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Biotechology Advances

Biotechology Advances. A perspective on diagnosis and research of Rabies Virus 生科系　宋帛軒　周儒筠. Introduction. g enus Lyssavirus , family Rhabdoviridae Neurotropic viruses Single-stranded, non-segmented negative-sense polarity RNA Five structural protiens are coded by the genome

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Biotechology Advances

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  1. Biotechology Advances A perspective on diagnosis and research of Rabies Virus 生科系　宋帛軒　周儒筠

  2. Introduction • genus Lyssavirus, family Rhabdoviridae • Neurotropic viruses • Single-stranded, non-segmented negative-sense polarity RNA • Five structural protiens are coded by the genome • Nucleoprotein (N) • Phosphoprotein (P) • Matrix protein (M) • Glycoprotein (G) • RNA-dependent RNA polymerase or large protein (L)

  3. Early Methods for Diagnosis • Direct fluorescent antibody test (DFAT) • Mouse inoculation test (MIT) • Both have high sensitivity and specificity. • Requires an expensive fluorescence microscope and time-consuming test, respectively. • Cannot detect the antigenic and genetic variability.

  4. Current Methods for Diagnosis-1 • Refined PCR techniques • Random amplification of polymorphic DNA (RAPD-PCR) • reverse transcription PCR (RT-PCR) • nested or hemi-nested PCR (hn-PCR) • PCR-enzyme-linked immunosorbent assay (ELISA) • multiplex PCR (mPCR) • real-time PCR

  5. Current Methods for Diagnosis-2 • Application of real-time RT-PCR • Quantification of viral RNA. • Discriminate the distinct strains of RABV by sequencing. • Important for epidemiological studies of human and/or animal rabies. • Involves amplification and detection occuring at the same time in a closed tube system.

  6. Current Methods for Diagnosis-3 • Real-time PCR assays • TaqManTM • SYBR Green

  7. Epideniological considerations • Viruses from a geographically restircted location have distinct nucleotide sequences in the nucleocapsid (N) genes. • Also can be used to identify the host range. • Designing universal oligonucleotide primers and probes for Lssyavirus RNA detection is problematic.

  8. Quantification of the RABV load • Ability of real-time quantitative RT-PCR • accurately quantify viral loads (amounts of viral RNA) • quantify the RABV loads in a wide variety of biological fluids and samples (e.g., saliva, cerebrospinal fuild, tears, skin biopsy sample, and urine)

  9. Antemortem diagnosis of rabies in human • Early and timely diagnosis during the acute phase of the disease can help in patient care and minimize the need for unnecessary treatments.

  10. Phylogeography of the Rabies Virus-1 • 主要在研究族群發育的生物地理學或一種族在時間和空間上的地理分布 • 可推斷出一個體過去的系統發育、未來擴散情形和其目前處於何種演化階段 • 同時研究其擴散情況及引發的相關疾病

  11. Phylogeography of the Rabies Virus-2 • MCMC method • BEAST package • 可用於推斷空間分布和演化狀況 • 結合流行病學監控

  12. Proteomics for Rabies Virus biomarker discovery-1 • 分析在不同環境條件下蛋白質在特定組織或細胞中的表現量及其型態 • 最常用的蛋白質分析方法是二維蛋白質電泳(2D-PAGE) • 病毒造成的胞內蛋白質改變可用蛋白質概況比較和辨別感染後病毒及細胞蛋白質在功能、結構及動態學上的改變而得知

  13. Proteomics for Rabies Virus biomarker discovery-2 • 病毒感染造成蛋白質在質和量上的改變 • 麻痺型和憤怒型的狂犬病 • 反相液相色譜法(RP-HPLC) • 二維螢光差異電泳(2D-DIGE)

  14. Monoclonal and recombinant anti-rabies antibodies for rabies diagnosis-1 • 1978AD,Wiktor and Koprowski-小鼠狂犬病毒單株抗體(mAbs) • mAbs-病毒nucleocapsid或 glycoprotein抗原辨識 • Jiang et al.-單株抗體的有效性 • 抗狂犬病毒nucleoprotein的單株抗體已被鑑別得知其為IgG2a的isotype • Direct rapid immunohistochemical test (dRIT) • Sandwich ELISA method(WELYSSA)

  15. Monoclonal and recombinant anti-rabies antibodies for rabies diagnosis-2 • Rapid immunodiagnostic assay (RIDA) • Type I & IItest • Yang et al.-double-antigen sandwich ELISA • Anti-rN and rPIgY • Recombinant single-chain Fv antibody fragments (scFv) • Diantibodies • Experiment of Nimmagadda et al.

  16. Conclusion-1 • 對公眾健康造成嚴重威脅的動物性疾病 • High cost of genomic methods • The immunological techniques currently recommended • Laboratory diagnosis in the first week of illness is rarely achieved • mAbs and recombinant antigens have been good alternatives

  17. Conclusion-2 • Phylogeographicresults, when used in conjunction with epidemiological surveillance, can predict the geographical movement of the agent and aid in the control of disease • Host molecular factors can be used as new biomarkers • These studies open new perspectives for molecular diagnosis and may help clinicians and their patients

  18. References-1 • Adams MJ, Carstens EB. Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses. Arch Virol 2012;157: 1411-22. • Altshuler EP, Serebryanaya DV, Katrukha AG. Generation of recombinant antibodies andmeans for increasing their affinity. Biochemistry 2010;75:1584-605. • Campos AC, Melo FL, Romano CM, Araujo DB, Cunha EM, Sacramento DR, et al. One-step protocol for amplification of near full-length cDNA of the rabies virus genome. J Virol Methods 2011;174:1-6. • Hipólito O. Raiva. In: Doenças dos Animais Transmissíveis ao Homem. Rio de Janeiro: Serviço de Informação Agrícola, Ministério da Agricultura Inc.; 1948.p. 31-7. • Mullis KB, Faloona F. Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol 1987;155:335-50.

  19. References-2 • Tordo N, Poch O, Ermine A, Keith G. Primary structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VSV. Nucleic Acids Res 1986;14:2671e83. • WacharapluesadeeS, Hemachudha T. Ante- and post-mortem diagnosis of rabies using nucleic acid-amplification tests. Expert Rev MolDiagn 2010;10: 207-18. • WiktorTJ, Koprowski H. Monoclonal antibodies against rabies virus produced by somatic cell hybridization: detection of antigenic variants. ProcNatlAcadSci 1978;75:3938-42. • WiktorTJ, Flamand A, Koprowski H. Use of monoclonal antibodies in diagnosis of rabies virus infection and differentiation of rabies and rabies-related viruses. J Virol Methods 1980;1:33-46. • WunnerHW. Rabies virus. In: Jackson AC, Wunner HW, editors. Rabies. San Diego: Academic Press Inc.; 2007. p. 23-68.

  20. Thanks for your listening

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