||The role of neutrophils in enterovirus 71 infection
||Department of Microbiology & Immunology
Enterovirus 71 (EV71) infection causes many clinical syndromes, including hand-foot-mouth disease, herpangina, aseptic meningitis, encephalitis, and paralysis, particularly, in infants and children. The relationship between disease progression and virus load is unclear. To investigate the virus load in serum samples collected from EV71 patients, I developed a reverse-transcription PCR assays to quantify EV71 genome in samples. These assays are specific for EV71. The detection limit is 103 copies genome per milliliter serum sample for both positive- and negative-stranded genome assays. When these assays were used to determine the amount of virus in peripheral blood mononuclear cells from 20 patients, no viruses can be detected in these samples. A mouse model was also established to investigate the pathogenesis of EV71 infection of intragastral (i.g.) or intraperitoneal (i.p.) inoculation. Results showed that the mouse survival rate was dependent on not only the age but also the body weight of mice. In EV71 infected-mice, viruses caused a systemic infection and their titers in neuronal organs were higher than those in non-neuronal organs during late infection. Immune cells, such as neutrophils, infiltrated into infected organs. Many reports indicated that neutrophils play an important in neurotropic virus infections. To investigate the role of neutrophils in EV71 infection, monoclonal antibody specific for granulocytes, RB6-8C5, was used to deplete neutrophils before and after oral infection with EV71. I found neutrophil depletion decreased mouse survival. The virus titer in the brain and spinal cord of neutrophil depleted mice was significantly higher than those of normal control mice at day 7 post infection. This result suggested that neutrophils play an important role in protecting mice from death and inhibiting virus growth after EV71 oral infection.
Chinese abstract i
English abstract iii
Material and methods 6
Virus and cells 6
Preparation of total RNA from patient PBMC and virus-infected cells 6
Quantitative reverse-transcription polymerase chain reaction (QR-PCR) 7
Infection of mice and tissue collection 8
Antibody purification 8
Neutrophil depletion during EV71 infection 9
Immunohistochemistry (IHC) staining 10
Histological examination 10
Cocultivation of EV71 and neutrophils in vitro 10
Statistical analysis 11
Determination of primers specificity 12
Establishment of RNA standards for QR-PCR 12
Analyses of PBMC from EV71 patients 13
Establishment of a mouse model for EV71 infection 13
The effect of neutrophil depletion on EV71-infected mice 16
Interaction of neutrophils and virus in vitro 17
Detection of viruses in patient PBMC samples 19
Establishment of mouse model foe EV71 infection 20
The role of neutrophils in EV71 infection 21
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