1 Departments of Microbiology & Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China
2 Departments of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China
3 Departments of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China
Correspondence
Huan-Yao Lei
hylei{at}mail.ncku.edu.tw
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ABSTRACT |
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INTRODUCTION |
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The pathogenesis of EV71 infection, especially the CNS involvement, is not clearly understood. Little is known of the factors contributing to the manifestation of CNS symptoms. We have reported that neonatal mice can be infected with EV71 experimentally. Intraperitoneal inoculation of 108 p.f.u. of EV71 induced death of 1-day-old ICR mice in an age- and dose-dependent manner (Yu et al., 2000). Since intraperitoneal injection is not the natural route for enterovirus infection, the application of this experimental infection model is limited. The objective of this study was to establish a murine EV71 model in which mice are orally infected and manifest symptoms that mimic the clinical situation in humans.
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METHODS |
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Enterovirus 71.
Local isolates of EV71 (4643, 6360, 6367, 6321, 5811 and 5101) were obtained from the Virological Center, National Cheng Kung University, Tainan, Taiwan. These EV71 isolates were derived from different sources: EV71 4643, 6360 and 6367 were from fatal cases with CNS involvement; 6321 was from a non-fatal case with CNS involvement; and 5811 and 5101 were from patients with HFMD. The mouse-adapted strain was obtained from brain tissues of mice injected with EV71 4643 strain by intraperitoneal administration. The virus was further propagated in the Vero cell line before use. In some experiments, the Caco-2 cell line-adapted EV71 virus was used. EV71 4643 was propagated in the Caco-2 cell line. Virus stocks were collected by freezing and thawing three times. The titre of each virus pool was determined by the standard method of plaque assay on Vero cells (see below) and found to be 1x107 p.f.u. ml-1. The sequence of the EV71 strains Tainan/4643/98 has been deposited in the GenBank database under accession number AF304458. (Yang et al., 2001). The sequence of the mouse-adapted 4643 isolate has 99 % homology with that of the parental 4643.
Plaque assay.
Vero cells (1x105 cells per well) were plated onto a 24-well plate, incubated overnight and infected with 200 µl of serially diluted virus suspension. After adsorption for 2 h, the virus suspension was replaced with DMEM containing 2 % FBS and 0·5 % methylcellulose (Sigma). The medium was removed at 72 h post-infection and the cells were stained with crystal violet solution (1 % crystal violet, 0·64 % NaCl and 2 % formalin) (Wen et al., 2003).
Infection of EV71 in Caco-2 and SK-N-SH cells.
Caco-2 and SK-N-SH cells were infected with EV71 4643 or mouse-adapted EV71 at an m.o.i. of 1 for 1 h. After washing with PBS, cell cultures were incubated at 37 °C and 5 % CO2. Virus titre was determined at various intervals using Vero cells as described above.
Infection of EV71 in intestinal organ cultures.
The organ culture of the small intestine was set up following the procedure described by Baumler et al. (1996). Briefly, a section of jejunum (1 cm) was dissected from a 1-day-old ICR mouse and placed in PBS. One end of the intestine was secured using a 6/0 braided silk suture (Chia Ho Silk Sutures Co., Taipei, Taiwan), while the other end was secured immediately after filling the lumen with 10 µl of EV71 (2x105 p.f.u.). The organ cultures were incubated in DMEM containing 10 % FBS at 37 °C for 24 h.
EV71 infection in mice.
For oral infection, groups of three to six 1-day-old pups were intragastrically inoculated with EV71 or mouse-adapted EV71 (1x106 or 1x107 p.f.u. per mouse) using a plastic feeding tube after fasting for 8 h. The mice were monitored daily for clinical signs. In some experiments, groups of 7-day-old mice were intragastrically inoculated with 2x106 p.f.u. of mouse-adapted EV71 per mouse. For virus isolation, various organs were weighed, homogenized and centrifuged. Clarified supernatants were inoculated into Vero cells after a 10-fold dilution. The titre was determined as described above and expressed as p.f.u. per 10 mg protein. For the determination of viraemia, 50 µl of blood sample was collected from each mouse at various times post-infection. The blood samples were diluted fourfold before infection of Vero cells. The titre was determined and expressed as p.f.u. ml-1 blood. The thresholds of detection for tissue and blood were 0 and 4 p.f.u., respectively.
Histological and immunohistochemical analysis.
Various tissue samples, including the intestines, spinal cord, heart and limb muscle, were removed from infected mice, embedded in OCT compound (Miles Inc.) and immediately frozen in liquid nitrogen. Four µm cryosections prepared with a Leica CM 1800 were placed on poly-L-lysine-coated glass slides and fixed with 3·7 % paraformaldehyde in PBS. Endogenous peroxidase was inhibited by 3 % H2O2 in PBS. EV71 was detected using mouse monoclonal anti-EV71 VP1 antibody (Chemicon International, Inc.). Vector M.O.M. agent was used in all incubation and washing steps to eliminate the background of mouse antibody on mouse tissues. The slides were then incubated with biotinylated anti-mouse antibody followed by Vectastain ABCperoxidase complex (Vector Laboratories). A red-coloured peroxidase stain was developed using aminoethyl carbazole (Zymed Laboratories) substrate and counterstained with Mayer's haematoxylin (Merck). Mouse organs were fixed with 3·7 % formaldehyde for 48 h and embedded in paraffin. Four µm sections were made and stained with haematoxylin and eosin Y for morphological examination.
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RESULTS |
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Replication of EV71 in intestinal organ cultures
As we aimed to develop a mouse model of EV71 infection via the oral route, we first tested whether EV71 could infect murine small intestine. Intestinal organ cultures (1 cm in length) were prepared from 1-day-old pups and infected with 2x105 p.f.u. EV71 in the lumens for 24 h. To ensure that infection had occurred, Caco-2-adapted 4643 and mouse-adapted 4643 were used in comparison with the clinical isolate of 4643. Immunohistochemical staining using anti-VP1 antibody showed that EV71 was present in the enterocytes of the organ cultures (Fig. 1). Under the same staining conditions, the most intense VP1 stain was noted in cultures infected with mouse-adapted 4643 rather than in those infected with Caco-2-adapted or the original 4643. This indicated that EV71 can infect the epithelial cells of the small intestine.
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DISCUSSION |
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We, along with other investigators, have reported the experimental infection of EV71 in mice (Wu et al., 2001; Yu et al., 2000
). Intraperitoneal or intracerebral administration of EV71 in suckling mice resulted in death. When the virus was administered intraperitoneally in the neonatal mice, the mice died in an age- and dose-dependent manner at day 710 post-injection. In an attempt to develop an EV71 infection model to mimic the clinical infection via the oral route, we first demonstrated that EV71 could infect epithelial cells in either the Caco-2 epithelial cell line or in small intestine organ cultures. Although various EV71 clinical isolates could replicate in cell lines such as Caco-2 or SK-N-SH, the replicative activity of the mouse-adapted EV71 was higher than those of the original isolate or the Caco-2-adapted strain. In suckling mice, oral administration of clinical isolate 4643 only induced mild skin lesions, whereas mouse-adapted 4643 caused hind limb paralysis. The mouse adaptation of EV71 increases its virulence in mice with a higher expression of VP1 in the small intestine and a higher mortality. The mouse adaptation of EV71 seems to increase virus replication and enhance cell damage. The adaptation procedure was repeated four times and it was found that more adaptation caused more virulence in mice (Fig. 4
, and unpublished observations).
In poliovirus infection, the virus first infects the gut, replicates in the epithelial cells lining the gut and is then released in the blood. Disseminated virus then replicates in skeletal muscle cells, finally reaching the peripheral nerves and then spreading to the CNS. Virus replication in skeletal muscle maintains a persisting viraemia. Poliovirus enters the CNS, probably through either penetration of the bloodbrain barrier or transmission via peripheral nerves using active retrograde axonal transport (Crotty et al., 2002; Ohka & Nomoto, 2001
; Ren & Racaniello, 1992
; Yang et al., 1997
). Paralytic poliomyelitis occurs as a result of neuronal destruction. In the reovirus serotype 3 infection in the newborn mouse model, reovirus first replicates in the lymphoid tissues of the gastrointestinal tract, then rapidly spreads to the mesenteric lymph nodes and produces viraemia (Mann et al., 2002
; Morrison et al., 1991
). Within 2 days of inoculation, viral titre increases in skeletal muscle and other extraneural sites and shortly thereafter is detected in the CNS. Viral antigen is detected by staining on the neurons of the dorsal motor nucleus of the vagus nerve in the brain stem. Reovirus enters a host via the lymphoid tissue of the alimentary tract, directly infecting nerves present in the muscle wall of the tract, and is then transported by them to the CNS. Thus, it spreads from the intestine to the CNS by vagus nerve fibres. In EV71 infection, our study has shown that EV71 initially replicates in the small intestine, followed by viraemia. The EV71 VP1 antigen was first detected in the thoracic segment of the spinal cord, later in the cervical segment of the spinal cord and then in the brain stem. EV71 might use the active retrograde axonal transport system to enter the CNS, similarly to poliovirus. Of course, trafficking across the bloodbrain barrier or by the peripheral vagus nerve is not excluded. The route by which EV71 spreads into the CNS is the subject of ongoing investigations. This example of CNS infection via the oral route provides a model to study the clinical manifestation of brain stem encephalitis, autonomous nerve system dysregulation and neurogenic pulmonary oedema caused by EV71 in humans (Huang et al., 1999
; Liu et al., 2000
; Wang et al., 1999
).
Furthermore, we found that EV71 can be transmitted between treated and non-treated littermates reared in the same cage. One-day old mice developed paralysis after oral inoculation of EV71. Surprisingly, mock-control mice developed mild skin lesions, indicating that EV71 might be shedding from the gut of treated mice and then being transmitted to their cagemates through contact with infected faeces. Anti-EV71 antibodies were present in lactating dams after nursing a litter of infected pups. The pups of the next litter became resistant to EV71 challenge (unpublished observations). This suggests that infective EV71 was released from the experimentally infected mice and transmitted to the dams and their siblings and that the anti-EV71 antibodies were passively transferred from the dams to protect the next generation of pups. This oral-to-faecal transmission mimics clinical EV71 infection. Moreover, the questions of whether respiratory droplets can transmit EV71 and by what molecular mechanism EV71 virus is spread to the CNS can be investigated by this model (Ho, 2000; McMinn, 2002
). Intravenous immunoglobulin has been used clinically to treat severe cases of EV71 infection with variable results the earlier the administration, the better the protection. This infectious murine model can be used for evaluation of the anti-EV71 antibody therapy. Moreover, it can also be used as an efficacy test for vaccine and anti-EV71 drug development.
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ACKNOWLEDGEMENTS |
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Received 12 June 2003;
accepted 10 September 2003.