| ObjectiveTo 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.Methods1 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.Results1 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.Conclusions1The 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.
|
PDF Full Text Request