우리는이 연구와 USDA에 사용 된 까마귀를 제공하기위한 S. 베르너에게 감사의 말씀을 전합니다, APHIS, 동물 관리 및 모니터링을위한 WS, NWRC 동물 관리 직원. 기재 또는 제품의 사용은 USDA의 보증을 의미하지는 않습니다. 이 연구를위한 자금 조달은 USDA, APHIS, 수의 서비스에 의해 제공되었다.

우리 프로토콜은 우리가 이전에 7을 발표 한에서 적용됩니다. 동물과 관련된 모든 절차는 미국 농무부 (USDA) 동식물 건강 검사 서비스 (APHIS), 야생 동물 서비스 (WS), 국립 야생 동물 연구 센터 (NWRC)의 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. 1. 까마귀 Gavaging <ol><li> 미국의 까마귀의 소화관을 통해 &#39;의사 뇌 물질&#39;의 통과 시간을 예상하고있다. <ol><li> 요리 (그림 1) 투여 바늘 2를 사용하여 청색 염료 및 위관 1 까마귀와 계란을 스크램블 5 ㎖를 혼합한다. </li><li> 블루 / 그린 묻은 대변 배설을 더 이상 제공하지 않습니다 때까지 까마귀에게 30 분마다 확인합니다. </li></ol></li><li> 감염되지 않은 및 말기 RML (챈들러 주)에 감염된 C57BL / 6 마​​우스의 뇌를 얻습니다. </li><li> 1X와 총알 블렌더 균질의 정상 20 % 중량 / 부피 감염된 마우스의 뇌 균질 생성인산염 완충 생리 식염수 (PBS)과 유리 구슬, 다음 큰 입자상 물질을 제거하기 위해 1 분 3,000 XG에 원심 분리기. 상층 액을 제거하고 멸균 PBS의 10 % 중량 / 부피를 생성하는 1X PBS의 동일한 볼륨을 넣어. 필요할 때까지 -80 ° C에서 동결. </li><li> 이전 위관 17 시간은 까마귀 펜에서, 물없는 음식을 제거 할 수 있지만. </li><li> 무작위로 치료 그룹에 까마귀를 할당하고 경구 투여 바늘에 2를 사용하여 정상 또는 감염 중 하나를 마우스의 뇌 균질 5 ㎖에 각 까마귀 위관. </li><li> 전송 개별 케이지에 까마귀와 4 시간 포스트 위관 (그림 2) 각 케이지 내의 모든 배설물을 수집하고 풀. </li><li> 균일 한 질감이 달성 될 때까지 유리 구슬 총알 믹서 균질화 각 까마귀에 대한 풀링 대변을 균질화. * 까마귀 배설물은 대부분 액체하고 쉽게 혼합 할 수 있습니다. </li><li> 각 까마귀를 들어, 10 ㎖의 총 부피 9.5 ㎖의 1X PBS로 대변 균질의 500 μl를 희석. </li><li> 배설물 homogena을 원심 분리기1,400 XG에 15 분 동안 테 상층 액을 추출합니다. </li><li> 2 차 세균 감염의 위험을 최소화 균질의 100 μL 당 100 단위의 μL / ㎖ 페니실린 및 100 ㎍ / ml의 스트렙토 마이신 (인비 트 로젠, NY)와 상층 액을 치료하는 다음 줄이기 위해 20 분 동안 실온에서 UV 빛의 밑에 배치 바이러스와 세균 오염의 위험이 있습니다. UV 노출 후, 남아있는 미생물의 세포막을 방해하는 30 초 70를 설정에서 3000 MP를 초음파기의 초음파 처리 샘플. </li></ol> 2. 마우스 접종 <ol><li> 무작위로 다음 치료 그룹 (표 1)에 마우스를 할당 : <ol><li> 그룹 1 - 긍정적 인 치료 마우스는 구두로 5 ㎖에 감염된 마우스의 뇌에 gavaged 까마귀의 배설물 균질 1 ㎖로 (IP)을 복강 내 주사. </li><li> 그룹 2 - 음의 치료 마우스는 경구 5ml는 정상 마우스의 두뇌와 gavaged 까마귀의 배설물 균질 1 ㎖로 IP를 접종. </li><li> infecte 희석1:100 W / 1X PBS에서 V에 그룹 3, 4 D와 일반 마우스의 뇌 균질. </li><li> 그룹 3 - 긍정적 인 제어 마우스는 감염된 마우스의 뇌 파쇄 액 1 ㎖로 IP를 접종. </li><li> 그룹 4 - 음성 대조군 마우스는 정상 마우스의 뇌 균질 물 1 ㎖로 IP를 접종. </li></ol></li></ol><table border="1"><tbody><tr><td></td><td> 치료 그룹 </td><td> 동물의 수 </td></tr><tr><td> 그룹 1 </td><td> 스크래피 + 까마귀 배설물 </td><td> (100) </td></tr><tr><td> 그룹 2 </td><td> 스크래피 - 까마귀 배설물 </td><td> 25 </td></tr><tr><td> 그룹 3 </td><td> 스크래피 + 마우스의 뇌 </td><td> 10 </td></tr><tr><td> 그룹 4 </td><td> 스크래피 - 마우스의 뇌 </td><td> 5 </td></tr></tbody></table> . 표 1 스크래피 접종의 상태 (음 양의 +, -)과 동물의 수는 7을 사용했다. <ol start="2"><li> 복강 마우스를 접종 : <ol><li> 부드럽게 목덜미의 엄지와 검지 손가락으로 등쪽 목 모피로 마우스와는 복부 측면을 노출 회전. </li><li> 머리가 약간 낮은 그래서 마우스의 후방 끝을 올립니다. </li><li> 1cm 피부, 중간 선 1 센티미터 측면을 통해 25 G 바늘을 삽입하고 1~2cm 전방 천장 관절에 바늘 잠금 주사기를 사용. </li><li> 1 ml의 마우스 체강 내에 접종 천천히 주입한다. </li></ol></li></ol> 3. 마우스 모니터링 <ol><li> 그들은 마우스 스크래피의 임상 증상을 표현할 때까지 매일 쥐를 모니터링합니다. 임상 적 증상이 포함될 수 있습니다 : 후만 변형, 운동 장애, 뻣뻣한 꼬리, 손질의 부족, 쇠약, 무기력합니다. </li><li> 0 표시 = 없음, 1 = 보통, 2 = 심한 곳에 흔적이 분명 할 때 6 임상 증상 각각에 대해 마우스를 점수. </li><li> 각 기호에 대한 일일 총 점수는 1 일 동안 ≥ 8에 도달 할 때 쥐를 안락사, ≥ 6 연속 3 일간, 또는 365에서 일 접종 (DPI)을 배치한다. </li><li> 수확의 두뇌 바로 다음에 euthan-70 ° C에서 아시아 및 저장 </li><li> , 스크래피의 진단을 확인하기 다음과 같이 희석 된 단백질 분해 효소-K 용액 (PK)의 50 ㎍ / ㎖의 3 μL와 뇌 샘플을 소화하기 : 3.1. μL PK, 500 mM의 EDTA의 12.5 μL, pH가 8, 45 ° C에서 30 분 동안 1X PBS의 109.39 μL은 다음 8 μL 로딩 버퍼를 추가하고 5 분 동안 95 ° C에서 샘플을 배양하여 PK를 비활성화. 12 % SDS-PAGE 젤, electrophorese 실온에서 1 시간 동안 PBS 0.2 % 트윈 20 Immobilon PVDF 막 5 % 무 지방 우유와 함께 블록에 전송 상에로드 샘플. 다음, 상온에서 1 시간 동안 슈퍼 블록에 희석 냉이 퍼 옥시 다제,에 접합 Bar224 안티의 PrP 단일 클론 항체로 프로브. PBS-0.2 % 트윈 20과 1 시간 동안 멤브레인을 씻어. 시각화, G-상자 젤 문서 시스템에 화학 발광 기판과 이미지로 웨스턴 블롯 5 분을 배양합니다. </li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guano+deposition+in+an+Oklahoma+crow+roost.">Guano deposition in an Oklahoma crow roost.</a> Condor. 81 (3), 247-250 (1979).</li><li>Aldous, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Winter+habits+of+crows+in+Oklahoma.">Winter habits of crows in Oklahoma.</a> Journal of Wildlife Management. 73 (4), 290-295 (1944).</li></ol>

관심 없음 충돌 선언하지 않습니다.

우리는 까마귀의 소화 시스템을 통해의 PrP 해상도 물질의 통과를 문서화하는 과정을 보여줍니다. 우리는 까마귀는 질병이없는 지리적 영역의 PrP 해상도 물질을 이동시키다 수있는 능력을 가지고 있는지 확인하기 위해 기존의 방법을 사용했다. 다른 사람은 그것을 제거하는 데 실패 둘 다 19 ~ 21과 설치류 (22, 23) 소화 체액을 반추하는의 PrP 해상도의 저항?...

전염성 해면상 뇌증 (TSE)는 야생 동물에 영향을 미치는 치명적인 전염성 신경 퇴행성 질환, 가축, 인간입니다. TSE 질병의 전염 물질은 프리온 단백질 1의 잘못 폴딩 또는 병원성 이소 PRP (해상도)이 나타납니다. 양과 염소의 스크래피,, 소 해면상 뇌증 (동물 TSE 질환 노새 사슴 (Odocoileus의 hemionus), 흰 꼬리 사슴 (Odocoileus의 virginianus), 엘크 (Cervus의의 elaphus), 그리고 사슴 (에 Alces alces)에 만성 소모성 질병 (CWD)를 포함 국내 가축의 BSE) 으 농장 밍크의 전염성 밍크 뇌증, 고양이의 고양이 해면상 뇌증, 인간이 아닌 영장류 8과 해면상 뇌증, 이국적인 동물원에 이국적인 유제 해면상 뇌증은 가족 Bovidae의 반추. 하나의 인간 TSE 질환, 변종 크로이츠 펠트 - 야콥 병은 희귀의 PrP RES-contamin 소비에 의해 인수 될 것으로 생각됩니다ated 음식 9. 오염 된 쇠고기가 10을 소비하는 경우 마찬가지로, BSE가 인간을 감염시킬 수 있습니다. 모든 TSE 질환, 스크래피와 CWD는 동물에서 동물 2,3,11뿐만 아니라 환경 소스를 4-6로 추정되는 자립 전염병 감염에 대한 근원을 가진 유일한 두 가지입니다. 연구는 대부분의 TSE 질환은 임상 증상의 발현에의 PrP 해상도 재료의 자연적인 노출 사건에서 주목할만한 확장 배양 기간을 필요로 2-4,6,8 및 명백한 종의 장벽을 최소화하지만, 종간 전송 12-14의 가능성을 제거하지 않는 것이 좋습니다 . 감염성 프리온 PRP (해상도) 물질의 확산 메커니즘을 식별 TSE 질환이 풍경을 가로 질러 이동하는 방법에 대한 질문에 대한 답변을 위해 매우 중요하다. 실험 조사는 15, 16, 가금류 및 돼지 17과 미국의 까마귀 (까마귀 풀어야 곤충 것을 제안했습니다hyrhynchos) 7,18가의 PrP 해상도 재료의 수동적 인 사업자 또는 분배기입니다. 까마귀의 소화 시스템을 통해의 PrP 해상도 물질의 통로는 최근에 그들이 TSE 질환 (7)의 분산에 재생할 수있는 역할을 시연, 설명되어 있습니다. 이러한 결과는, 까마귀, 헤매다가 발생할 수 있음이 그럴듯하게 소비하고, 질병이없는 지역에, 대변 증착을 통해 전염성 물질을 수송한다. 우리가 여기에서 설명하는 절차는 까마귀의 소화 시스템을 통해의 PrP 해상도 물질의 통과를 문서화하는 데 사용되었습니다 크게 관련 미래 연구의 다른 폐품과 육식 동물의 종 - 특정 모델에 이러한 방법의 적용을 용이하게합니다. 본 연구에서는 기존의 방법의 PrP 해상도 물질의 확산 및 전반적인 부담에 기여할 수의 PrP 해상도 재료를 인신 매매의 틀에 얽매이지 않는 방법을 연구하는 데 사용되었습니다.

<table border="0" cellpadding="0" cellspacing="0" width="545">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RML Chandler strain mouse-adapted scrapie</td>
			<td style="width:112px;">Rocky Mountain Laboratories</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">RC57BL/6 mice</td>
			<td style="width:112px;">Hilltop Lab Animals</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">American crows</td>
			<td style="width:112px;">wild captured</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Pen/Strep</td>
			<td>Invitrogen</td>
			<td style="width:137px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Phosphate buffered Saline</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">70011-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Sonicator</td>
			<td>Misonix</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Proteinase-K solution</td>
			<td style="width:112px;">Roche</td>
			<td align="right" style="width:137px;">3115887001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Loading buffer</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0007 and 0009</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Bis-tris SDS PAGE 12% gel</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:137px;">NP0342</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Immobilon PVDF membrane</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">1SEQ00010</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">Tween 20</td>
			<td style="width:112px;">Sigma Aldrich</td>
			<td style="width:137px;">P2287</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bullet blender homogenizer</td>
			<td style="width:112px;">Braintree Scientific</td>
			<td style="width:137px;">BBX24B</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">2.3 mm Zirconia/silica beads</td>
			<td style="width:112px;">BioSpec Products</td>
			<td style="width:137px;">11079125Z</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Bar224 anti-PrP monoclonal antibody</td>
			<td style="width:112px;">Cayman Chemical</td>
			<td align="right" style="width:137px;">10009035</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">Superblock</td>
			<td style="width:112px;">Thermo Scientific</td>
			<td align="right" style="width:137px;">37517</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:217px;">chemiluminescent substrate</td>
			<td style="width:112px;">Millipore</td>
			<td style="width:137px;">WBKLS0500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:217px;">G-box gel documentation system</td>
			<td style="width:112px;">Syngene</td>
			<td style="width:137px;"></td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

procedures for identifying infectious prions after passage through the digestive system of an avian species

감염성 프리온 PRP (해상도) 재료는 가능성이 치명적인 신경 퇴행성 전염성 해면상 뇌증 (TSE) 질병 (1)의 원인이된다. 만성 소모성 질병 (CWD) 등 TSE 질병의 전송은, 동물 2,3에 동물에서뿐만 아니라 환경 소스를 4-6로 추정된다. 청소부와 육식 소비와 CWD 오염 된 썩은 고기의 배설물을 통해의 PrP 해상도 물질을 이동시키다하는 가능성이있다. 최근 작품은 미국의 까마귀의 소화 시스템 (까마귀 brachyrhynchos), 공통 북미 헤매다 ~ 7의 PrP 해상도 물질의 통과를 기록했다. 우리는 미국의 까마귀를 통해 고해상도의 PrP 물질의 통과를 문서화하는 데 사용되는 절차에 대해 설명합니다. 크로우는 RML 변형 마우스 적응 스크래피와 gavaged하고, 배설물은 4 시간 포스트 위관을 수집 하였다. 까마귀 배설물로 다음 풀링 및 복강 주사 하였다C57BL / 6 마​​우스. 그들은 마우스 스크래피의 임상 증상을 표현하고, 그 후 안락사 될 때까지 마우스는 매일 관찰 하였다. 증상이 마우스는 365 일 후 접종 할 때까지 감시했다. 웨스턴 블롯 분석은 질병 상태를 확인하기 위하여 실시 하였다. 결과 프리온 테스트 마우스에있는 질병을 일으키는 원인이 까마귀의 소화 시스템을 통해 여행 후 감염을 유지하고 배설물에 존재하는 것으로 나타났다.

사용 절차는 까마귀의 소화 시스템이 스크래피 뇌 균질 7의 경구 위관 이후의 PrP 해상도 감염 4 시간을 제거하지 않음을 보여줍니다. 의 PrP 해상도 재료로 gavaged 된 모든 스물 까마귀 이후 마우스에 대변을 통해의 PrP 해상도의 자료를 전송. 병에 걸린 마우스는 임상 마우스 스크래피 증상과 질병의 확인이 웨스턴 블롯 분석에 의해 완성되었다의 발현에 의해 확인되...

Watch this Scientific Journal Video about Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species at JoVE.com

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

청소부는 질병이없는 지역에 자신의 배설물에 전염성 전염성 해면상 뇌증의 프리온을 이동시키다 수있는 가능성이있다. 우리 마우스 적응 스크래피 프리온은 미국의 까마귀의 소화 기관 (까마귀 brachyrhynchos), 죽은 동물의 일반적인 소비자 비록 통과 후 감염성 유지 여부를 확인하는 데 사용 세부 방법.

조류 종의 소화 기관을 통과 한 후 감염성 프리온을 확인하기위한 절차

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

procedures-for-identifying-infectious-prions-after-passage-through

Research

JoVE Journal

Immunology and Infection

11.0K 조회수.  USDA.  청소부는 질병이없는 지역에 자신의 배설물에 전염성 전염성 해면상 뇌증의 프리온을 이동시키다 수있는 가능성이있다. 우리 마우스 적응 스크래피 프리온은 미국의 까마귀의 소화 기관 (까마귀 brachyrhynchos), 죽은 동물의 일반적인 소비자 비록 통과 후 감염성 유지 여부를 확인하는 데 사용 세부 방법. 

비디오: Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

에 대한 부정적인 선택 마커의 개발 Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

연구

면역학 및 감염병학

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

전파와 녹색 형광 단백질을 표현 Maedi - visna 바이러스의 감염성 분자 클론을 검출

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions의 단백질 Misfolding의 순환 증폭

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

사용하여 전통과 소설 오버레이 시스템 : 플라크 분석 실험을 통해 바이러스 농도 결정

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 : 1. 바이오 안전성 레벨 4 정장 연구소 스위트 출입국 절차

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 2. 일반 사례

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 4. 의료 영상 절차

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 3. Aerobiology

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol releases or unexpected natural aerosol transmission. Specific laboratories designed to perform these experiments require tremendous engineering controls to provide a safe and secure working environment and constant systems maintenance to sustain functionality. Class III biosafety cabinets, also referred to as gloveboxes, are gas-tight enclosures with non-opening windows. These cabinets are maintained under negative pressure by double high-efficiency-particulate-air (HEPA)-filtered exhaust systems and are the ideal primary containment for housing aerosolization equipment. A well planned workflow between staff members within high containment from, for instance, an animal biosafety level-4 (ABSL-4) suit laboratory to the ABSL-4 cabinet laboratory is a crucial component for successful experimentation. For smooth study execution, establishing a communication network, moving equipment and subjects, and setting up and placing equipment, requires staff members to meticulously plan procedures prior to study initiation. Here, we provide an overview and a visual representation of how aerobiology research is conducted at the National Institutes of Health, National Institute of Allergy and Infectious Diseases Integrated Research Facility at Fort Detrick, Maryland, USA, within an ABSL-4 environment.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 3. Aerobiology

As high-consequence pathogens can potentially infect subjects through airborne particles, aerobiology has been increasingly applied in pathogenesis research and medical countermeasure development. We present a detailed visual demonstration of aerobiology procedures during an aerosol challenge in nonhuman primates in an animal biosafety level 4 maximum containment environment.

(A)의 안전주의 사항 및 운영 절차 BSL-4 실험실 3. 공중 생물학

Aerosol or inhalational studies of high-consequence pathogens have recently been increasing in number due to the perceived threat of intentional aerosol ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika 바이러스 감염 세포 배양 시스템과<em&gt; 체외</em인터페론의&gt; 예방 효과

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

마우스 작은 장의 면역 시스템에서 세포를 분리

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...