腸病毒71型(enterovirus 71；EV71)屬於小RNA病毒科(Picornaviridae)，腸病毒屬(enterovirus genus)。腸病毒71型感染會引起輕微的手足口症、腦炎、以及肺水腫。細胞自噬(autophagy)，為自我吞噬 (self-eating)的過程，屬於真核細胞中第二型的計畫性細胞死亡(Type II programmed cell death ;PCD)；為細胞中，許多生命周期長的蛋白(long-lived)分解過程所必需。先前的報導指出，這種系統會發生在許多生理過程中，例如飢餓反應、細胞死亡、胞器和蛋白翻轉、細胞分化、疾病產生。除此之外，細胞自噬似乎與RNA病毒感染細胞及神經疾病有關。本研究因此假設腸病毒71型感染細胞會引起細胞自噬。經由DNA轉殖方式表現自噬體(autophagosome)的特異性標記，LC3，其氨基末端的帶有綠色螢光蛋白(GFP)。LC3在腸病毒71型感染之細胞會有螢光聚集的情況。在西方點墨法的結果中，腸病毒71型感染的細胞跟沒有被感染的細胞相比，LC3- II會增加，表示自噬體形成，或是自噬體與溶酶體(lysosome)的融合減少。除此之外，在電子顯微鏡的結果，我們也看到感染腸病毒71型的 SK-N-SH細胞中有自噬體的空泡存在，部分病毒顆粒甚至包裹在其中。本研究中證明腸病毒71型感染細胞會引起細胞自噬。利用3-MA抑制細胞自噬時，LC3螢光的聚集會減少，而且細胞外病毒的產量也會下降。相反的，若感染的細胞中，以血清飢餓(serum starvation)處理或加入tamoxifen、rapamycin引發細胞自噬，則LC3螢光的聚集會增加，且細胞外病毒的產量也會增加。綜述之，腸病毒71型感染細胞的確會引起細胞自噬，而且細胞自噬的機制，能夠促進病毒複製，似乎有利於病毒生長。
血管內皮生長因子(vascular endothelial growth factor；VEGF)是在胚胎發育時，形成新血管的血管生長因子，在許多病理情況下會過度表現。報導指出VEGF在血管通透性改變上扮演重要的角色，於肺部過度表現會引發肺水腫。本研究發現VEGF在腸病毒71型病人血液中的含量比正常人要高兩倍。因此，推論VEGF在腸病毒感染病人之致病性可能扮演的重要角色。為了決定感染腸病毒71型的細胞是否分泌血管內皮生長因子，以酵素免疫法(ELISA)偵測細胞外，以及西方點墨法偵測細胞內之VEGF表現量。我們的結果顯示，被感染的細胞無論是細胞外或細胞內之VEGF表現量，均比未感染之細胞要來的少；而感染細胞中之VEGF的RNA表現量則比未感染的高。因此，VEGF在腸病毒71型感染之病人體內是否確有過度表現以及其角色還需要進一步釐清。

Enterovirus 71 (EV71), which belongs to enterovirus genus in the family of Picornaviridae, consists of a non-enveloped capsid and a positive single-stranded RNA genome approximately 7.5 kb in size. EV71 infection causes mild hand foot and mouth disease, polio-like paralysis, fatal encephalitis, and pulmonary edema. Autophagy, type II programmed cell death (PCD) in eukaryotes, means ‘self-eating’ process. It is responsible for the degradation of most long-lived proteins in order to survive. Previous reports indicate that the system has been implicated in various physiological processes including starvation response, cell death, organelle and protein turnover, cellular differentiation, and pathogenesis. Moreover, autophagy was also detected in the cells infected by RNA viruses. It is interesting to know whether the cells infected by EV71 infected cells could induce autophagy. The autophagic marker protein LC3 was aggregated in EV71 infected cells, indicating that EV71 infections indeed could induce autophagy in the cells. Consistently, the cells infected by EV71 showed increased levels of LC3-II by Western blotting, indicating that the formation of autophagosome is increased and fusions of autophagosome-lysosome is decreaed, compared to control cells. Furthermore, autophagosomes vesicles were clearly detected in neuron SK-N-SH cells infected with EV71 by electron microscopy. Interestingly, EV71 infection induced autophagy was inhibited by the inhibitor 3-MA. Accordingly, LC3 aggregation was decreased and virus titer was also decreased. In contrast, autophagy in EV71 infected cells was further enhanced by tamoxifen and rapamycin, LC3 aggregation and virus titer were accordingly increased. In summary, EV71 infection could induce autophagy, which further enhances virus replication. In conclusion, authphagy in infected cells was beneficial for virus replication.
Vascular endothelial growth factor (VEGF) is an angiogenic factor essential for the formation of new blood vessels during embryogenesis and is overexpressed under many pathological conditions. Recent reports indicate that VEGF may play an important role in vascular permeability change, and overexpression of VEGF in lung can induce pulmonary edema. We found that the secretion of VEGF in EV71 patients was 2-fold higher than in normal control. Whether VEGF involves in the pathogenesis of EV71 infection is noteworthy for investigation. Initially, we determine whether VEGF is overexpressed in EV71 infected cells, the secretion of extracellular VEGF as well as the intracellular VEGF expression were detected by ELISA and Western blotting, respectively. In contrast to our expectation, only RNA expression of VEGF in the infected cells was increased. VEGF expression at protein level in either form from the infected cells was decreased when compared to the control cells. The underlying mechanisms of VEGF expression in EV71 infected cells and patients remain unclear, further study is required.

Bartholdi, D., Rubin, B. P., and Schwab, M. E. (1997). VEGF mRNA induction correlates with changes in the vascular architecture upon spinal cord damage in the rat. Eur J Neurosci 9, 2549-2560.

Belsham, G. J., and Sonenberg, N. (1996). RNA-protein interactions in regulation of picornavirus RNA translation. Microbiol Rev 60, 499-511.

Blomberg, J., Lycke, E., Ahlfors, K., Johnsson, T., Wolontis, S., and von Zeipel, G. (1974). Letter: New enterovirus type associated with epidemic of aseptic meningitis and-or hand, foot, and mouth disease. Lancet 2, 112.

Chang, L. Y., Lin, T. Y., Huang, Y. C., Tsao, K. C., Shih, S. R., Kuo, M. L., Ning, H. C., Chung, P. W., and Kang, C. M. (1999). Comparison of enterovirus 71 and coxsackie-virus A16 clinical illnesses during the Taiwan enterovirus epidemic, 1998. Pediatr Infect Dis J 18, 1092-1096.

Chen, Y. C., Yu, C. K., Wang, Y. F., Liu, C. C., Su, I. J., and Lei, H. Y. (2004). A murine oral enterovirus 71 infection model with central nervous system involvement. J Gen Virol 85, 69-77.

Chu, P. Y., Lin, K. H., Hwang, K. P., Chou, L. C., Wang, C. F., Shih, S. R., Wang, J. R., Shimada, Y., and Ishiko, H. (2001). Molecular epidemiology of enterovirus 71 in Taiwan. Arch Virol 146, 589-600.

Chumakov, M., Voroshilova, M., Shindarov, L., Lavrova, I., Gracheva, L., Koroleva, G., Vasilenko, S., Brodvarova, I., Nikolova, M., Gyurova, S., et al. (1979). Enterovirus 71 isolated from cases of epidemic poliomyelitis-like disease in Bulgaria. Arch Virol 60, 329-340.

Dice, J. F. (1990). Peptide sequences that target cytosolic proteins for lysosomal proteolysis. Trends Biochem Sci 15, 305-309.

Dunn, W. A., Jr. (1994). Autophagy and related mechanisms of lysosome-mediated protein degradation. Trends Cell Biol 4, 139-143.
Ferrara, N., Gerber, H. P., and LeCouter, J. (2003). The biology of VEGF and its receptors. Nat Med 9, 669-676.

Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., and Semenza, G. L. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol 16, 4604-4613.

Grugel, S., Finkenzeller, G., Weindel, K., Barleon, B., and Marme, D. (1995). Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells. J Biol Chem 270, 25915-25919.

Hagiwara, A., Tagaya, I., and Yoneyama, T. (1978). Epidemic of hand, foot and mouth disease associated with enterovirus 71 infection. Intervirology 9, 60-63.

Hayashi, T., Abe, K., Suzuki, H., and Itoyama, Y. (1997). Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke 28, 2039-2044.

Hernandez, L. D., Pypaert, M., Flavell, R. A., and Galan, J. E. (2003). A Salmonella protein causes macrophage cell death by inducing autophagy. J Cell Biol 163, 1123-1131.
Herold, J., and Andino, R. (2001). Poliovirus RNA replication requires genome circularization through a protein-protein bridge. Mol Cell 7, 581-591.

Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu Rev Biochem 67, 425-479.

Ho, M., Chen, E. R., Hsu, K. H., Twu, S. J., Chen, K. T., Tsai, S. F., Wang, J. R., and Shih, S. R. (1999). An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med 341, 929-935.

Iliopoulos, O., Levy, A. P., Jiang, C., Kaelin, W. G., Jr., and Goldberg, M. A. (1996). Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci U S A 93, 10595-10599.

Jackson, W. T., Giddings, T. H., Jr., Taylor, M. P., Mulinyawe, S., Rabinovitch, M., Kopito, R. R., and Kirkegaard, K. (2005). Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol 3, e156.
Juhasz, G., Csikos, G., Sinka, R., Erdelyi, M., and Sass, M. (2003). The Drosophila homolog of Aut1 is essential for autophagy and development. FEBS Lett 543, 154-158.

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. Embo J 19, 5720-5728.

Kennett, M. L., Birch, C. J., Lewis, F. A., Yung, A. P., Locarnini, S. A., and Gust, I. D. (1974). Enterovirus type 71 infection in Melbourne. Bull World Health Organ 51, 609-615.

Kuo, R. L., Kung, S. H., Hsu, Y. Y., and Liu, W. T. (2002). Infection with enterovirus 71 or expression of its 2A protease induces apoptotic cell death. J Gen Virol 83, 1367-1376.

Li, M. L., Hsu, T. A., Chen, T. C., Chang, S. C., Lee, J. C., Chen, C. C., Stollar, V., and Shih, S. R. (2002). The 3C protease activity of enterovirus 71 induces human neural cell apoptosis. Virology 293, 386-395.

Liang, C. C., Sun, M. J., Lei, H. Y., Chen, S. H., Yu, C. K., Liu, C. C., Wang, J. R., and Yeh, T. M. (2004). Human endothelial cell activation and apoptosis induced by enterovirus 71 infection. J Med Virol 74, 597-603.

Liang, X. H., Kleeman, L. K., Jiang, H. H., Gordon, G., Goldman, J. E., Berry, G., Herman, B., and Levine, B. (1998). Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 72, 8586-8596.

Lin, T. Y., Hsia, S. H., Huang, Y. C., Wu, C. T., and Chang, L. Y. (2003). Proinflammatory cytokine reactions in enterovirus 71 infections of the central nervous system. Clin Infect Dis 36, 269-274.

Lum, L. C., Wong, K. T., Lam, S. K., Chua, K. B., Goh, A. Y., Lim, W. L., Ong, B. B., Paul, G., AbuBakar, S., and Lambert, M. (1998). Fatal enterovirus 71 encephalomyelitis. J Pediatr 133, 795-798.

Monacci, W. T., Merrill, M. J., and Oldfield, E. H. (1993). Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues. Am J Physiol 264, C995-1002.

Nagy, G., Takatsy, S., Kukan, E., Mihaly, I., and Domok, I. (1982). Virological diagnosis of enterovirus type 71 infections: experiences gained during an epidemic of acute CNS diseases in Hungary in 1978. Arch Virol 71, 217-227.

Nakagawa, I., Amano, A., Mizushima, N., Yamamoto, A., Yamaguchi, H., Kamimoto, T., Nara, A., Funao, J., Nakata, M., Tsuda, K., et al. (2004). Autophagy defends cells against invading group A Streptococcus. Science 306, 1037-1040.

Nicholson, R., Pelletier, J., Le, S. Y., and Sonenberg, N. (1991). Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies. J Virol 65, 5886-5894.

Nishino, I., Fu, J., Tanji, K., Yamada, T., Shimojo, S., Koori, T., Mora, M., Riggs, J. E., Oh, S. J., Koga, Y., et al. (2000). Primary LAMP-2 deficiency causes X-linked vacuolar cardiomyopathy and myopathy (Danon disease). Nature 406, 906-910.

Ogunshola, O. O., Antic, A., Donoghue, M. J., Fan, S. Y., Kim, H., Stewart, W. B., Madri, J. A., and Ment, L. R. (2002). Paracrine and autocrine functions of neuronal vascular endothelial growth factor (VEGF) in the central nervous system. J Biol Chem 277, 11410-11415.

Otto, G. P., Wu, M. Y., Kazgan, N., Anderson, O. R., and Kessin, R. H. (2003). Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum. J Biol Chem 278, 17636-17645.

Pelletier, J., and Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320-325.

Pestova, T. V., Shatsky, I. N., and Hellen, C. U. (1996). Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Mol Cell Biol 16, 6870-6878.

Petersen, A., Larsen, K. E., Behr, G. G., Romero, N., Przedborski, S., Brundin, P., and Sulzer, D. (2001). Expanded CAG repeats in exon 1 of the Huntington's disease gene stimulate dopamine-mediated striatal neuron autophagy and degeneration. Hum Mol Genet 10, 1243-1254.

Pringle, C. R. (1999). Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Arch Virol 144, 2065-2070.

Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., et al. (2003). Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 112, 1809-1820.

Robinson, C. J., and Stringer, S. E. (2001). The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci 114, 853-865.

Safran, M., and Kaelin, W. G., Jr. (2003). HIF hydroxylation and the mammalian oxygen-sensing pathway. J Clin Invest 111, 779-783.

Salo, R. J., and Cliver, D. O. (1976). Effect of acid pH, salts, and temperature on the infectivity and physical integrity of enteroviruses. Arch Virol 52, 269-282.

Sarnow, P., Bernstein, H. D., and Baltimore, D. (1986). A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome. Proc Natl Acad Sci U S A 83, 571-575.

Schlegel, A., Giddings, T. H., Jr., Ladinsky, M. S., and Kirkegaard, K. (1996). Cellular origin and ultrastructure of membranes induced during poliovirus infection. J Virol 70, 6576-6588.

Schmidt, N. J., Lennette, E. H., and Ho, H. H. (1974). An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 129, 304-309.

Senger, D. R., Galli, S. J., Dvorak, A. M., Perruzzi, C. A., Harvey, V. S., and Dvorak, H. F. (1983). Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219, 983-985.

Shih, S. R., Ho, M. S., Lin, K. H., Wu, S. L., Chen, Y. T., Wu, C. N., Lin, T. Y., Chang, L. Y., Tsao, K. C., Ning, H. C., et al. (2000). Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998. Virus Res 68, 127-136.

Tagaya, I., and Tachibana, K. (1975). Epidemic of hand, foot and mouth disease in Japan, 1972-1973: difference in epidemiologic and virologic features from the previous one. Jpn J Med Sci Biol 28, 231-234.

Tanida, I., Tanida-Miyake, E., Ueno, T., and Kominami, E. (2001). The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. J Biol Chem 276, 1701-1706.

Teckman, J. H., and Perlmutter, D. H. (2000). Retention of mutant alpha(1)-antitrypsin Z in endoplasmic reticulum is associated with an autophagic response. Am J Physiol Gastrointest Liver Physiol 279, G961-974.

Tsukada, M., and Ohsumi, Y. (1993). Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 333, 169-174.

Wang, J. R., Tuan, Y. C., Tsai, H. P., Yan, J. J., Liu, C. C., and Su, I. J. (2002). Change of major genotype of enterovirus 71 in outbreaks of hand-foot-and-mouth disease in Taiwan between 1998 and 2000. J Clin Microbiol 40, 10-15.

Wileman, T. (2006). Aggresomes and autophagy generate sites for virus replication. Science 312, 875-878.