1、Techniques in Virology,This slide is download on http:/ (中国病毒学自由学术论坛)Shared by Victor (wwwkkk83),Original slides (PPT files) are download on the internet. Thanks for authors of these slides.,1,viruses are usually detected by indirect methods,(i) multiplication in a suitable culture system and detect
2、ion of the virus by the effects it causes; (ii) serology, which makes use of the interaction between a virus and antibody directed specifically against it; (iii) detection of viral nucleic acid.,2,Sampling Injection in animal models (diseases), or eggs Virus culture in mosquito or mammalian cells De
3、tection of virus replication Cytopathic effect (syncitia, inclusion bodies, cell rounding, apoptosis, microarrays) Hemadsorption Antigen detection (immunocapture, Dot-blot, Western blot, Immunofluorescence, Immunohistochemistry, ELISA Electron microscopy Genome detection (Electrophoresis, in situ or
4、 membrane Hybridization, RT-PCR, multiplex or real time PCR, NASBA, Hybrid RNA-DNA, microarrays, Sequencing,substraction sequencing, random amplification-cloning-high throughput sequencing-blast) Reporter gene in genetically engineered cell lines,3,Specimens:Blood: serum, plasma, PBMC Swabs: Trachea
5、l, nasopharyngeal, cutaneous Fluids: CSF, urine, bronchoalveolar lavage, tracheal aspirate, tears Tissues: Organs, skin,4,Sampling Injection in animal models (diseases), or eggs Virus culture in mosquito or mammalian cells Detection of virus replication Cytopathic effect (syncitia, inclusion bodies,
6、 cell rounding, apoptosis, microarrays) Hemadsorption Antigen detection (immunocapture, Dot-blot, Western blot, Immunofluorescence, Immunohistochemistry, ELISA Electron microscopy Genome detection (Electrophoresis, in situ or membrane Hybridization, RT-PCR, multiplex or real time PCR, NASBA, Hybrid
7、RNA-DNA, microarrays, Sequencing,substraction sequencing, random amplification-cloning-high throughput sequencing-blast) Reporter gene in genetically engineered cell lines,5,tracheal organ cultures,Sections through tracheal organ cultures: (a) uninfected;(b) infected with a rhinovirus for 36 hours.
8、Note the disorganization of the ciliated cells (uppermost layer) after infection. (Courtesy of Bertil Hoorn.),8,Growing viruses in the lab,Normal cells,Virus infected cells,Embryonated egg inoculation,9,Cell culture,10,Animal virus plaques in a cell culture,Plaques formed by porcine reproductive and
9、 respiratory syndrome virus in a cell culture From Lee and Yoo (2005) Journal of General Virology, 86, 3091.,11,Phage plaques in a lawn of bacterial cells,Plaques formed by phage MS2 in Escherichia coli cells Courtesy of Kathryn Newton.,12,Plaques formed by a phage in a bacterial lawn. The control p
10、late on the left was inoculated with only the bacterial host. The plate on the right was inoculated with phage and bacterial host.,13,14,Quantification of viruses,Plaque assay technique for quantification of bacterial viruses.,Plaques formed by West Nile virus in a cell monolayer,Both flasks were in
11、oculated with virus. Plaque formation in the flask on the right has been inhibited by West Nile virus-specific antibody. Courtesy of Dr. Elieen Ostlund, National Veterinary Services Laboratories, US Department of Agriculture.,A cell monolayer is inoculated with virus and overlaid with agarose.,15,ht
12、tp:/,Plaques of viruses,(a) Plaques of a bacteriophage on a lawn of Escherichia coli.,(c) Plaques of influenza virus on a monolayer culture of chick embryo fibroblast cells.,(b) Local lesions on a leaf of Nicotiana caused by tobacco mosaic virus,16,17,Quantification of animal viruses,18,Efficiency o
13、f Plating,Plaque assay has low efficiency: With bacteriophages: 50% With animal virues: 0.1-1%Therefore, to quantify virus, it is accurate to express the concentration (called titer) of the virus suspension not as the absolute number of virion units, but as the number of plaque-forming units.,19,Pri
14、nciples of virus infection,Multiplicity of infection (defective particles) Time and temperature of INCUBATION Subpassages Cytopathic effect (round cells, lysis, fusion, inclusion bodies in nucleus or cytoplasm) pH of culture medium Virus recovery Virus congelation Virus titration,plaque assay,Serial
15、 10-fold dilutions of HSV to determine the titer of virus in a stock solution.,20,A plaque reduction neutralization test showing the ability of a test antibody to reduce the infectivity of the virus (for convenience of illustration, 10 PFU are shown, although a larger inoculum is usually employed).
16、The 50% endpoint is calculated as the dilution of antibody that would reduce the plaque count from the control level of 10 to 5 PFU. In this example the endpoint is 1/3750.,plaque reduction neutralization test,21,Outcomes of virus infection of a host,22,23,Sites of virus morphogenesis,Nucleus Cytopl
17、asm RER Golgi Membrane,Incorporation of nucleic acid material Post-assembly modifications and virus release Facilitation of release and prevention to reinfection (downregulation of receptors),Cytopathic effects caused by replication of poliovirus and herpes simplex virus in cultures of Vero (monkey
18、kidney) cells. (a) Uninfected Vero cells. (b) Infected with poliovirus. (c), (d) Infected with herpes simplex virus. (a), (b) and (d) were viewed at 400 magnification; (c) was viewed at 100 magnification. The cells were stained with haematoxylin and eosin.,24,Herpes virus CMV RSV,Cytopathic effect o
19、n cell lines,25,Purkinje cell - Negri bodies,Jackson et al. ( NEJM 1996),26,Fusion activity of Nipah virus in Vero cells,27,YFV 17D induces apoptosis in hepatoma HepG2 cells 2 days post-infection,YFV 17D induces apoptotis,Lefeuvre et al., 2006,28,Hippocampus (CA1) Cerebral Cortex,Rabies virus in mic
20、e,29,30,CPE caused by an influenza A virus and human respiratory syncytial virus (HRSV) in confluent cell monolayers,(a),(b),31,HSV-induced changes in the properties of actin microfi laments of a cultured monkey fi broblast,Uninfected cells,HSV infected cells,The cell was stained with a fluorescent
21、dye that reacts with actin fibers so that they can be visualized in ultraviolet light.,The left panel shows parallel arrangement of the microfibrils in the uninfected cell, while HSV infection (right panel) results in disassociation of the fibrils and diffusion of the actin throughout the cytoplasm.
22、 At the same time, the cell loses its spindle-shaped morphology and becomes rounded. The arrows indicate junctions between cells that are also rich in actin fibrils and are not disrupted by HSV infection at this time,32,常用的细胞培养,33,细胞病变(Cytopathic effect, CPE) 包涵体（Inclusion Bodies）,34,检测病毒,正常细胞,病变细胞,
23、http:/,狂犬病毒包涵体（Negri body）,35,巨细胞病毒包涵体,36,one-step growth curve of bacteriophage,During the eclipse phase (1), the infectivity of the cell-associated, infecting virus is lost as it uncoats; during the maturation phase (2) infectious virus is assembled inside cells (cell-associated virus), but not ye
24、t released; and the latent phase (3) measures the period before infectious virus is released from cells into the medium. Total virus is the sum of cell-associated virus + released virus. Cell-associated virus decreases as cells are lysed. This classic experiment shows that phages develop intracellul
25、arly.,37,38,39,http:/,Example of a TCID50 assay,40,41,Aliquots of the indicated stock virus dilutions were pipetted into the cultures and the plate was incubated for 48 hours and then developed with a stain that indicates black for virus-infected cells. Any well that received at least 1 PFU of virus
26、 stained black (two separate experiments are shown). The percentage of positive (infected) wells is shown at each dilution.,Quantal (endpoint dilution) assay of HSV in tissue culture wells.,42,In the graph, one can estimate that a dilution at which 50% of the wells would be infected is about 4 103;
27、therefore, the TCID50 was 4 103 in the original sample. More accurate measures of the ID50 of a virus stock can be obtained by using statistical methods such as the method of Reed and Muench, which is described in a variety of basic statistical texts.Although ID50 is a measure of dilution, an ID50 u
28、nit is directly related to PFU; 1 ID50 unit measures a dilution required to ensure that 50% of the aliquots in that dilution have infectious virus in them. This will only occur if there are 0.7 PFU (average) per aliquot, or 7 PFUs in 10 ml in the above example.,43,44,45,病毒数量和感染性测定,1、蚀斑试验,2、ID50 或 TC
29、ID50,病毒感染鸡胚、动物或细胞后，引起 50% 发生死亡或病变的最小量。,病毒感染单层细胞后，产生的局限性病灶，称蚀斑，蚀斑是由一个感染性病毒体复制形成的，称蚀斑形成单位，病毒悬液中的感染性病毒量以每毫升的蚀斑形成单位PFU来表示。,46,neutralization test,Virus A loses its infectivity after combining with A-specific antibody (it is neutralized). A-specific antibody does not bind to virus B, so infectivity of vi
30、rus B is unaffected. The complete test requires the reciprocal reactions.,47,Hemagglutination titration,Here an influenza virus is serially diluted from left to right in wells in a plastic plate. Red blood cells (RBCs) are then added to 0.5% v/v and mixed with each dilution of virus. Where there is
31、little or no virus, RBCs settle to a button (from 1/128) indistinguishable from RBCs to which no virus was added (row 3). Where sufficient virus is present (up to 1/64), cells agglutinate and settle in a diffuse pattern. (Photograph by Andy Carver.),48,Assay of infl uenza virus by hemagglutination,4
32、9,hemagglutinationinhibition test,In the hemagglutinationinhibition test, antibody is diluted from left to right. Four hemagglutination units (HAUs) of an influenza virus are added to each well. The antibodyvirus reaction goes to completion in 1 hour at 20C. Red blood cells are then added to detect
33、virus that has not bound antibody. In this test, hemagglutination is inhibited up to an antibody dilution of 1/3200. (Photograph by Andy Carver.),50,Fluorescent antibody staining,An antibody covalently bound to a fluorescent dye has been used to detect an antigen present mainly in the nucleus (arrow
34、s) of influenza virus-infected cells.,51,(A) Nonvirus infected, methanol-fixed whole MRC-5 cells immunofluorescence- labeled for tubulin fibers. Original magnification 25. (B) Permeabilized, HSV-1-infected whole MRC-5 cells immunofluorescence-labeled for gC situated in cytoplasmic vesicles, in a Gol
35、gi-like region (arrow) close to the nucleus and in cellular adhesion areas (arrowhead). Original magnification 25.,52,Identification of an unknown virus by ELISA with a specific antiserum.,The unknown virus would be used in parallel with a control virus preparation that is known to react with the an
36、tibody. The unknown virus is positively identified when the antibody reaction is identical to that using the control virus.,53,54,http:/,55,中国病毒学自由学术论坛,Detection of human papillomavirus (HPV)16 E6/E7 mRNAs by RT-PCR. Atypical squamous cells of undetermined significance (lane 1), cervical cancers (la
37、nes 2 and 5), high-grade squamous intraepithelial lesion (lane 3), low-grade squamous intraepithelial lesion (lane 4), and normal cervical tissue (lane 6).,56,Detection of virus replication in cell lines,57,Antigen detection tests:,Type A reactive Enzyme Immunoassay kits A only (DirectigenFluA) A+B
38、(does not differentiate) (e.g. QuickVue Influenza test) Differentiates A from B (DirectigenFlu A+B; QuickVue Influenza A+B etc.),58,Immune detection of soluble NS1 protein,59,DNA Microarray Technique for Detection and Identification of Viruses,Principle of the microarray assay for virus detection an
39、d identification.,60,61,Hantavirus microarray constructed with 500-nucleotide fragment probes . Closely related strains of Puumala virus could be identified and distinguished.,62,The response of peripheral blood mononuclear cells in monkeys infected with variola virus. Using microarray to identify t
40、he expression of a large number of host genes that participate in the innate immune response to this acute infection.,Microarrays to identify genes that are up- or down-regulated and the application of bioinformatics to identify patterns of gene expression,63,Electron microscopy,Rabies virus,Paramyx
41、ovirus,64,Electron micrograph of the rotavirus.,65,66,Transmission electron micrograph of urine sediment from the same patient as in Figure 4-73, showing intranuclear icosahedral viral capsids. (A, 15,500; B, 39,000). (Electron photomicrographs courtesy of Julie Wen.),中国病毒学自由学术论坛,# of deaths,pfu/ani
42、mal (i.p.),LD50=102,32,Days,Infection of golden hamsters with Nipah virus,67,How to prevent carryover contamination in RT-PCR (false positive)?Use of dUTP and Uracil-N-glycosylase Use of fuocoumarin (psoralens) and UVTip with filters, plugged tips Cleaning of the micropipettes UV irradiation after w
43、ork Use small working aliquots Cleaning of equipment and laboratory surfaces with 10% bleach Physical separation of the laboratories1. Treatment of the specimen (NA extraction)2. Preparation of the reaction tubes 3. RT-PCR reaction4. Tube opening and detection of amplification products Change lab co
44、at (overshoes) in the detection lab.,68,Transcription assay Specific PCR Multiplex PCR Quantitative (competitive) PCR Real-time PCR- Fluorogenic 5 exonuclease assay (Taqman): the probe contains a fluorescent dye at its 5 (reporter), and 3 (quencher) extremities.- Hybridization probe assay: fluoresce
45、nce resonance energy transfer using two oligos labeled at 5 (accepter) or 3 (donor) ends- SYBR green assay (discrimination based on melting point)Use of multiple fluorescent dyes (Cy3, Cy5, Texas red),69,Detection of amplified productsAgarose gel and Bet Agarose gel and hybridization using labeled p
46、robes 5-labeled primer (P32, Biotine ) Branched chain DNA assay Hybrid capture assay,70,One-tube multiplex PCR,71,Branched chain DNA assay,72,Hybrid captureassay,73,74,75,Identification of new viruses,Representational difference analysis (RDA)- Substractive hybridation by difference enrichment and i
47、dentification of the sequences from the genomes of unknown virus (N.A. Lisitsyn, TIG, 1995)Systematic screening- Treatment of the sample with DNase I- Genome extraction- Random priming with taged exanucleotide- Klenow digestion- Amplification Taq po- EcoRV digestion- Cloning- High throughput sequenc
48、ing,76,Serology Detection of antibodies ELISA (colorimetric, radiometric, fluorescent). IgM, IgG, IgA Indirect histology (immunofluorescence or immunohistochemistry) Hemagglutination inhibition Complement fixation Immunochromatography Neutralizing antibodies (PRNT, DCID50, use of pp) Western blot,DIAGNOSTIC (cont.),Cell activation profileTranscriptome or proteome of infected cells,77,Viral isolation. On Vero E6, inoculation with biological samples. Method of reference, but tedious (when immunodetection), and requires a BSL-4 containment,
