LATEST KNOWLEDGE ON PEDV AND SWINE DELTACORONAVIRUS DIAGNOSTICS 猪流行性腹泻病毒和 δ- 冠状病毒诊断的最新进展 J. Christopher-Hennings DVM, MS Professor, Head and Director

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1 LATEST KNOWLEDGE ON PEDV AND SWINE DELTACORONAVIRUS DIAGNOSTICS 猪流行性腹泻病毒和 δ- 冠状病毒诊断的最新进展 J. Christopher-Hennings DVM, MS Professor, Head and Director Veterinary & Biomedical Sciences Department Animal Disease Research & Diagnostic Laboratory South Dakota State University Brookings, SD 简克里斯托弗 - 亨宁斯, 南达科他州立大学 Disclosure: I do not, benefit, but my institution, SDSU benefits from sale of diagnostic reagents

2 Swine Enteric Coronavirus Disease 猪肠冠状病毒病 Where the Pigs Are Located in US 美国养猪分布图密度等级极高很高高中等无

3 May 2013, PEDV in U.S 年 5 月猪流行性腹泻病毒在美国出现 Sun Mon Tues Wed Thurs Fri Sat Part 1 PEDv Timeline May st Phone Call (ISU) 6 1 st Indiana Sow Farm 首例印第安纳州 7 2nd Indiana Sow 第二例印第安纳州 8 9 *Received samples at SDSU for TGE testing 接样检测传染性胃肠炎 NVSL Announces PEDv 宣布发现 PEDV *gelbased PCR on line at SDSU June: SDSU: real-time PCR, Kim et al. modified primers (J. Virol. Methods 146: ; 2007) NVSL: Virus Isolation, Sequence KF267450

4 Multiplex nested RT-PCR for the detection of porcine enteric viruses 检测猪肠病毒的多重 PCR Salem A. et al. Journal of Virological Methods 165 (2010) Importance of : 重要在于 1) published, refereed papers 发表, 参考的文章 2) GenBank accessions 基因库中的序列 Lane 1: 100bp ladder 泳道 1:100 碱基梯度标记 2: Neg 阴性 3: Neg 阴性 4: Positive 阳性 5: Positive 阳性 6: Neg 阴性 7: Positive 阳性 8: Positive 阳性 9: Neg Intestine 阴性小肠 10: Neg Intestine 阴性小肠 11: Neg Intestine 阴性小肠 12: PEDV Positive Control (291 bp) 猪流行性腹泻病毒阳性对照 (291 个碱基 )

5 Modified protocol for real-time PCR 改进的实时 PCR 操作规程 Kim H-S et. al. Multiplex real-time RT-PCR for the simultaneous detection and quantification of transmissible gastroenteritis virus and porcine epidemic diarrhea virus. Kim 等. 同时检测传染性胃肠炎和猪流行性腹泻病毒的多重实时 PCR J. Virological Methods 病毒学方法杂志 146: , 2007

Real-time PCR 实时 PCR EZ-PEDV/PDCoV/TGEV MPX Multiplex with PEDV, TGEV & PDCoV (Tetracore, Inc.)http://tetracore.

6 Real-time PCR 实时 PCR EZ-PEDV/PDCoV/TGEV MPX Multiplex with PEDV, TGEV & PDCoV (Tetracore, Inc.) 猪流行性腹泻病毒, 传染性胃肠炎和猪新型冠状病毒的同时检测 Can be semi-quantitative 半定量 Does not differentiate infectious from inactivated virus 不能区分传染性和灭活病毒 Internal control (checks for inhibitors) 内控 ( 抑制剂排查 ) Total run time of 1:22 h 总运行时间 1 小时 22 分钟 Specificity: no reactivity with: 特异性 : 与以下病原无交叉反应 PRCV 猪呼吸道冠状病毒 Porcine Rota A,B,C 猪轮状病毒 A,B,C 型 Sensitivity: Lawsonia intracellularis 胞内劳森氏菌 FFU/ml Ct level 4 x 10e x 10e x10e x.10e x 10e x 10e x10e x 10e x 10e x 10e-3 negative

7 Detection of PEDv strains in various sample types from multiple states via PCR 多个州从不同样品中用 PCR 检测到猪流行性腹泻病毒 Samples: feces, pooled feces, intestine, oral fluid, pooled tissue, cell culture. 样品 : 粪, 粪混样, 小肠, 口腔液, 组织混样, 细胞培养经典型猪流行性腹泻病毒变异型猪流行性腹泻病毒

8 Swine Enteric Coronavirus Disease Diagnostic 猪肠冠状病毒诊断 Comparison of CT Values of 50 Positive Oral Fluid Samples provided by ISU ISU 对 50 个阳性口腔液样本用不同 PCR 方法得到的 CT 值的比较

9 PEDv sequencing 猪流行性腹泻病毒测序 the full-length S gene, the S1 portion, or the S2 portion S 基因全序列,S1 片段或者 S2 片段 appears to be able to reflect the genetic diversity observed at the whole-genome level. 似乎可以代表全基因组水平上的观察到基因变异 Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States. 与 2013 年美国疫情有关的猪流行性腹泻病毒的分离和鉴定 Chen Q et al. J. Clin. Micro 临床微生物学杂志 2014; 52:

10 NAHLN (62 laboratories currently) 国家动物健康实验室联盟 (62 个 ) Currently, requesting whole genome sequencing of PEDv 目前正在对猪流行性腹泻病毒全基因组测序 Rinderpest

SDSU developed Monoclonal Antibodies for PEDv and PDCoV

of virus infected cells by fluorescent antibody staining

Fluorescent focus neutralization (FFN) assay 荧光噬斑中和试验

11 SDSU developed Monoclonal Antibodies for PEDv and PDCoV Diagnostics SDSU 开发的诊断 PEDV 和 PDCV 的单抗 Used for detection of virus infected cells by fluorescent antibody staining 荧光抗体染色的病毒感染细胞 Verification of virus isolation 病毒分离的证实 Fluorescent focus neutralization (FFN) assay 荧光噬斑中和试验 Immunohistochemistry 免疫组化 Staining of antigen in infected tissues 感染组织的抗原染色

12 SDSU Monoclonal Antibodies for PEDv 针对 PEDV 的单抗 Clone 克隆 Protein 蛋白 Ig isotype 同型抗体 SD-6-24 NP IgG 1 SD NP IgG 1 SD-6-29 NP IgG 1 SD NP IgG 1 SD NP IgG 1 SD NP IgG 1 SD NP IgG 1 SD NP IgG 1 SD NP IgM SD NP IgM SDH S1 IgG 2a SD S1 IgM

13 Diagnostic serology tests 血清学检测方法 Indirect Fluorescent Antibody (IFA) 间接荧光抗体 Indirect NP-ELISA 间接针对 NP 的 ELISA Detects antibody against PEDV-NP 针对核蛋白 Licensed for commercial distribution 已商业化 Blocking ELISA 阻断 ELISA Greater specificity in theory 特异性更高 Measures ability of sample Ab to block binding of a labeled mab to the PEDV- NP antigen 测量样品抗体阻断标记单抗结合 PEDV-NP 的能力 Fluorescent Microsphere Immunoassay (FMIA) 荧光微球免疫试验 Allows for multiplexing to detect antibody against multiple target antigens at the same time 多重抗原的同时检测 Virus neutralization (FFN) 病毒中和试验 Detects antibodies that neutralize PEDV in vitro 检测抗体体外中和 PEDV 的能力 May correlate with protection of piglets 与保护力也许有相关性

14 IFA Serology (indirect fluorescent antibody assay) IFA 血清学 - 间接荧光抗体试验 Detects antibody against PEDV due to prior exposure 检测接触 PEDV 后产生的抗体 Detectable antibody by 7 to 14 days post-exposure 接触 PEDV 后 7-14 天的抗体可以检出 Can run dilution series some ability to quantify 可以倍比稀释能部分定量

15 Indirect Fluorescent Antibody Test (IFA) 间接荧光抗体检测 Anti-Swine IgG /FITC Antibody FITC 标记的抗猪的二抗 Serum Antibody 血清抗体 PEDv Virus PED 病毒 Ab positive serum PEDV 抗体阳性血清 Vero-76 cells Vero-76 细胞系 Animations courtesy of Neal Ferrin

16 Indirect ELISA 间接 ELISA ABTS Substrate 显色底物 Anti Swine IgG/ HRPO Conjugate 偶联辣根过氧化物酶的抗猪的二抗 Serum Antibody 血清抗体 Ab Positive Serum PEDV 抗体阳性血清 PEDv Ag (NP expressed protein) PED 病毒的抗原 ( 表达的 NP 蛋白 )

17 PEDV-NP antibody response as detected by indirect ELISA (serum samples from experimentally infected pigs) 间接 ELISA 检测到的针对 PEDV-NP 蛋白的抗体 ( 试验感染的猪的血清 ) 接种后天数 Serum samples courtesy of Dr. Dick Hesse, Kansas State University

18 SDSU PED Blocking ELISA (belisa) PED 阻断 ELISA Enzyme Substrate 显色底物 Strepavidin-HRP Enzyme Conjugate 偶联链亲和素 - 辣根过氧化物酶的抗猪的二抗 Biotinylated anti-pedv mab 生物素 -PEDV 单抗 BSA Blocking agent BSA 封闭液 Ab Negative Serum PEDV 抗体阴性血清 PEDv Ag NP (expressed protein) PED 病毒的抗原 ( 表达的 NP 蛋白 )

19 The Total Luminex 200 Fluid Analyzer System for Fluorescent Microsphere Immunoassay (FMIA) 多功能流式荧光点阵仪 ---- 荧光微球免疫试验

20 Fluorescent Microsphere Immunoassay (FMIA) 荧光微球免疫试验 The Luminex xmap technology allows for the (theoretical) testing of up to 100 analytes in the same test well. 可对同一个孔内样品的 100 个指标同时检测 This ability to multiplex greatly reduces the time and cost of serological testing. 大大节省检测时间和成本

How it Works 工作原理 The Luminex System uses 5.6-6.

2 微米大小的聚苯乙烯微球或者磁性微球 Each bead set contains a unique concentration of dye identifiable by one laser 每个微球含有可被某种激光识别的特定浓度的染料 Beads can be coated with

21 How it Works 工作原理 The Luminex System uses micron polystyrene or magnetic microsphere beads 采用微米大小的聚苯乙烯微球或者磁性微球 Each bead set contains a unique concentration of dye identifiable by one laser 每个微球含有可被某种激光识别的特定浓度的染料 Beads can be coated with specific antigen targets that bind antibodies in the sample 微球上包被抗原来吸附样品中的特定抗体 Bound antibody detected by another dye and 2 nd laser 结合的抗体被另外一种染料和另一种激光检测到 Beads coated with PEDV NP 包被 PEDV NP 抗原的微球 Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. JVDI 25: , J. Christopher-Hennings et al.

Anti-antigen Antibodies 识别抗原的抗体 Addition of microspheres to antibodypositive serum 加入磁珠到阳性血清 Microsphere 磁珠 Laser 激光 Biotinylated Anti-swine antibody 生物素标记的抗猪抗体

22 Anti-antigen Antibodies 识别抗原的抗体 Addition of microspheres to antibodypositive serum 加入磁珠到阳性血清 Microsphere 磁珠 Laser 激光 Biotinylated Anti-swine antibody 生物素标记的抗猪抗体 Antigen 抗原 Reporter Addition of Biotinylated anti-swine antibody to serum 加生物素标记二抗 Addition of reporter (PE*) Streptavidin to serum 加荧光 PE 标记的链亲和素 Designed by Mike Dunn, SDSU

23 Seroconversion 7-10 days post inoculation 接种后 7-10 天血清转阳

24 Assay validation 试验验证 (establishment of cut-off levels, diagnostic sensitivity & specificity 临界值, 敏感性, 特异性的确定 )

25 Fluorescent Focus Neutralization Assay (FFN) 荧光噬斑中和分析 Measurement of neutralizing antibodies measures a biological function of the antibodies 检测中和抗体也就是检测抗体的一个生物功能

26 Neutralizing Antibodies: 中和抗体 One arm of the immune system 免疫系统的一支 Rhorer J et al. 2009; Vaccine 27:1101 Prevent binding to receptor 阻断与受体结合 Block uptake into cells 阻断进入细胞 Prevent uncoating of the genomes in endosomes 阻断在内含体释放基因组 Cause aggregation of virus particles 引发病毒颗粒聚集 Enveloped viruses---may be lysed when antiviral antibodies and serum complement disrupt membranes 抗体和补体联合作用裂解 ( 带囊膜 ) 病毒

27 PEDV Fluorescent Focus Neutralization (FFN) PEDV 荧光噬斑中和分析 well plate format 96 孔板 can be used with serum or rennet treated milk or colostrum 可用于血清, 凝乳酶处理的常乳或者初乳 2. Sample dilution series (2X) 倍比稀释 In MEM + 2µg/ml TPCK-treated trypsin (100µl/well) 3. Add indicator virus (eg. PEDv) 加指示病毒 ( 如 PEDv) (100µl/well at FFU/100µl) and incubate 1 h at 37C 共反应 1 小时 4. Transfer to washed confluent monolayers of Vero-76 cells 转移到 Vero-76 单层细胞 incubate 1 h at 37C 温育 1 小时 5. Wash and add replacement media with trypsin 洗盘, 加含胰蛋白酶的溶液 incubate h 温育小时

28 PEDV Fluorescent Focus Neutralization (FFN) 6. Acetone fix 丙酮固定 7. Stain with FITC-conjugated PEDV mab SD6-29 用 FITC 标记的单抗染色 8. Read by fluorescence microscopy 荧光显微镜观察 Endpoints are interpreted as the greatest serum/milk dilution showing a 90% reduction in fluorescent foci/well relative to negative control wells 终点解读为 90% 噬斑减少孔的最大的稀释度 (Ex.) If negative control wells show ~100 foci/well, the greatest sample dilution showing 10 or fewer foci/well is considered the endpoint. 例子 : 阴性对照有 100 个噬斑, 那么 10 个噬斑对应的稀释度就是终点 PEDV FFN results may not be identical in magnitude to CPE-based virus neutralization results obtained by other methods However, they should show relative correlation in most cases 细胞病变基础的中和试验不完全一致, 但是大多数情况下有可比性

29 FMIA tests targeting IgG, IgA or IgM antibodies to the NP or spike protein were compared to virus neutralization results 检测针对核蛋白和 S 蛋白抗体与病毒中和结果比较 (n=10, 6-week old pigs following PEDV challenge, serum provided by U of MN, Dr. Torremorell) IgM (NP) in serum: 血清中抗 NP 的 IgM peak at 7 DPI 感染后 7 天到高峰 IgG (NP) in serum: 血清中抗 NP 的 IgG + by 7-14 DPI 感染后 7-14 天检出 IgG, IgM, IgA (spike) in serum: 血清中抗 S 蛋白的 IgG, IgM, IgA low levels compared to Ab response to NP S 蛋白产生的抗体较 NP 为低 Serum neutralizing antibody (FFN): Days post inoculation + by 14 DPI & rising at 20 DPI 中和抗体 : 感染后 14 天出现, 上升到 20 天

30 Application of the FFN to the field FFN 在生产中的应用 Duration and level of PEDv neutralizing antibodies for clinical protection using feedback protocols 返饲后有临床保护力的 PEDV 中和抗体的持续时间和维持水平 1. Determine the level of neutralizing antibody in sows 母猪中和抗体的确定 Serum levels 血清水平 Colostrum/milk levels (intestinal immunity) 初乳 / 奶中水平 ( 肠道免疫力 ) 2. Duration of neutralizing antibody in sows 母猪中和抗体的持续时间 Funding through the National Pork Board, #13-263

Neutralizing Antibodies: 中和抗体 One arm of the immune system 免疫系统的一支 Prevent binding to receptor 阻断与受体结合 Block uptake into cells 阻断进入细胞 Prevent uncoating of the genomes in

31 Neutralizing Antibodies: 中和抗体 One arm of the immune system 免疫系统的一支 Prevent binding to receptor 阻断与受体结合 Block uptake into cells 阻断进入细胞 Prevent uncoating of the genomes in endosomes 阻断在内含体释放基因组 Cause aggregation of virus particles 引发病毒颗粒聚集 Enveloped viruses---may be lysed when antiviral antibodies and serum complement disrupt membranes 抗体和补体联合作用裂解 ( 带囊膜 ) 病毒

32 PEDV Outbreaks 流行性腹泻病毒的爆发 Site A: 4,000 head farrowing to wean sow farm 场 A: 4000 头分娩到断奶母猪场 Initial infection January 11, 2014 初次感染 2014 年 1 月 11 日 Initial feedback (gestation/farrowing) 初次返饲 ( 怀孕 / 分娩 )2014 年 1 月 11 日,12 日 January 11, 12, 2014 Site B: 4,304 breeding animals 场 B: 4304 头种猪 Initial infection- January 31, 2014 初次感染 2014 年 1 月 31 日 Initial feedback 初次返饲, 月 5 日 February 5,

33 Site A Feedback A 场反饲 Week 1: 第一周 Feedback all sows and gilts (4oz top-dressing 3X). 反饲所有母猪和后备 (3 倍稀释的上清毫升 ) Week 2: 第二周 Repeat 3 feedbacks to all animals (gestation, farrowing, gilts) 重复 3 次返饲 ( 怀孕, 哺乳, 后备 ) Weeks 3-6: 3-6 周 Feedback all gilts that are not yet bred 2X per week and the breeding/gestation/open pool of sows 1X per week. 未配种后备每周两次, 配种怀孕空怀母猪每周 1 次 Week 7: 第七周 If 90% of gilts and sows sick and pigs are not showing clinical signsdiscontinue feedback. 若 90% 后备和母猪发病, 仔猪不发病 - 停止返饲

34 Site B Feedback B 场反饲 Week 1: 第一周 February 5, 2014 首次返饲 2 月 5 日 All sows not showing clinical signs hours were fed again. 不发病母猪小时后再次返饲 Week 2: 第二周 2 nd feedback February 10, 2014 第二次返饲 2 月 10 日

35 PCR on Feedback material to check for PEDv 返饲材料中 PED 病毒的含量 Site A 场 A Concentrate Ct= 浓缩液 Ct= Feedback Mill Ct=22.31 返饲研磨液 Ct=22.31 Site B 场 B Feedback 1 Ct= 返饲 1 Ct= Feedback 2 Ct= 返饲 2 Ct= 17.97

36 Site A and B Feedback 场 A 和 B 的返饲步骤 1. Stop all traffic 停止所有运输 2. Perform herd closure 封闭猪群 3. Humanely euthanize poor piglets 安乐死病弱仔猪 -Spread diarrhea through farrowing units to infect pigs if needed. 必要时人为感染产房仔猪 4. Create feedback material 制备返饲材料 Garbage disposal, blender, or meat grinder with water 研磨打浆机 5. Feed every sow top-dress to their feed 逐头撒在母猪料上 4-8 oz 毫升 6. Mark all sows/ sow cards of animals that are showing signs of illness. 标记所有发病母猪

37 Clinical signs/status 临床症状 / 状态 3 weeks 3 周后 significant clinical signs, mortality 发病明显, 死亡 6-10 weeks 6-10 周后 Site A, PED in farrowing at 10 weeks 场 A 10 周发 PED Site B, clinical signs not cleaning up as well 场 B, 临床症状没有消失 PEDV PCR positives in piglets 仔猪 PEDVPCR 阳性 Checked sequence to confirm wasn t new introduction 序列分析排除新的感染 Checked PDCoV status by PCR-Negative 仔猪 PDCoV 阴性 6-7 months 6-7 个月后 Site A and B, sentinels tested negative, quarantine lifted 场 A 和 B, 哨兵猪阴性, 隔离解除

38 Site B-Variability in Clinical Signs 场 B 临床症状各异

39 1:20 FFN Titer in Sows 母猪中和抗体水平

40 FFN Titer Site B-4 groups of sows 场 B4 组母猪中和抗体水平

41 Site A-FFN Titers on milk & serum from individual sows 场 A 若干母猪血清和乳中中和抗体水平 1:1280 1:640 1:80 1:40 1:20 动物标示

42 FFN Titers- Site B Mean Colostrum Milk Samples 场 B 母猪血清初乳和常乳样品的中和抗体水平 1:640 1:320 1:80 1:40 n=38 n=34 n=29 血清初乳奶

43 Comparison of FFN titers of Site A and B: Serum Milk/Colostrum Samples A 场与场 B 母猪血清初乳和常乳样品的中和体水平的比较 >1:2560 1:1280 1:640 1:320 1:80 1:40 1:20

44 PEDV neutralizing antibodies in colostrum/milk and serum from field study using feedback + vaccine 生产试验中返饲 + 疫苗处理的母猪的初乳 / 常乳血清样品的中和抗体水平 1: Means (error bars: 95% CI for mean) 1: :6406 1:3205 1:1604 1:80 3 1:402 Colostrum wk 1 Milk_wk_2Sow_Serum_wk_2Milk_wk_3Sow_Serum_wk_3 Colostrum Serum Milk Serum Milk Serum Week 1 Week 2 Week 3 Feedback followed by Harrisvaccines (PEDv Vaccine, RNA) given 1 wk pre-farrowing (n=27) 返饲 + 产前 1 周免疫 Harris 腹泻疫苗 Samples courtesy of Dr. Rebecca Robbins, Seaboard Foods, USA

45 Site A-# of Piglets FFN Titer 90% (27/30) piglets with FFN titer by 9 wks PI 场 A 感染 9 周后的仔猪体内中和抗体水平分布图 (90% 阳性 )

46 Site B-# of Piglets FFN Titer 52% (15/29) piglets with FFN Titer by 10 wks. PI Piglets were shedding virus Ct range from 场 B 感染 10 周后的仔猪体内中和抗体水平分布图 (52% 阳性 )

47 Field Conclusions (Site A, B) 生产总结 (A B 场 ) FFN test after feedback 返饲后的中和抗体 Validated in a real world setting. 在真实的环境下验证 % positive FFN titers combined for Sites A and B 3 weeks 97% Sow serum samples were positive 3 周 -97% 母猪血清阳性 6 weeks 97% Sow Serum samples were positive 6 周 -97% 母猪血清阳性 6 months 100% Sow Serum samples were positive 6 个月 -100% 母猪血清阳性 Neutralizing antibodies were higher in colostrum than serum 初乳中的中和抗体高于血清 Neutralizing antibodies were similar between serum and milk (indicator of protection?) 常乳与血清的中和抗体接近 ( 意味着保护?)

48 Porcine Deltacoronavirus (PDCoV) Diagnostics 猪新冠状病毒的诊断

(FFN) assay 荧光噬斑中和试验 IFA with PDCoV monoclonal antibody 单抗免疫荧光试验 PDCoV-NP mab SD 55-24 Immunohistochemistry using SD 55-24 免疫组化

49 PDCoV diagnostic reagents 诊断试剂 Monoclonal antibodies (SDSU) and specific antisera 单抗与特异的抗血清 Used for detection of virus infected cells by fluorescent antibody staining 用荧光抗体染色来确定感染的细胞 Verification of virus isolation 病毒分离的确认 Fluorescent focus neutralization (FFN) assay 荧光噬斑中和试验 IFA with PDCoV monoclonal antibody 单抗免疫荧光试验 PDCoV-NP mab SD Immunohistochemistry using SD 免疫组化 Staining of antigen in infected tissues 感染组织抗原染色 Sample courtesy of Drs. Sarah Vitosh & Bruce Brodersen (University of Nebraska-Lincoln)

50 PDCoV serological assay performance 猪新冠状病毒血清学检测结果 125 field samples of known serostatus for PDCoV were used for initial test optimization to show relative difference between positive and negative samples. 用 125 个已知阳性状态的样品来对检测方法进行优化, 更好的揭示阳性与阴性样品的相对差别

51 Summary 小结 Real-time PCR is commercially available 实时 PCR 试剂盒已经上市 Serology testing is comparable 血清学检测方法可以相互比较 IFA, indirect ELISA, belisa, FMIA ELISA quicker, high throughput FMIA can be used for multiplexing for surveillance purposes for other diseases Full-genome sequencing now used for surveillance 全基因组测序被用来监测病原的演变 USDA NAHLN program Monoclonal antibodies are commercially available 单抗已上市 IHC, VI, titrations, FFN FFN may be a measure of protection FFN 也许作为衡量保护力的一种方法 transmission from serum to colostrum/milk for piglet intestinal protection? 从血清到初乳 / 奶传播, 保护仔猪肠道

52 Diagnostic Take away points 诊断. 关键点 Early identification of new diseases 新发疾病的早期确诊 Need for surveillance tests 亟需监测方法 What if clinical signs look similar to common disease? (eg. PEDv & other diarrheal diseases in finisher pigs?) 如果临床症状与一般疾病相似怎么办?( 育肥猪腹泻疾病与猪流行性腹泻病毒 ) Need for pan-family PCR assays for sequencing or next generation sequencing for unknowns 亟需对未知病原测序的泛家族 PCR 方法或者新一代的测序方法 For very quick identification of new diseases 新发疾病的快速诊断

53 Diagnostic Take away points 诊断关键点 Need for GenBank Accessions for designing new & updated assays 亟需基因库的序列来设计新的实验方法 Need for refereed papers describing clinical signs, pathogenesis, when virus may be found and in what tissues, sample types and at what level 亟需参考文献谈及临床症状, 致病机理, 病毒在组织的分布规律, 样品种类及其病毒含量等

54 Cooperating producers & practitioners SDSU Dr. Eric Nelson Dr. Steve Lawson Faten Okda Julie Nelson ADRDL & ARW staff KSU (sample sets) Dr. Dick Hesse & staff U of MN Acknowledgements 致谢 Dr. Montserrat Torremorell Dr. Michael Murtaugh NVSL (virus isolates) Dr. Sabrina Swenson Melinda Jenkins-Moore Funding sources NPB Grant NPB Grant NPB Grant South Dakota AES SD-ADRDL Commercial collaborators Tetracore Inc., Rockville, MD Biovet, St-Hyacinthe, QC, Canada Medgene, Brookings, SD

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<4D6963726F736F667420576F7264202D20B2CCA3BA4542B2A1B6BE52746120496747BFB9CCE5D3EBB1C7D1CAB0A9B7D6C6DAB5C4B9D8CFB52E646F63> EB 病毒 Rta/IgG 抗体与鼻咽癌分期的关系蔡永林, 郑裕明, 成积儒, 李军, 莫永坤, 钟青燕 ( 广西梧州市红十字会医院, 梧州市肿瘤防治研究所, 广西梧州 543002) 摘要 : 目的探讨 EB 病毒 Rta/IgG 抗体与鼻咽癌临床分期的关系方法用酶