial structure and function,suggesting mitochondria might be a target for EV71 infection.