中国西部地区WNV感染初步流行病学研究以及WNV和NiV主要蛋白的免疫信息学分析
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摘要
目的:
调查西尼罗病毒(West nile virus, WNV)在中国西部地区人和动物中的自然感染及流行情况,以获得我国西部地区WNV的流行病学资料,评估我国西部地区的生物安全现状。预测WNV和Nipah病毒( Nipah virus, NiV)主要蛋白的二级结构和B细胞表位,为WNV和NiV的免疫预防和免疫治疗提供理论依据。
方法:
1对课题组前期开发的WNV一步法实时RT-PCR检测方法进行活病毒验证。提取WNV、登革热病毒(dengue virus,DENV)、日本乙型脑炎病毒(Japanese encephalitis virus,JEV)细胞培养液上清和WNV接种鼠脑组织的RNA。对WNV细胞培养液上清所提的RNA倍比稀释,然后进行一步法Real time RT-PCR反应。对WNV接种的鼠脑组织提取的RNA倍比稀释,并进行一步法Real time RT-PCR反应。对DEN、JEV细胞培养液提取RNA,进行一步法real time RT-PCR反应。
2自2007年5月起,收集重庆地区发热患者外周血标本100份并采集重庆地区饲养的猪外周血标本300份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。收集重庆地区病毒性脑炎患者脑脊液22份,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
3自2007年6月起,采集新疆地区驴外周血标本200份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
4自2008年6月起,采集宁夏地区羊外周血标本200份和牛外周血标本100份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
5根据WNV的E蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对E蛋白的二级结构中的柔性区域的分析,预测WNV的E蛋白的B细胞表位。
6根据NiV的G蛋白和F蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对G蛋白和F蛋白的二级结构中的柔性区域的分析,预测NiV的G蛋白和F蛋白的B细胞表位。
结果:
1WNV一步法实时RT-PCR方法能检测的WNV细胞培养液上清RNA的浓度范围为103～10-2 PFU/ml,能检测的WNV接种鼠脑组织RNA的浓度范围为10-3～10-1(0原液起始浓度100)。该方法检测DEN、JEV细胞培养液上清RNA结果为阴性。
2对重庆地区收集的人血、脑脊液和采集的猪血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
3对新疆地区采集的驴血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
4对宁夏采集的羊血和牛血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
5 WNV的E蛋白N端第41-56, 68-73, 129-139, 209, 214-220, 239-250, 261-267, 285-296, 365-371, 412-416, 468和471-472区域形成α螺旋可能大;N端第32-34, 61-65, 140-143, 157-159, 166-170, 185-189, 201-207, 210-213, 301-304, 322-328, 238-344, 354-358, 406-411, 434-436, 442-447, 450-453, 460-463和482-496区域形成β折叠的可能大;E蛋白的柔性结构区域位于N端第8, 15-17, 27, 36-38, 100-101, 103-104, 109-112, 145-147, 152-155, 173, 175, 181, 192-195, 226-230,257, 276-277, 298-299, 317-320, 334-335, 377-381, 389, 399-401, 430-432和439区段。综合分析推测WNV的E蛋白B细胞表位最有可能位于N端第35-42, 147-156, 191-198, 226-249, 329-337和375-382区段,另外E蛋白N端第275-284, 313-320和396-403区段也可能存在B细胞表位。
6 Nipah病毒G蛋白N端第2-6区域形成α螺旋可能大;N端第24-26, 47-73, 80-84, 87-95, 110-112, 117-120, 178-182, 200-210, 215-219, 229-231, 245-256, 264-269, 279-285, 290-301, 362-374, 384-386, 398-401, 406-409, 427-430, 435-436, 450-456, 462-472, 483-485, 511-517, 519-526, 560-567, 573-581, 586-589, 592-596和600-602区域形成β折叠的可能大;G蛋白的柔性结构区域位于N端第13-19, 76-78, 106-107, 140-146, 151-153, 162-165, 174-175, 185-186, 194, 227, 240-243, 257, 259-260, 275-277, 288, 309-312, 324-329, 343-346, 352-354, 379-382, 390-394, 402-404, 419-421, 423-424, 432-434, 440-441, 481-482, 488-490, 494-500, 527-530, 541-543, 555-558和582-585区段。综合分析推测Nipah病毒G蛋白的B细胞表位最有可能位于N端第140-153, 270-278和401-408区段,另外G蛋白N端第7-23, 72-79, 162-175, 192-198, 255-262, 340-348, 373-394, 416-424, 432-448, 479-489, 527-534, 540-547和552-561区段也可能存在B细胞表位。
Nipah病毒F蛋白N端第92-96, 108-111, 122-127, 131-139, 157-165, 194-203, 251, 352, 355, 467-469和474-479区域形成α螺旋可能大;N端第1-4, 11-19, 37-41, 44-47, 57-60, 78, 82-88, 102-107, 112-117, 121, 128-130, 170-179, 184-190, 209-214, 226-231, 240-246, 263-271, 276-285, 292-299, 308-311, 316-321, 324-331, 338-340, 368-377, 382-386, 390-391, 396-401, 408-412, 416, 421-429, 447-460, 480-484, 491-515和541-546区域形成β折叠的可能大;F蛋白的柔性结构区域位于N端第52, 66-67, 98-100, 216-218, 222, 236-239, 303-306, 334-336, 343, 348-351, 357-359, 380-381, 402-405, 439, 462-463, 470-472, 523-524和537-539区段。综合分析推测Nipah病毒F蛋白的B细胞表位最有可能位于N端第95-105和519-539区段,另外F蛋白N端第42-54, 216-225, 301-307, 356-365和470-482区段也可能存在B细胞表位。
结论:
1本课题组前期开发的西尼罗病毒一步法Real-time RT-PCR方法敏感性高、特异性好,能准确定量,适用于大规模的流行病学调查和病毒性疾病检测。
2初步流行病学研究提示我国西部重庆地区部分人群和猪群中暂未发现WNV感染。
3初步流行病学研究提示我国西部新疆地区部分驴群中暂未发现WNV感染。
4初步流行病学研究提示我国西部宁夏地区部分羊群和牛群中暂未发现WNV感染。
5应用生物信息学方法分析了WNV主要抗原蛋白E蛋白的二级结构B细胞表位,为进一步研究WNV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。
6应用生物信息学方法分析了NiV主要抗原蛋白G蛋白和F蛋白的二级结构B细胞表位,为进一步研究NiV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。
Objective
To investigate the natural infection and epidemic of West Nile Virus (WNV) in human and animal in western region of China, obtain the epidemiologic data of WNV in western region of China and assess of the biosafety status in these areas. To predict the secondary structure and B-cell epitopes of major proteins of WNV and nipah virus (NiV), provide a basis for immunoprophylaxis and immunotherapy of WNV and NiV.
Methods
1 The sensitivity and specificity of the established WNV One-step real-time RT-PCR was verified by live virus. WNV RNA of cell culture and mice brain was extracted using Qiagen RNeasy Mini Kit. Serial 10-fold dilutions of WNV RNA extracted from cell culture and WNV RNA extracted from mice brain were performed One-step real-time RT-PCR to assess the sensitivity. DEN and JEV RNA extracted from cell culture was performed One-step real-time RT-PCR to assess the specificity.
2 Peripheral blood samples of 100 patients with fever and 300 domestic pigs, and cerebrospinal fluid of 22 patients with Viral Encephalitis were collected from Chongqing since May, 2007. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
3 Peripheral blood samples of 200 donkeys were collected from Xinjiang Uygur Autonomous Region since June, 2007. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
4 Peripheral blood samples of 200 sheep and 100 cattle were collected from Ningxia Hui Autonomous Region since June, 2008. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
5 Based on the amino acid sequence of the E protein of WNV, three methods was used to find the probable secondary structure of the E protein including Garnier-Robson method, Chou-Fasman method and Karplus-Schulz method. Based on the flexible regions of E protein, probable B-cell epitopes of the E protein of WNV were predicted by the above parameters, Kyte-Doolittle method was used for hydrophilicity prediction, Emini method was used for accessibility prediction and Jameson-Wolf method was used for antigenicity prediction.
6 Based on the amino acid sequence of the G protein and the F protein of NiV, three methods was used to find the probable secondary structure of the G protein and the F protein including Garnier-Robson method, Chou-Fasman method and Karplus-Schulz method. Based on the flexible regions of G protein and the F protein, probable B-cell epitopes of the G protein and the F protein of NiV were predicted by the above parameters, Kyte-Doolittle method was used for hydrophilicity prediction, Emini method was used for accessibility prediction and Jameson-Wolf method was used for antigenicity prediction.
Results
1 The quantitative ranges of one-step real-time RT-PCR for WNV RNA extracted from cell culture were 103～10-2 PFU/ml, and the quantitative ranges for WNV RNA extracted from mice brain were10-3～10-10 (The initial concentration of the RNA was 100). Nonspecific PCR amplification with DEN and JEV RNA extracted from cell culture.
2 Nucleic acid detections searching for WNV were successfully performed in blood samples of patients with fever and domestic pigs, and cerebrospinal fluid of patient with Viral encephalitis collected from Chongqing, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
3 Nucleic acid detections searching for WNV were successfully performed in blood samples of donkeys collected from Xinjiang Uygur Autonomous Region, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
4 Nucleic acid detections searching for WNV were successfully performed in blood samples of sheep and cattle collected from Ningxia Hui Autonomous Region, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
5 As to the E protein of WNV, it showed that the N-terminal No. 41-56, 68-73, 129-139, 209, 214-220, 239-250, 261-267, 285-296, 365-371, 412-416, 468 and 471-472 were theα-helix regions; the N-terminal No. 32-34, 61-65, 140-143, 157-159, 166-170, 185-189, 201-207, 210-213, 301-304, 322-328, 238-344, 354-358, 406-411, 434-436, 442-447, 450-453, 460-463 and 482-496 were theβ-helix regions; and the flexible regions located at the N-terminal No. 8, 15-17, 27, 36-38, 100-101, 103-104, 109-112, 145-147, 152-155, 173, 175, 181, 192-195, 226-230, 257, 276-277, 298-299, 317-320, 334-335, 377-381, 389, 399-401, 430-432 and 439 regions. The results indicated the most probable B-cell epitopes of E protein were located within or nearby its N-terminal No. 35-42, 147-156, 191-198, 226-249, 329-337 and 375-382. The N-terminal No. 275-284, 313-320 and 396-403may be the possible B cell epitopes.
6 As to the G protein of NiV, it showed that the N-terminal No. 2-6 was theα-helix regions; the N-terminal No. 24-26, 47-73, 80-84, 87-95, 110-112, 117-120, 178-182, 200-210, 215-219, 229-231, 245-256, 264-269, 279-285, 290-301, 362-374, 384-386, 398-401, 406-409, 427-430, 435-436, 450-456, 462-472, 483-485, 511-517, 519-526, 560-567, 573-581, 586-589, 592-596 and 600-602were theβ-helix regions; and the flexible regions located at the N-terminal No. 13-19, 76-78, 106-107, 140-146, 151-153, 162-165, 174-175, 185-186, 194, 227, 240-243, 257, 259-260, 275-277, 288, 309-312, 324-329, 343-346, 352-354, 379-382, 390-394, 402-404, 419-421, 423-424, 432-434, 440-441, 481-482, 488-490, 494-500, 527-530, 541-543, 555-558 and 582-585 regions. The results indicated the most probable B-cell epitopes of NiV G protein were located within or nearby its N-terminal No. 140-153, 270-278 and 401-408, the N-terminal No. 7-23, 72-79, 162-175, 192-198, 255-262, 340-348, 373-394, 416-424, 432-448, 479-489, 527-534, 540-547 and 552-561 may be the possible B-cell epitopes.
As to the F protein of NiV, it showed that the N-terminal No. 92-96, 108-111, 122-127, 131-139, 157-165, 194-203, 251, 352, 355, 467-469 and 474-479 was theα-helix regions; the N-terminal No. 1-4, 11-19, 37-41, 44-47, 57-60, 78, 82-88, 102-107, 112-117, 121, 128-130, 170-179, 184-190, 209-214, 226-231, 240-246, 263-271, 276-285, 292-299, 308-311, 316-321, 324-331, 338-340, 368-377, 382-386, 390-391, 396-401, 408-412, 416, 421-429, 447-460, 480-484, 491-515 and 541-546 were theβ-helix regions; and the flexible regions located at the N-terminal No. 52, 66-67, 98-100, 216-218, 222, 236-239, 303-306, 334-336, 343, 348-351, 357-359, 380-381, 402-405, 439, 462-463, 470-472, 523-524 and 537-539 regions. And the most probable B-cell epitopes of NiV F protein were located within or nearby its N-terminal No. 95-105 and 519-539, the N-terminal No. 42-54, 216-225, 301-307, 356-365 and 470-482 may be the possible B-cell epitopes.
Conclusions
1The WNV one-step real-time RT-PCR is sensitive and reliable and allows rapid detection and quantitation of WNV in field and experimental materials used for epidemiological surveillance and specific diagnosis.
2 Until now, we found no infections of WNV in either patients or pigs blood samples or patients cerebrospinal fluid sample collected from Chongqing.
3 Until now, we found no infections of WNV in donkey blood samples collected from Xinjiang Uygur Autonomous Region.
4 Until now, we found no infections of WNV in either sheep or cattle blood samples collected from Ningxia Hui Autonomous Region.
5 The secondary structure and B cell epitopes of E protein on multi-parameters were predicted successfully, which would establish the basis for studies of the characterization of the protein , development of epitope based vaccine , and preparation of monoclonal antibody against E protein.
6 The secondary structure and B cell epitopes of G protein and F protein of NiV on multi-parameters were predicted successfully, which would establish the basis for studies of the characterization of the G protein and F protein, development of epitope based vaccine, and preparation of monoclonal antibody against G protein and F protein.
调查西尼罗病毒(West nile virus, WNV)在中国西部地区人和动物中的自然感染及流行情况,以获得我国西部地区WNV的流行病学资料,评估我国西部地区的生物安全现状。预测WNV和Nipah病毒( Nipah virus, NiV)主要蛋白的二级结构和B细胞表位,为WNV和NiV的免疫预防和免疫治疗提供理论依据。
方法:
1对课题组前期开发的WNV一步法实时RT-PCR检测方法进行活病毒验证。提取WNV、登革热病毒(dengue virus,DENV)、日本乙型脑炎病毒(Japanese encephalitis virus,JEV)细胞培养液上清和WNV接种鼠脑组织的RNA。对WNV细胞培养液上清所提的RNA倍比稀释,然后进行一步法Real time RT-PCR反应。对WNV接种的鼠脑组织提取的RNA倍比稀释,并进行一步法Real time RT-PCR反应。对DEN、JEV细胞培养液提取RNA,进行一步法real time RT-PCR反应。
2自2007年5月起,收集重庆地区发热患者外周血标本100份并采集重庆地区饲养的猪外周血标本300份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。收集重庆地区病毒性脑炎患者脑脊液22份,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
3自2007年6月起,采集新疆地区驴外周血标本200份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
4自2008年6月起,采集宁夏地区羊外周血标本200份和牛外周血标本100份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。
5根据WNV的E蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对E蛋白的二级结构中的柔性区域的分析,预测WNV的E蛋白的B细胞表位。
6根据NiV的G蛋白和F蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对G蛋白和F蛋白的二级结构中的柔性区域的分析,预测NiV的G蛋白和F蛋白的B细胞表位。
结果:
1WNV一步法实时RT-PCR方法能检测的WNV细胞培养液上清RNA的浓度范围为103～10-2 PFU/ml,能检测的WNV接种鼠脑组织RNA的浓度范围为10-3～10-1(0原液起始浓度100)。该方法检测DEN、JEV细胞培养液上清RNA结果为阴性。
2对重庆地区收集的人血、脑脊液和采集的猪血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
3对新疆地区采集的驴血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
4对宁夏采集的羊血和牛血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。
5 WNV的E蛋白N端第41-56, 68-73, 129-139, 209, 214-220, 239-250, 261-267, 285-296, 365-371, 412-416, 468和471-472区域形成α螺旋可能大;N端第32-34, 61-65, 140-143, 157-159, 166-170, 185-189, 201-207, 210-213, 301-304, 322-328, 238-344, 354-358, 406-411, 434-436, 442-447, 450-453, 460-463和482-496区域形成β折叠的可能大;E蛋白的柔性结构区域位于N端第8, 15-17, 27, 36-38, 100-101, 103-104, 109-112, 145-147, 152-155, 173, 175, 181, 192-195, 226-230,257, 276-277, 298-299, 317-320, 334-335, 377-381, 389, 399-401, 430-432和439区段。综合分析推测WNV的E蛋白B细胞表位最有可能位于N端第35-42, 147-156, 191-198, 226-249, 329-337和375-382区段,另外E蛋白N端第275-284, 313-320和396-403区段也可能存在B细胞表位。
6 Nipah病毒G蛋白N端第2-6区域形成α螺旋可能大;N端第24-26, 47-73, 80-84, 87-95, 110-112, 117-120, 178-182, 200-210, 215-219, 229-231, 245-256, 264-269, 279-285, 290-301, 362-374, 384-386, 398-401, 406-409, 427-430, 435-436, 450-456, 462-472, 483-485, 511-517, 519-526, 560-567, 573-581, 586-589, 592-596和600-602区域形成β折叠的可能大;G蛋白的柔性结构区域位于N端第13-19, 76-78, 106-107, 140-146, 151-153, 162-165, 174-175, 185-186, 194, 227, 240-243, 257, 259-260, 275-277, 288, 309-312, 324-329, 343-346, 352-354, 379-382, 390-394, 402-404, 419-421, 423-424, 432-434, 440-441, 481-482, 488-490, 494-500, 527-530, 541-543, 555-558和582-585区段。综合分析推测Nipah病毒G蛋白的B细胞表位最有可能位于N端第140-153, 270-278和401-408区段,另外G蛋白N端第7-23, 72-79, 162-175, 192-198, 255-262, 340-348, 373-394, 416-424, 432-448, 479-489, 527-534, 540-547和552-561区段也可能存在B细胞表位。
Nipah病毒F蛋白N端第92-96, 108-111, 122-127, 131-139, 157-165, 194-203, 251, 352, 355, 467-469和474-479区域形成α螺旋可能大;N端第1-4, 11-19, 37-41, 44-47, 57-60, 78, 82-88, 102-107, 112-117, 121, 128-130, 170-179, 184-190, 209-214, 226-231, 240-246, 263-271, 276-285, 292-299, 308-311, 316-321, 324-331, 338-340, 368-377, 382-386, 390-391, 396-401, 408-412, 416, 421-429, 447-460, 480-484, 491-515和541-546区域形成β折叠的可能大;F蛋白的柔性结构区域位于N端第52, 66-67, 98-100, 216-218, 222, 236-239, 303-306, 334-336, 343, 348-351, 357-359, 380-381, 402-405, 439, 462-463, 470-472, 523-524和537-539区段。综合分析推测Nipah病毒F蛋白的B细胞表位最有可能位于N端第95-105和519-539区段,另外F蛋白N端第42-54, 216-225, 301-307, 356-365和470-482区段也可能存在B细胞表位。
结论:
1本课题组前期开发的西尼罗病毒一步法Real-time RT-PCR方法敏感性高、特异性好,能准确定量,适用于大规模的流行病学调查和病毒性疾病检测。
2初步流行病学研究提示我国西部重庆地区部分人群和猪群中暂未发现WNV感染。
3初步流行病学研究提示我国西部新疆地区部分驴群中暂未发现WNV感染。
4初步流行病学研究提示我国西部宁夏地区部分羊群和牛群中暂未发现WNV感染。
5应用生物信息学方法分析了WNV主要抗原蛋白E蛋白的二级结构B细胞表位,为进一步研究WNV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。
6应用生物信息学方法分析了NiV主要抗原蛋白G蛋白和F蛋白的二级结构B细胞表位,为进一步研究NiV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。
Objective
To investigate the natural infection and epidemic of West Nile Virus (WNV) in human and animal in western region of China, obtain the epidemiologic data of WNV in western region of China and assess of the biosafety status in these areas. To predict the secondary structure and B-cell epitopes of major proteins of WNV and nipah virus (NiV), provide a basis for immunoprophylaxis and immunotherapy of WNV and NiV.
Methods
1 The sensitivity and specificity of the established WNV One-step real-time RT-PCR was verified by live virus. WNV RNA of cell culture and mice brain was extracted using Qiagen RNeasy Mini Kit. Serial 10-fold dilutions of WNV RNA extracted from cell culture and WNV RNA extracted from mice brain were performed One-step real-time RT-PCR to assess the sensitivity. DEN and JEV RNA extracted from cell culture was performed One-step real-time RT-PCR to assess the specificity.
2 Peripheral blood samples of 100 patients with fever and 300 domestic pigs, and cerebrospinal fluid of 22 patients with Viral Encephalitis were collected from Chongqing since May, 2007. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
3 Peripheral blood samples of 200 donkeys were collected from Xinjiang Uygur Autonomous Region since June, 2007. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
4 Peripheral blood samples of 200 sheep and 100 cattle were collected from Ningxia Hui Autonomous Region since June, 2008. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of WNV with one-step real-time RT-PCR methods established before. And epidemiologic reports were submitted.
5 Based on the amino acid sequence of the E protein of WNV, three methods was used to find the probable secondary structure of the E protein including Garnier-Robson method, Chou-Fasman method and Karplus-Schulz method. Based on the flexible regions of E protein, probable B-cell epitopes of the E protein of WNV were predicted by the above parameters, Kyte-Doolittle method was used for hydrophilicity prediction, Emini method was used for accessibility prediction and Jameson-Wolf method was used for antigenicity prediction.
6 Based on the amino acid sequence of the G protein and the F protein of NiV, three methods was used to find the probable secondary structure of the G protein and the F protein including Garnier-Robson method, Chou-Fasman method and Karplus-Schulz method. Based on the flexible regions of G protein and the F protein, probable B-cell epitopes of the G protein and the F protein of NiV were predicted by the above parameters, Kyte-Doolittle method was used for hydrophilicity prediction, Emini method was used for accessibility prediction and Jameson-Wolf method was used for antigenicity prediction.
Results
1 The quantitative ranges of one-step real-time RT-PCR for WNV RNA extracted from cell culture were 103～10-2 PFU/ml, and the quantitative ranges for WNV RNA extracted from mice brain were10-3～10-10 (The initial concentration of the RNA was 100). Nonspecific PCR amplification with DEN and JEV RNA extracted from cell culture.
2 Nucleic acid detections searching for WNV were successfully performed in blood samples of patients with fever and domestic pigs, and cerebrospinal fluid of patient with Viral encephalitis collected from Chongqing, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
3 Nucleic acid detections searching for WNV were successfully performed in blood samples of donkeys collected from Xinjiang Uygur Autonomous Region, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
4 Nucleic acid detections searching for WNV were successfully performed in blood samples of sheep and cattle collected from Ningxia Hui Autonomous Region, using One-step real time RT-PCR. We found no“takeoff points”in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative.
5 As to the E protein of WNV, it showed that the N-terminal No. 41-56, 68-73, 129-139, 209, 214-220, 239-250, 261-267, 285-296, 365-371, 412-416, 468 and 471-472 were theα-helix regions; the N-terminal No. 32-34, 61-65, 140-143, 157-159, 166-170, 185-189, 201-207, 210-213, 301-304, 322-328, 238-344, 354-358, 406-411, 434-436, 442-447, 450-453, 460-463 and 482-496 were theβ-helix regions; and the flexible regions located at the N-terminal No. 8, 15-17, 27, 36-38, 100-101, 103-104, 109-112, 145-147, 152-155, 173, 175, 181, 192-195, 226-230, 257, 276-277, 298-299, 317-320, 334-335, 377-381, 389, 399-401, 430-432 and 439 regions. The results indicated the most probable B-cell epitopes of E protein were located within or nearby its N-terminal No. 35-42, 147-156, 191-198, 226-249, 329-337 and 375-382. The N-terminal No. 275-284, 313-320 and 396-403may be the possible B cell epitopes.
6 As to the G protein of NiV, it showed that the N-terminal No. 2-6 was theα-helix regions; the N-terminal No. 24-26, 47-73, 80-84, 87-95, 110-112, 117-120, 178-182, 200-210, 215-219, 229-231, 245-256, 264-269, 279-285, 290-301, 362-374, 384-386, 398-401, 406-409, 427-430, 435-436, 450-456, 462-472, 483-485, 511-517, 519-526, 560-567, 573-581, 586-589, 592-596 and 600-602were theβ-helix regions; and the flexible regions located at the N-terminal No. 13-19, 76-78, 106-107, 140-146, 151-153, 162-165, 174-175, 185-186, 194, 227, 240-243, 257, 259-260, 275-277, 288, 309-312, 324-329, 343-346, 352-354, 379-382, 390-394, 402-404, 419-421, 423-424, 432-434, 440-441, 481-482, 488-490, 494-500, 527-530, 541-543, 555-558 and 582-585 regions. The results indicated the most probable B-cell epitopes of NiV G protein were located within or nearby its N-terminal No. 140-153, 270-278 and 401-408, the N-terminal No. 7-23, 72-79, 162-175, 192-198, 255-262, 340-348, 373-394, 416-424, 432-448, 479-489, 527-534, 540-547 and 552-561 may be the possible B-cell epitopes.
As to the F protein of NiV, it showed that the N-terminal No. 92-96, 108-111, 122-127, 131-139, 157-165, 194-203, 251, 352, 355, 467-469 and 474-479 was theα-helix regions; the N-terminal No. 1-4, 11-19, 37-41, 44-47, 57-60, 78, 82-88, 102-107, 112-117, 121, 128-130, 170-179, 184-190, 209-214, 226-231, 240-246, 263-271, 276-285, 292-299, 308-311, 316-321, 324-331, 338-340, 368-377, 382-386, 390-391, 396-401, 408-412, 416, 421-429, 447-460, 480-484, 491-515 and 541-546 were theβ-helix regions; and the flexible regions located at the N-terminal No. 52, 66-67, 98-100, 216-218, 222, 236-239, 303-306, 334-336, 343, 348-351, 357-359, 380-381, 402-405, 439, 462-463, 470-472, 523-524 and 537-539 regions. And the most probable B-cell epitopes of NiV F protein were located within or nearby its N-terminal No. 95-105 and 519-539, the N-terminal No. 42-54, 216-225, 301-307, 356-365 and 470-482 may be the possible B-cell epitopes.
Conclusions
1The WNV one-step real-time RT-PCR is sensitive and reliable and allows rapid detection and quantitation of WNV in field and experimental materials used for epidemiological surveillance and specific diagnosis.
2 Until now, we found no infections of WNV in either patients or pigs blood samples or patients cerebrospinal fluid sample collected from Chongqing.
3 Until now, we found no infections of WNV in donkey blood samples collected from Xinjiang Uygur Autonomous Region.
4 Until now, we found no infections of WNV in either sheep or cattle blood samples collected from Ningxia Hui Autonomous Region.
5 The secondary structure and B cell epitopes of E protein on multi-parameters were predicted successfully, which would establish the basis for studies of the characterization of the protein , development of epitope based vaccine , and preparation of monoclonal antibody against E protein.
6 The secondary structure and B cell epitopes of G protein and F protein of NiV on multi-parameters were predicted successfully, which would establish the basis for studies of the characterization of the G protein and F protein, development of epitope based vaccine, and preparation of monoclonal antibody against G protein and F protein.
引文
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[1] Lanciotti RS, Ebel GD,Deubel V, et al. Complete genome sequences andphylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East [J]. Virology, 2002, 298 (1): 96-105.
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[15] Mukhopadhyay S, Kim BS, Chipman PR, et al. Structure of West Nile virus [J]. Science, 2003, 302(5643): 248.
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[4] Beasley DW, Holbrook MR, Travassos Da Rosa AP, et al. Use of a recombinant envelope protein subunit antigen for specific serological diagnosis of West Nile virus infection[J]. J Clin Microbiol. 2004, 42(6):2759-65.
[5] Volk DE, Beasley DW, Kallick DA, et al. Solution structure and antibody binding studies of the envelope protein domain III from the New York strain of West Nile virus[J]. Biol Chem. 2004, 279(37):38755-61.
[6] Lee JW, Chu JJ, Ng ML. Quantifying the specific binding between West Nile virus envelope domain III protein and the cellular receptor alphaVbeta3 integrin [J]. J Biol Chem. 2006, 281(3):1352-60.
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[27]邓永强,秦鄂德.虫媒黄病毒包膜E蛋白的研究进展.军事医学科学院院刊,2006, 30(6):575-579.
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