Production of Avian Influenza Vaccines Using Insect Larvae

Recently, the baculovirus/insect cell expression system has beingincreasingly used for the production of vaccine candidate. However, cultivation ofinsect cells using the conventional fermentation method was restricted by thedifficulties of the scale-up step and the high cost of culture medium. It is ofeconomically importance to minimize the cost for the production of vaccines,especially for the animal vaccines. To overcome the difficulties of fermentorscale-up and avoid the usage of expensive culture medium, larvae of Trichoplusia ni,a lepidopterous insect, is a promising host to express foreign proteins. Ourresearch proposal is to develop a production platform of animal vaccines by usingbaculovirus/insect larvae system, and to establish an efficient downstreampurification process. The avian influenza has threatened the poultry industry andeven the human beings worldwide. It has been reported that the HA protein ofavian influenza virus (AIV) produced by the baculovirus expression system canprotect avians from the disease. The HA proteins of H6 and H7 subtypes andVLPs containing H7 HA have been expressed and produced using baculovirusexpression system. Preliminary experiments demonstrated subunit vaccinescomprising VLPs can elicit an effective humoral immune response in SPF chickens.Our research mainly focuses on using insect larva instead of cell culture as afeasible and economic tool for AIV HA production. We will express H5 HA byfollowing the strategy on H7 HA in order to confirm our previous results could beapplied to other AIV subtypes. The three-year proposal will accomplish theoptimization of the infectious conditions in T. ni larva using DsRed fluorescence asa probe and the following purification processes in the first year. The second yearwill evaluate antigen immunogenecity in specific-pathogen-free chickens. Thethird years will establish the scale-up process of expression and purification of HAprotein in insect larvae and evaluate antigen immunogenecity in farm chickens.近年來，桿狀病毒表現系統在疫苗研發上的重要性日漸增加，傳統發酵槽因為大量生產的困難性及複雜性加上商用細胞培養液的成本昂貴，且在農業應用上的成本預算遠低於人類健康醫療；因此利用具有成本低廉及操作簡便特性的桿狀病毒表現系統生產動物用疫苗就相對具有高度經濟競爭力。本計劃將開發利用昆蟲幼蟲生產動物用疫苗之高效率生產及純化流程，以便將此生產平台落實於動物疫苗之生產。由於禽流感對於台灣甚至於全世界之養雞工業威脅日益嚴重，本實驗室已成功利用昆蟲桿狀病毒的系統表現禽流感病毒弱毒株H6 及H7 亞型的HA 蛋白及H7 亞型相關似病毒顆粒，初步免疫試驗已證實了H7 亞型相關似病毒顆粒疫苗可以在SPF 雞隻中產生16 倍以上的HI 抗體以及至少40 倍以上的病毒中和能力。在這個計畫當中我們將應用先前的研究成果並以昆蟲幼蟲生產H6及H7 亞型的HA 蛋白以代替先前細胞的生產模式，發展可行且具經濟價值的生產工具，另外我們也將進行H5 亞型 HA 的表現，擴展我們的系統應用於其他亞型或綜合多價疫苗的發展。我們預計在三年內完成本計劃，第一年的工作重點為 藉由紅色螢光蛋白DsRed 在昆蟲幼蟲中的表現來偵測HA 的表現以建立蟲體感染的最適化條件，以及建立HA 之似流感病毒顆粒(influenza virus-like particles, VLPs)生產與純化方法之確立。第二年的工作重點為蟲體生產之VLPs (內含HA)的雞隻免疫效果。第三年的工作重點為大規模蟲體生產抗原流程及田間雞隻免疫效果之確立

生物性農業資材開發與利用之方向

目前生物性農業資材之開發與利用，大致而言可有下列之四個大方向。一、配方有機質肥料與有機液肥之開發與利用：一般堆肥及有機質肥料之三要素濃度的比例，不能符合作物生長最適合的需求，且肥效反應較慢，故開發配方有機質肥料與有機液肥，應用于有機農業栽培，是有必要性與實用性。二、多功能生物性肥料與複合功能生物性有機質肥料之開發與利用：化學肥料製造過程需要耗費大量能源與礦源，而石油能源與礦是珍貴與有限的，故各國正推動減少或取代化學肥料之施用，而開發微生物肥料菌株與有機質肥料複合之多功能肥料，施用於增進作物生長與品質，既省工又省時，可顯著減少化學肥料施用量。三、生物性農藥與天敵之開發與利用：化學農藥對生物與生態的殺傷與破壞力是非常強的，所以如何減少化學農藥施用，開發並利用生物性農藥劑，以防治作物病蟲害，是目前研發的重點之一。四、生物性可分解塑膠之開發與利用：傳統塑膠材料雖有輕便、耐用及價格低等優點，但塑膠性質太穩定不易分解，累積之後，對農業環境會造成嚴重之污染，且導致農田生產力降低，目前生物可分解塑膠取代傳統塑膠的國內潛在市場有很大的需求性，故以生物技術開發生物分解性塑膠是值得發展的方向

Establish the Cultivation Practices for Safe Quality of Vegetables and Fruits

1.建構長豇豆安全生產體系：本年度計畫預期完成長豇豆施肥策略和合理化施肥模式的建立，達到降低施肥成本、減少農藥使用、生產無農藥殘留超過容許量且高品質的長豇豆，並在增加農民收益的同時，建立他們正確的病蟲害防治觀念及技術。2.建構萵苣安全生產體系：完成建構萵苣安全生產體系計畫並繳交報告。3.建構芹菜安全生產體系：完成建構芹菜安全生產體系計畫並繳交報告。4.建構青蔥安全生產體系：甜菜夜蛾(Spodoptera exigua)可危害本省作物種類極多，對化學性殺蟲劑極易產生抗性，更是威脅青蔥栽培最重要之害蟲。多種蟲生病原被證實具抑制該蟲之作用，一種核多角體病毒已被開發成有效之生物殺蟲劑，包括綠殭菌(Nomuraea rileyi)、白殭菌(Beauveria bassiana)及黑殭菌(Metarhizium anisopliae)也有相同之作用。此蟲對苦楝和棉仔萃取之油類具敏感性，礦物油也具殺卵及殺幼蟲作用。費洛蒙除偵測及誘殺作用外，干擾成蛾交配也被證實，本計畫即擬整合多種防治技術，建立有效安全之綜合防治策略。5.建構印度棗安全生產體系：無農藥殘毒之安全農產品是國人健康之保障，也是農產品外銷之重要品質之一。 台灣全年都有各種重要水果上市，這是台灣人民的可貴資源，也是台灣農業可以 傲視國際之處，因此建立各種水果之安全生產體系是相當重要，本計畫擬於三年 中完成印度棗之安全生產體系。各種水果生產都有專責專家組成之研究團隊，進 行安全生產體系各技術之整合。包括品種及安全種苗之繁殖，合理安全之肥培管 理，包括生物控制及農藥控制之安全病蟲害防治等建立各種水果安全生產行事曆。6.建構木瓜安全生產體系：台灣木瓜品種肉質鮮美且可終年生產，極具外銷潛力，由於病蟲害的發生為木瓜生產不可避免的問題之一，且安全的果品為國內外市場需共同要求的要件，因此精確而適當化學藥劑使用方法應明確建立與建議，此外適當的土壤管理為高品質生產與永續農業的重要條件，因此本計畫將建立安全種苗、合理施肥及合理用藥的標準安全生產作業流程，並舉辦觀摩會向農友解釋及推廣木瓜安全生產流程。7.建構豬草監測與防除體系：豬草為原產美洲的外來入侵雜草，亦是有毒雜草，於開花期會引起花粉過敏症，由於生長迅速，繁殖力強，大量滋生易形成優勢雜草，造成人類健康及農業、生態上的危害。外來入侵植物之調查、監測及建立防疫管理技術是目前植物檢防疫上重要的工作，本計畫擬針豬草在本省各地進行監測調查，並於嚴重大面積發生的地區進行防治工作，藉此提供外來入侵植物監測、防治、管理的方法。1.建構長豇豆安全生產體系：This year, the strategy of reasonable fertilization on asparagus bean is ready to be founded to lower the prime cost of fertilization and pesticide and asparagus bean of high quality will be yielded. At the other hand, the aim of having farmers gain the correct concepts and skills on controlling the plant diseases and pests will be able to be estimated while their income increases.2.建構萵苣安全生產體系：Complete the project of establishment of the cultivation practices for lettuce in plastic pipe-house production system and hand in the report.3.建構芹菜安全生產體系：Complete the project of establishment of the cultivation practices for celery production system and hand in the report.4.建構青蔥安全生產體系：The beet armyworm (Spodoptera exigua) has a wide host range, occurring as a serious pest of green onion. Insecticide resistance is a major problem in management of this insect. Several insect pathogens may prove to be useful for suppression of beet armyworm. A nuclear polyhedrosis virus isolated from beet armyworm is effective as a bioinsecticide. Entomopathogenic fungi Nomuraea rileyi, Beauveria bassiana and Metarhizium anisopliae have the same attributes. Beet armyworm larvae are susceptible to neem products. Eggs can be killed with petroleum oil, and both eggs and young larvae can be controlled with cottonseed oil. Pheromones can also be used disrupt mating and inhibit or eliminate reproduction. The objectives of this study are designed to set up a beet armyworm control system for safer green onion production.5.建構印度棗安全生產體系：The agricultural products with high quality and no pesticide residue by the appropriate proudction systems are the guarantee for peoples health ware and one of the important properties of products fot export. There are wide varieties of fruits on market produced through all year round produced in Taiwan. This is a very gloried resource for Taiwan in economical view and in academic research. Therefore, it is very crucial to esablish standard operation processes(SOP) for each fruit tree production to meet the requirement in producing high quality and safety fruits. This project intends to establishthe SOP for Indina jujube. The SOP for each fruit production will be conducted by separate research team on safety seedling, appropriate soil management, precisionfertilization, proper pesticides use, and post harvest management as well. The SOP will be written on production calendar for farmers essily followed.6.建構木瓜安全生產體系：The papaya fruits produced in Taiwan are high quality and able to be offered all yearround. It is strongly potential to expand its markets to foreign countries. Besides, safety product with qualified pesticide residue is one of the important properties for domestic and export markets. Because it is inevitable that some diseases and pests, if without control, will reduce the qualities of papaya, the efficient and appropriate pesticide use should be set down and suggested. Furthermore, appropriate soil management is also important to produce high quality products and maintain sustainable agriculture. Therefore, the project intends to establish a standard operation processes including safety seedling, appropriate fertilization, and proper pesticide use. Field demonstrations about the standard operation processes will be held to explain the research results and show the effects for farmers to follow.7.建構豬草監測與防除體系：Common ragweed (Ambrosia artemisiifolia L.) originated from New World are serious invasive weeds in Taiwan, This Asteraceae was found infesting non-cultivated fields, open disturbed habitats, and roadsides. Common ragweed induce dermatitis and may causes hay fever symptoms in some peoples. The monitoring survey and effective control of common ragweed will be investigated

Establish the Cultivation Practices for Safe Quality of Vegetables and Fruits

1.建構長豇豆安全生產體系：This year, the strategy of reasonable fertilization on asparagus bean is ready to be founded to lower the prime cost of fertilization and pesticide and asparagus bean of high quality will be yielded. At the other hand, the aim of having farmers gain the correct concepts and skills on controlling the plant diseases and pests will be able to be estimated while their income increases.2.建構萵苣安全生產體系：Complete the project of establishment of the cultivation practices for lettuce in plastic pipe-house production system and hand in the report.3.建構芹菜安全生產體系：Complete the project of establishment of the cultivation practices for celery production system and hand in the report.4.建構青蔥安全生產體系：The beet armyworm (Spodoptera exigua) has a wide host range, occurring as a serious pest of green onion. Insecticide resistance is a major problem in management of this insect. Several insect pathogens may prove to be useful for suppression of beet armyworm. A nuclear polyhedrosis virus isolated from beet armyworm is effective as a bioinsecticide. Entomopathogenic fungi Nomuraea rileyi, Beauveria bassiana and Metarhizium anisopliae have the same attributes. Beet armyworm larvae are susceptible to neem products. Eggs can be killed with petroleum oil, and both eggs and young larvae can be controlled with cottonseed oil. Pheromones can also be used disrupt mating and inhibit or eliminate reproduction. The objectives of this study are designed to set up a beet armyworm control system for safer green onion production.5.建構印度棗安全生產體系：The agricultural products with high quality and no pesticide residue by the appropriate proudction systems are the guarantee for peoples health ware and one of the important properties of products fot export. There are wide varieties of fruits on market produced through all year round produced in Taiwan. This is a very gloried resource for Taiwan in economical view and in academic research. Therefore, it is very crucial to esablish standard operation processes(SOP) for each fruit tree production to meet the requirement in producing high quality and safety fruits. This project intends to establishthe SOP for Indina jujube. The SOP for each fruit production will be conducted by separate research team on safety seedling, appropriate soil management, precisionfertilization, proper pesticides use, and post harvest management as well. The SOP will be written on production calendar for farmers essily followed.6.建構木瓜安全生產體系：The papaya fruits produced in Taiwan are high quality and able to be offered all yearround. It is strongly potential to expand its markets to foreign countries. Besides, safety product with qualified pesticide residue is one of the important properties for domestic and export markets. Because it is inevitable that some diseases and pests, if without control, will reduce the qualities of papaya, the efficient and appropriate pesticide use should be set down and suggested. Furthermore, appropriate soil management is also important to produce high quality products and maintain sustainable agriculture. Therefore, the project intends to establish a standard operation processes including safety seedling, appropriate fertilization, and proper pesticide use. Field demonstrations about the standard operation processes will be held to explain the research results and show the effects for farmers to follow.7.建構豬草監測與防除體系：Common ragweed (Ambrosia artemisiifolia L.) originated from New World are serious invasive weeds in Taiwan, This Asteraceae was found infesting non-cultivated fields, open disturbed habitats, and roadsides. Common ragweed induce dermatitis and may causes hay fever symptoms in some peoples. The monitoring survey and effective control of common ragweed will be investigated.1.建構長豇豆安全生產體系：本年度計畫預期完成長豇豆施肥策略和合理化施肥模式的建立，達到降低施肥成本、減少農藥使用、生產無農藥殘留超過容許量且高品質的長豇豆，並在增加農民收益的同時，建立他們正確的病蟲害防治觀念及技術。2.建構萵苣安全生產體系：完成建構萵苣安全生產體系計畫並繳交報告。3.建構芹菜安全生產體系：完成建構芹菜安全生產體系計畫並繳交報告。4.建構青蔥安全生產體系：甜菜夜蛾(Spodoptera exigua)可危害本省作物種類極多，對化學性殺蟲劑極易產生抗性，更是威脅青蔥栽培最重要之害蟲。多種蟲生病原被證實具抑制該蟲之作用，一種核多角體病毒已被開發成有效之生物殺蟲劑，包括綠殭菌(Nomuraea rileyi)、白殭菌(Beauveria bassiana)及黑殭菌(Metarhizium anisopliae)也有相同之作用。此蟲對苦楝和棉仔萃取之油類具敏感性，礦物油也具殺卵及殺幼蟲作用。費洛蒙除偵測及誘殺作用外，干擾成蛾交配也被證實，本計畫即擬整合多種防治技術，建立有效安全之綜合防治策略。5.建構印度棗安全生產體系：無農藥殘毒之安全農產品是國人健康之保障，也是農產品外銷之重要品質之一。 台灣全年都有各種重要水果上市，這是台灣人民的可貴資源，也是台灣農業可以 傲視國際之處，因此建立各種水果之安全生產體系是相當重要，本計畫擬於三年 中完成印度棗之安全生產體系。各種水果生產都有專責專家組成之研究團隊，進 行安全生產體系各技術之整合。包括品種及安全種苗之繁殖，合理安全之肥培管 理，包括生物控制及農藥控制之安全病蟲害防治等建立各種水果安全生產行事曆。6.建構木瓜安全生產體系：台灣木瓜品種肉質鮮美且可終年生產，極具外銷潛力，由於病蟲害的發生為木瓜生產不可避免的問題之一，且安全的果品為國內外市場需共同要求的要件，因此精確而適當化學藥劑使用方法應明確建立與建議，此外適當的土壤管理為高品質生產與永續農業的重要條件，因此本計畫將建立安全種苗、合理施肥及合理用藥的標準安全生產作業流程，並舉辦觀摩會向農友解釋及推廣木瓜安全生產流程。7.建構豬草監測與防除體系：豬草為原產美洲的外來入侵雜草，亦是有毒雜草，於開花期會引起花粉過敏症，由於生長迅速，繁殖力強，大量滋生易形成優勢雜草，造成人類健康及農業、生態上的危害。外來入侵植物之調查、監測及建立防疫管理技術是目前植物檢防疫上重要的工作，本計畫擬針豬草在本省各地進行監測調查，並於嚴重大面積發生的地區進行防治工作，藉此提供外來入侵植物監測、防治、管理的方法