98 Ev Mp4

98 Ev Mp4

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Institute of Basic Medical Sciences,1 Departments of Microbiology and Immunology,2 Pediatrics,3 Pathology,4 Clinical Medicine,5 Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China6

Mouse-adapted EV71 grows more rapidly and is more cytotoxic than the parental strain. (A) One-step growth curves of the parental EV71 strain Tainan/4643/98 and the mouse-adapted strain MP4 in RD, SK-N-SH, and Caco-2 cells were determined as described in Materials and Methods. (B) SK-N-SH cells (2 104) grown in 96-well plates were infected with 4643 or MP4, and cell viability was determined at the times indicated. Data represent the means SEM of results of three independent experiments performed in triplicate (A) and of two experiments with eight duplicates (B). *, P < 0.05.

EV71 induces apoptosis of Caco-2 and SK-N-SH cells. (A) Caco-2 or SK-N-SH cells were infected with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at an MOI of 1. The expressions of both the proform and the cleavage form of caspase 9 in whole-cell lysates were determined by Western blotting. (B) Apoptosis of EV71-infected cells was determined by flow cytometry after staining with Annexin V. Values are the means SEM of results of three independent experiments. *, P value of

The mouse-adapted EV71 strain is more virulent than the parental strain in 1-day-old mice. One-day-old ICR mice were inoculated i.p. with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at 3 106 PFU/mouse (A) or 101 to 107 PFU/mouse (B). Survival was then monitored daily after infection. Control mice were given cell lysate instead of virus suspension. Each group contained six to eight mice.

Mouse-adapted EV71 induces death in mice after oral inoculation. Seven-day-old ICR mice were orally inoculated with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain at 1 104 to 5 106 PFU/mouse (A) or 5 106 PFU/mouse (B), both at 200 μl, with or without i.p. administration of mouse anti-EV71 serum (50 μl). Survival was then monitored daily after infection. Control mice were given cell lysate instead of virus suspension. Each group contained six to eight mice.

Representative histology of various tissues of EV71-infected mice. Seven-day-old ICR mice were orally inoculated with cell lysate (mock control) or the mouse-adapted EV71 strain (MP4) (5 106 PFU/mouse), and tissues were examined histopathologically at 6 dpi as described in Materials and Methods. Typical lesions seen in MP4-infected mice include neuronal loss in ventral horns of the spinal cord (A and B), severe necrotizing myositis (C and D), decreased hematopoiesis in the liver (E and F), splenic atrophy (G and H), and villous blunting in the small intestine (I and J; 1 dpi). Hematoxylin and eosin stain. Magnifications, 400 (A, B, I, and J), 100 (C to F), and 40 (G and H).

Tissue viral titers in EV71-infected mice. Seven-day-old mice were inoculated with the parental (Tainan/4643/98) or mouse-adapted (MP4) EV71 strain (5 106 PFU/mouse, 200 μl). Viral titers were assessed by plaque assays of tissues collected at the times indicated. Results represent the mean virus titer (log10 PFU) per milligram of tissue or per milliliter of blood SEM (six mice per group).

The parental EV71 strain 4643, an isolate from throat swabs of an 18-month-old patient with encephalitis, has been used in studies of its pathogenesis in mice (31). Inoculation i.p. of strain 4643 caused paralysis and death in 1-day-old mice. However, as demonstrated in this study, strain 4643 was nonpathogenic to 7-day-old mice when it was acquired via oral infection. There was only a transient colonization of the virus in the villous epithelium. In contrast, after oral inoculation, strain MP4 rapidly spread to and replicated in the CNS. It is likely that a mutant(s) develops during the adaptation process, resulting in significant variations in the pathogenesis of MP4 compared to that of the natural virus isolate. However, it is unlikely that the MP4 variant acquires the capacity to recognize a surface molecule of neurons as the virus receptor, since the degree of virulence of 4643 in mice appears to be less than that of MP4 only when delivered via p.o. inoculation. Strain 4643 maintains its neurovirulence, since it was able to induce apoptosis of SK-N-SH cells as MP4 did.

How MP4 induced mortality is not known. The 2A and 3C protease activities of EV71 have been reported to induce apoptosis of human fibroblasts (11) and glioblastoma cells (12), respectively. We demonstrated that EV71 triggered apoptosis of SK-N-SH cells in vitro and that EV71 infection of mice resulted in apoptosis of neural cells. Concomitantly, CNS tissues had a much higher viral load and displayed more severe tissue damage than other internal tissues. Thus, apoptosis may be involved in the pathogenesis of EV71 infection in mice. The finding that SK-N-SH cells were more susceptible than Caco-2 cells to EV71-induced apoptosis further supported the idea that EV71 was neurotropic. Mononuclear cell infiltration in the CNS was not prominent in EV71-infected mice. It was probably due to the severe depletion of leukocytes after infection. Pulmonary edema has been reported to be frequently associated with fatal EV71 infections in children (8, 18); however, it was not observed in mice with brain stem lesions. Furthermore, the lung as well as the heart tissues of EV71-infected mice contained very little virus. On the other hand, the muscles contained a high viral load and displayed severe necrotizing myositis. Whether the muscle lesions contribute to death is not known. Interestingly, skeletal muscle involvement is seldom reported in the literature concerning EV71 pathogenesis. To our knowledge, this is the first report showing that EV71 displays strong myotropism.

There are some other important observations from this study. First, during active infection, the virus is present in the stools. Transmission of EV71 between infected and noninfected littermates occurred after close contact, as indicated by the occurrence of seroconversion and the presence of viable virus in the stool samples of noninfected littermates. These results support the idea that the common transmission route of EV71 is fecal-oral. Second, disease in EV71-infected mice was readily attenuated by anti-EV71 Ab, indicating that humoral immunity is protective in this model system.

This work was supported by Chang Gung Memorial Hospital, Taoyuan, Taiwan (BMRP416, CMRPD1E0041-3,CMRPD1G0301-3, and CMRPD1F0581-3), the Ministry of Science and Technology of Taiwan (106-2320-B-182-004-MY3, 106-2811-B-182-011, 106-2632-B-182-001, and 107-2811-B-182-512), the Research Center for Emerging Viral Infections from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan, and the Ministry of Science and Technology, Taiwan (MOST 109-2634-F-182-001).

As a neurotropic virus, enterovirus A71 (EV-A71) emerge and remerge in the Asia-Pacific region since the 1990s, and has continuously been a threat to global public health, especially in children. Annually, EV-A71 results in hand-foot-and-mouth disease (HFMD) and occasionally causes severe neurological disease. Here we reviewed the global epidemiology and genotypic evolution of EV-A71 since 1997. The natural selection, mutation and recombination events observed in the genetic evolution were described. In addition, we have updated the antigenicity and virulence determinants that are known to date. Understanding EV-A71 epidemiology, genetic evolution, antigenicity, and virulence determinants can expand our insights of EV-A71 pathogenesis, which may benefit us in the future. 781b155fdc