- 學位論文
感染豬生殖與呼吸綜合症誘發抗體依賴性增強現象之探討
Observation of antibody-dependent enhancement in pigs infected with porcine reproductive and respiratory syndrome
摘要
抗體依賴性增強現象 (Antibody dependent enhancement, ADE) 被發現在許多病毒感染時因有抗體之出現而可增強病毒感染之能力，諸如登革熱病毒 (dengue virus, DV)、貓傳染性腹膜炎病毒 (feline infectious peritonitis virus, FIPV) 以及豬生殖與呼吸綜合症病毒 (porcine reproductive and respiratory syndrome virus, PRRSV)。豬生殖與呼吸綜合症為豬場常見之疾病，可引發保育豬肺炎及母豬繁殖障礙，造成豬場嚴重經濟損失。為了解PRRSV感染豬隻後發生之ADE現象，本研究以兩群PRRS-free SPF豬進行動物試驗，各群均將其分為4組：對照組、疫苗免疫組、PRRS病毒攻毒組及疫苗免疫後PRRSV攻毒組。第一群豬隻取PRRS MLV活毒疫苗於豬隻4週齡肌肉免疫1劑，PRRS病毒 (1#westeur024#106/mL TCID50) 於8週齡以鼻腔接種攻毒，各組豬隻於4至14週齡間每二週採血檢測；第二群豬隻以PRRS次單位疫苗於豬隻3週齡及6週齡施打，PRRS病毒於8週齡以鼻腔接種，所有豬隻於3、6、8、9、10、11週齡採血。兩群動物試驗所採集之血液經分離血清後篩檢PRRS病毒、活體外ADE試驗、病毒中和抗體及PRRSV ELISA抗體力價檢測。結果顯示，兩組試驗豬群於不同時間可見血中PRRS病毒量消長趨勢不同，疫苗免疫組在施打MLV疫苗後6週齡病毒量明顯上升，8週齡後下降，而施打次單位疫苗則皆呈陰性反應，PRRS病毒攻毒組皆在攻毒後1至2週檢測到最高病毒量，疫苗免疫後PRRSV攻毒組之病毒量則於攻毒後僅測到少量PRRSV。ADE試驗第一群豬隻之疫苗免疫組10週齡血清稀釋4倍和14週齡血清稀釋64倍，以及疫苗免疫後PRRSV攻毒組12週齡血清稀釋16倍檢測病毒量與對照組相比有顯著增加 (p<0.05)。第二群豬隻之疫苗免疫組6週齡血清稀釋64倍和9週齡血清稀釋256倍，以及PRRS病毒攻毒組11週齡血清稀釋256倍與疫苗免疫後PRRSV攻毒組在6週齡血清稀釋64倍、9週齡血清稀釋256倍、10週齡血清稀釋4及64倍病毒量也都顯著高於對照組 (p<0.05)；兩群動物試驗於各組不同週齡僅在部分血清稀釋倍數下，可見血液中病毒量顯著增加之狀況。豬隻免疫MLV活毒疫苗，兩週後即產生PRRSV特異性抗體，反之施打次單位疫苗豬隻於試驗期間皆無特異性抗體產生，而PRRSV病毒組與疫苗免疫後PRRSV攻毒組相比較，施打疫苗的豬隻在受到PRRSV感染後較快刺激豬隻免疫反應產生。綜上所述，試驗期間豬隻不論是施打PRRSV MLV疫苗和次單位疫苗或是直接以PRRS攻毒，僅在部分稀釋倍數下有發生ADE反應，均無產生一致性ADE反應之變化趨勢，且當豬隻遭受PRRSV感染後，疫苗效力可縮短豬隻產生具保護力抗體反應之時間。 關鍵字：豬生殖與呼吸綜合症、抗體依賴性增強、疫苗
並列摘要
The phenomenon of antibody-dependent enhancement (ADE) is found for many viruses and can enhance the ability of the viral infection. A number of human and animal viruses, such as dengue virus, feline infectious peritonitis virus, and porcine reproductive and respiratory syndrome virus (PRRSV), can induce the ADE phenomenon. PRRSV causes devastating economic losses due to late-term reproductive failure and severe pneumonia in neonatal pigs. However, the correlation between the antibody dynamics and ADE in PRRSV is still not clear. Furthermore, in Taiwan, pigs still show typical PRRS symptoms after they are vaccinated. Therefore, this study aims to discover the correlation between the ADE phenomenon and PRRSV infection. Four groups of PRRS-free SPF pigs, including a control group, vaccination group, challenge group and vaccination/challenge group, were used in two trials in this study. The trial-1 pigs were vaccinated with the MLV vaccine at 4 weeks of age, and challenged with the virus at 8 weeks of age. Blood samples were collected from each pig at 4, 6, 8, 10, 12 and 14 weeks of age. The trial-2 pigs were vaccinated with a subunit vaccine at 3 and and 6 weeks of age and challenged with the virus at 8 weeks of age. Blood samples were collected from each pig at 3, 6, 8, 9, 10 and 11 weeks of age. The serum was separated for screening for PRRSV viremia and in vitro ADE. A PRRSV neutralization test was performed, and the PRRSV ELISA specific antibody titer was obtained. The results showed that the vaccination group had a significantly increased viral load after the application of the MLV vaccine after 6 weeks, while the application of the subunit vaccine produced all negative reactions. The highest viral load was detected in the challenge group 1-2 weeks after the challenge. The lowest viral load was detected in the vaccination/challenge group following the challenge. In the ADE assay of the trial-1 pigs, the viral load was higher in 1:4 diluted serum at 10 weeks and 1:64 diluted serum at 14 weeks in the vaccination group, as well as serum diluted 1:16 at 12 weeks of age in the vaccination/challenge group, than in the control group. In the ADE assay of the trial-2 pigs, the viral load was significantly higher in 1:64 diluted serum at 6 weeks and 1:256 diluted serum at 9 weeks in the vaccination group; 1:256 diluted serum at 11 weeks in the challenge group; 1:64 diluted serum at 6 weeks, 1:256 diluted serum at 9 weeks, and 1:4 and 1:64 diluted serum at 10 weeks in the vaccination/challenge group than in the control group. In the two trials of the ADE assay, the viral load was significantly increased in each group at serum dilutions in different weeks. PRRSV-specific antibodies were detected in pigs that were immunized with MLV vaccine for two weeks, whereas non-specific antibodies were detected in pigs immunized with the subunit vaccine. The vaccinated pigs showed a faster immune response compared to non-vaccinated pigs after they were infected with PRRSV. In conclusion, PRRSV enhanced the ADE phenomenon at different times in the vaccination group and challenge group without consistent trends during the test. Neither the PRRSV MLV vaccine nor the subunit vaccine injection were correlated with the ADE phenomenon in pigs or could shorten the response time for producing protective antibodies. Keywords : porcine reproductive and respiratory syndrome virus、antibody-dependent enhancement、vaccine