Indirect Examination

Transcript Indirect Examination

IN THE NAME OF GOD

Department of Microbiology, Islamic Azad University, Falavarjan Branch

Advanced Virology

Detection Methods in Virology

By: Keivan Beheshti Maal

Studying Animal Viruses

 studying animal viruses is more expensive and more time consuming than the study of bacterial viruses  since viruses multiply inside a living cell, that is where they must be studied  an animal cell cycle is not nearly as rapid as that of a bacterial cell  acquiring enough viruses to study is the challenge to studying animal viruses 2

Studying Animal Viruses

 Some viruses can only be grown in living animals  There is an ethical issue regarding purposely infecting an animal with a virus just for study  Other viruses can be cultivated in tissue culture  This technique involves small pieces of tissue being removed from the animal and cultured in the lab as a growth media for the virus 3

Diagnostic Methods in Virology

1. Direct Examination 2. Indirect Examination (Virus Isolation) 3. Serology

1 2 3 4

Direct Examination

Antigen Detection Electron Microscopy 1) Immunofluorescence 2) ELISA 3) RIA 4) Immunodiffusion 1) Morphology of virus particles 2) Immune Electron Microscopy Light Microscopy Viral Genome Detection 1) Histological Appearance 2) Inclusion Bodies 1) Hybridization with specific nucleic acid probes 2) Polymerase Chain Reaction (PCR)

Indirect Examination

Cell Culture

1) Cytopathic Effect (CPE) 2) Haemabsorption 3) Immunofluorescence 1) Pocks on CAM 2 Eggs 3

Animals

1) Disease or Death 2) Inclusion Bodies

Three main principles:

Direct detection of: Isolation on: Serology using: Virus particles Tissue culture Viral antigen Chicken embryo Viral nucleic acid Laboratory animals Cytopathology Baterial Culture

IF HI NT ELISA 7

What does an antibody look like?

-Immunoglobulin domains -Complementarity-determining Regions (CDRs) -Hinge -Fc= Fragment crystalline -Fab= Fragment antigen binding -F(ab)’2= Protease digestion still useful to bind antigen 8

Polyclonal Antibodies

Monoclonal antibodies

FLUORESCENCE MICROSCOPY

IMMUNOFLUORESCENCE

Immunofluorescence



Immunofluorescence

with fluorescent dyes.

is the labeling of antibodies or antigens  This technique is sometimes used to make viral plaques more readily visible to the human eye.

 Immunofluorescent labeled tissue sections are studied using a fluorescence microscope.

 Fluorescein is a dye which emits greenish fluorescence under UV light. It can be tagged to immunoglobulin molecules.

Immunofluorescence

 There are two ways of doing IF staining   Direct immunofluorescence Indirect immunofluorescence   

1. Direct immunofluorescence

 Ag is fixed on the slide  Fluorescein labeled Ab’s are layered over it Slide is washed to remove unattached Ab’s Examined under UV light in an fluorescent microscope The site where the Ab attaches to its specific Ag will show apple green fluorescence 

Use: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples

Direct immunofluorescence



2. Indirect Immunofluorescence:

Indirect test is a double-layer technique  The unlabelled antibody is applied directly to the tissue substrate  Treated with a fluorochrome-conjugated anti immunoglobulin serum 16



Advantage indirect IF over direct IF

 Because several fluorescent anti-immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test.

 It is also more time-efficient since it is only one signal labelled reagent, the anti-immunoglobulin, is prepared during the lengthy conjugation process 17

Immunofluorescence

IF staining of rabies in infected brain cells

ELISA

Enzyme-Linked Immunosorbent Assay

What is ELISA?

 Biochemical technique used mainly in immunology.

 First and most basic test to determine if an individual is positive for a selected pathogen, such as HIV.

 8 x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter.

Types of ELISA



Qualitative ELISA

 Postive or Negative results 

Quantitative ELISA

 optical density or fluorescent units of the sample is interpolated into a standard curve, which is typically a serial dilution of the target.

Applications of ELISA

       

Serum Antibody Concentrations Detecting potential food allergens

(milk, peanuts, walnuts, almonds and eggs)

Disease outbreaks- tracking the spread of disease

e.g. HIV, bird flu, common colds, cholera,

Detections of antigens

e.g. pregnancy hormones, drug allergen, mad cow disease  

Detection of antibodies in blood sample for past exposure to disease

e.g. HIV, bird flu 24

Basic principles of ELISA

 The Ab fixed to a solid surface, tube such as the inner surface of a test  A preparation of the same Ab coupled to an enzyme.

 e.g.

β-galactosidase

that produces substrate.

a colored product from a colorless 25

Types of immunodetection systems

1. Direct immunodetection Primary antibody conjugated with enzyme system 2. Indirect immunodetection Secondary antibody conjugated with enzyme system HRP HRP HRP HRP Ag Ag 3. Sandwich indirect immunodetection Antigen applied in soluble form HRP HRP HRP Ag Ag HRP Ag Ag HRP 4. Indirect immunodetection with biotin linkers Biotinylated primary antibodies HRP HRP HRP HRP

Streptavidin

Ag Ag

Simple ELISA protocol (For screening monoclonal antibodies) 1. Coat antigen onto microplate 2. Allow protein adsorption and block unoccupied sites with neutral protein 3. Add antibody solution (hybridoma supernatant) into each well 4. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate 5. Read absorbance in spectrophotometer with appropriate filter and quantitate relative antigen levels

Sandwich ELISA protocol

1. Coat primary antibody onto microplate 1a. Allow antibody adsorption and block unoccupied sites with neutral protein (BSA) 2. Add antigen to be detected in biological (clinical) material into each well 3. Add second primary antibody against antigen into each well 4. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate 5. Read absorbance in ELISA spectrophotometer with appropriate filter and quantitate relative antigen levels

Types of today’s immunodetection systems

Streptavidin

Ag Ag

Radioimmunoassay (RIA)

 Separation of a protein using the specificity of

antibody antigen

binding and quantification using

radioactivity

 1960: Berson and Yalow as an assay for the concentration of insulin in plasma  First time that hormone levels in the blood could be detected by an in vitro assay.

RIA Technique

 A mixture is prepared of  radioactive antigen  Iodine atoms can be introduced into tyrosine residues in a protein,

the radioactive isotopes 125 I or 131 I are often used

 antibodies against that antigen.

 Known amounts of unlabeled ("cold") antigen are added to samples of the mixture. [These compete for the binding sites of Abs] 31

RIA Technique

 At increasing concentrations of unlabeled antigen, an increasing amount of radioactive antigen is displaced from the antibody molecules.

  The antibody-bound antigen is separated from the free antigen in the supernatant fluid, and the radioactivity of each is measured.

Gamma Counter

Serology

Detection of rising titres of antibody between acute and convalescent stages of infection, or the detection of IgM in primary infection.

Classical Techniques Newer Techniques

Complement fixation tests (CFT) 2.

Haemagglutination inhibition tests 3.

Immunofluorescence techniques (IF) 4.

Neutralization tests 5.

Counter-immunoelectrophoresis 1.

Radioimmunoassay (RIA) 2.

Enzyme linked immunosorbent assay (EIA) 3.

Particle agglutination 4.

Western Blot (WB) 5.

RIBA, Line immunoassay 35

Virus Isolation

Cell Cultures are most widely used for virus isolation, there are 3 types of cell cultures: 1. Primary cells - Monkey Kidney 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK Primary cell culture are widely acknowledged as the best cell culture systems available since they support the widest range of viruses. However, they are very expensive and it is often difficult to obtain a reliable supply.

Continuous cells are the most easy to handle but the range of viruses supported is often limited.

Cell Cultures

Growing virus may produce 1. Cytopathic Effect (CPE) - such as the ballooning of cells or syncytia formation, may be specific or non-specific.

2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells.

Confirmation of the identity of the virus may be carried out using neutralization, haemadsorption-inhibition or immunofluorescence tests.

Resource Samples for Phage Isolation

 Wastewater before treatment  Lake water  Ocean water  River water  Soil, Animal and Plant samples 38

Isolation and enrichment of bacteriophage

 Sampling of water and wastewater resources  Centrifuge in 8000g for 10 minutes  Filtration of supernatant using 0.22

filters μ m syringe membrane  Addition of 10 ml of filtrates to 40 ml M-17 Broth and culture of 100 μ l active overnight

E. faecalis

 Incubation at 37oC with 120 RPM aeration speed 39

Incubation of Oral Streptococci with Isolated Bacteriophages

Phage Plaques of

S. mutans

Phage Plaques of

S. sobrinus

Spot Titration of Phage

 Gathering of 10 ml of enriched M-17 Broth media  Centrifuge at 8000g for 10 minutes  Filtration of supernatant using 0.22 μ m syringe filter  Making dilution of filtered solution using λ -Dil solution from 10 -1 to 10 -10  Culturing of

E.faecalis

ALH533 on M-17 Agar media using overlay method (top agar with 0.5% agar)  Spotting the diluted phage suspension on top agar  Incubation of M-17 Agar at 37oC overnight 43

Full Plate Titration of Phage

E.faecalis

ALH533-diluted phage (100 μ l + 100 μ l) on M-17 Agar media using overlay method (top agar with 0.5% agar)  Incubation at 37oC overnight 44

Spot Titration of

E. faecalis

Phage EFCPT-1

Spot Titer (PFU/ml) = Spot plaques X 1/Spot Volume X 1/Dilution

1.9 x 10 12 = 19 x 10 2 x 10 9

Whole Plate Titration of

E. faecalis

Phage EFCPT-1

Whole Plate Titer (PFU/ml) = Plate plaques X 1/Phage Volume X 1/Dilution

2.6 x 10 11 = 260 x 10 x 10 8

Purification of Phage

 Isolation of a well isolated bacteriophage plaque from cultures using a sterile Pasteur pippet  Transferring the plaque to an eppendorf containing 1 ml λ -Dil buffer and incubation at 4oC for 2 h  Centrifuge at 8000g for 10 minutes  Addition of 100 μ l of supernatant to 900 μ l of λ -Dil buffer and making dilutions up to 10 -10  Repeat of culture as previously described using overlay method 47



Preparation of Phage for DNA Extraction

Making Plate lysates using phage with 10 -1 dilution  Mixing 100 μ l of phage with 100 μ l

E. faecalis

melted M-17 top agar and transferring to a  Making 5 M-17 Agar with the same method  Incubation at 37oC overnight  Crashing the top soft agar of all 5 M-17 Agar plates and washing with 8 ml λ -Dil buffer for each plate  Transferring of crashed top agar (40 ml) to sterile falcon  Centrifuge at 8000g for 10 minutes and filter sterilization using 0.22 μ m syringe filter 48

DNA Extraction of phage, Restriction Map and Sequencing

 DNA extraction using Promega Wizard Phage DNA cleanup kit  Examination of DNA concentration using UV spectrophotometer at 260 nm  Digestion of phage DNA using 5 R.E, Incubation at 37oC for 16 h  Sending 10 μ l of extracted DNA of

E. faecalis

bacteriophage to University of Duke, North Carolina 49

Restriction Map of

E. Faecalis

Phage DNA

TEM of bacteriophage

 Use of the same phage purified suspension used for DNA Extraction with phage titer of 2.6 x 10 11 PFU/ml  Make a 50% dilution of phage suspension using λ -Dil buffer  Negative staining with uranyl acetate and observation of grids in TEM (JEOL-JEM-1200EX, USA) 51

TEM of

E. faecalis

Phage: EFCPT-1

Enterococcus faecalis

phage, EFCPT-1, a Siphovirus, with a hexagonal head of 57.14-71.42 nm and a flexible noncontractile tail with 200 242.85 nm in length 52

Complete DNA Sequencing of EFCPT-1 Genome

          Analysis of DNA using ARTEMIS version 14.0.0 Sequencing of 4 contigs with 8912 bp, 1909 bp, 1786 bp and 6670 bp EFCPT-1 MW: 19276 bp Circular dsDNA GC%: 35.75% A: 29.79% C: 19.32% G: 16.41% T: 34.45% 30 ORF and 30 genes 

Coding for 12 proteins of phage assembly, 12 hypothtical conserved proteins and 5 hypothetical novel proteins

Restriction Map of EFCPT-1 DNA

1, 8: DNA control 2, 9: AvaI 3, 10: AvrII 4, 11: ClaI 5, 12: NcoI 6, 13: SacI 7 &14: ladder 10kb 54

Gene Map of EFCPT-1 DNA and ORFs

EFCPT-1 Genomic DNA BLAST

EFCPT-1 Proteins with Conserved Domains

BLASTP of 1st EFCPT-1 Protein with Conserved domain     First protein from gene 1, N-terminal domain of tail protein, with 94 a.a, MW: 10707 like COG4722 super family in phage related protein, pfam 055709 family of phage tail protein consisting of several

Siphovirus

aphi3626-gp14-N, as N-terminal domain of putative phage tail component proteins 58

Phylogenetic Distance Tree of 1th conserved Protein 59

BLASTP of 2nd EFCPT-1 Protein with Conserved domain 60

Phylogenetic Distance Tree of 2nd conserved Protein 61

BLASTP of 3rd EFCPT-1 Protein with Conserved domain     3rd protein, gene 4, major tail protein, 188 a.a MW: 19710 phi13 family of phage major tail protein pfam04630 family of phage major tail protein cL12297 super families of phage major tail proteins 62

Phylogenetic Distance Tree of 3rd conserved Protein 63

Cytopathic Effect (1)

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells . (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town) 64

Cytopathic Effect (2)

Syncytium formation in cell culture caused by RSV (top), and measles virus (bottom).

(courtesy of Linda Stannard, University of Cape Town, S.A.) 65

Haemadsorption

Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.) 66

Problems with cell culture

 Long period (up to 4 weeks) required for result.

 Often very poor sensitivity, sensitivity depends on a large extent on the condition of the specimen.

 Susceptible to bacterial contamination.

 Susceptible to toxic substances which may be present in the specimen.

 Many viruses will not grow in cell culture e.g. Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

Rapid Culture Techniques

Rapid culture techniques are available whereby viral antigens are detected 2 to 4 days after inoculation. The CMV DEAFF test is the best example, whereby  The cell sheet is grown on individual cover slips in a plastic bottle.

 Following inoculation, the bottle then is spun at a low speed for one hour (to speed up the adsorption of the virus) and then incubated for 2 to 4 days.

 The cover slip is then taken out and examined for the presence of CMV early antigens by immunofluorescence.

DEAFF test for CMV

(Virology Laboratory, Yale-New Haven Hospital) 69

Viruses Isolated by Cell Culture

Viruses readily isolated by cell culture

Herpes Simplex Cytomegalovirus Adenoviruses Polioviruses Coxsackie B viruses Echoviruses Influenza Parainfluenza Mumps Respiratory Syncytial Virus

Less frequently isolated viruses

Varicella-Zoster Measles Rubella Rhinoviruses Coxsackie A viruses 70

Electron Microscopy

10 6 virus particles per ml required for visualization,  50,000 - 60,000 magnification normally used. Viruses may be detected in the following specimens.

Faeces

Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus

Vesicle Fluid Skin scrapings

HSV VZV papillomavirus, orf molluscum contagiosum 71

Electronmicrographs

Adenovirus Rotavirus (courtesy of Linda Stannard, University of Cape Town, S.A.) 72

Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy. There are two variants: Classical Immune electron microscopy (IEM) - the sample is treated with specific anti-sera before being put up for EM.

Viral particles present will be agglutinated and thus congregate together by the antibody.

Solid phase immune electron microscopy (SPIEM) - the grid is coated with specific anti-sera. Virus particles present in the sample will be absorbed onto the grid by the antibody.

Problems with Electron Microscopy



Expensive equipment



Expensive maintenance



Require experienced observer



Sensitivity often low

Serology

Criteria for diagnosing Primary Infection     4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera Presence of IgM Seroconversion A single high titre of IgG (or total antibody) - very unreliable Criteria for diagnosing Reinfection   fold or more increase in titre of IgG or total antibody between acute and convalescent sera Absence or slight increase in IgM 75

Typical Serological Profile After Acute Infection

Note that during reinfection, IgM may be absent or present at a low level transiently 76

Complement Fixation Test

Complement Fixation Test in Microtiter Plate.

Rows 1 and 2 exhibit complement fixation obtained with acute and convalescent phase serum specimens, respectively. (2-fold serum dilutions were used) The observed 4-fold increase is significant and indicates recent infection.

ELISA for HIV antibody

Microplate ELISA for HIV antibody: coloured wells indicate reactivity 78

Western Blot

HIV-1 Western Blot     Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate  Sample C: Positive 79

Usefulness of Serological Results

    How useful a serological result is depends on the individual virus.

For example, for viruses such as rubella and hepatitis A, the onset of clinical symptoms coincide with the development of antibodies. The detection of IgM or rising titres of IgG in the serum of the patient would indicate active disease.

However, many viruses often produce clinical disease before the appearance of antibodies such as respiratory and diarrhoeal viruses. So in this case, any serological diagnosis would be retrospective and therefore will not be that useful.

There are also viruses which produce clinical disease months or years after seroconversion e.g. HIV and rabies. In the case of these viruses, the mere presence of antibody is sufficient to make a definitive diagnosis.

Problems with Serology

    Long period of time required for diagnosis for paired acute and convalescent sera.

Mild local infections such as HSV genitalis may not produce a detectable humoral immune response.

Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV, Japanese B encephalitis and Dengue, may lead to false positive results.

immunocompromised patients often give a reduced or absent humoral immune response.

  Patients with infectious mononucleosis and those with connective tissue diseases such as SLE may react non-specifically giving a false positive result.

Patients given blood or blood products may give a false positive result due to the transfer of antibody.

CSF antibodies

 Used mainly for the diagnosis of herpes simplex and VZV encephalitis  CSF normally contain little or no antibodies  presence of antibodies suggest meningitis or meningoencephalitis CSF antibody titre Serum antibody titre > _1_ is indicative of meningitis 100  Diagnosis depends on the presence of an intact blood-brain barrier 82

Rapid Diagnosis Based on the Detection of Viral Antigens

Nasopharyngeal Aspirate Faeces Skin Blood RSV Influenza A and B Parainfluenza Adenovirus Rotaviruses Adenoviruses Astrovirus HSV VZV CMV (pp65 antigenaemia test) 83

Immunofluorescense

(Virology Laboratory, Yale-New Haven Hospital) Positive immunofluorescence test for rabies virus antigen. (Source: CDC) 84

CMV pp65 antigenaemia test

(Virology Laboratory, Yale-New Haven Hospital) 85

Advantages and Disadvantages

Advantages  Result available quickly, usually within a few hours.

Potential Problems  Often very much reduced sensitivity compared to cell culture, can be as low as 20%. Specificity often poor as well.

 Requires good specimens.

 The procedures involved are often tedious and time consuming and thus expensive in terms of laboratory time.

Specimens for Routine Tests

Clinical Category

Meningitis 2.

Encephalitis 3.

Paralytic disease 4.

Respiratory illness 5.

Hepatitis 6.

Gastroenteritis 7.

Congenital diseases 8.

Skin lesions 9.

Eye lesions 10.

Myocarditis 11.

Myositis 12.

Glandular fever 13.

Post Mortem

Blood

+ + + + + + + + + + +

Throat swab

+ + + +

Faeces

+ + +

CSF Other

+ + + Brain biopsy Nasopharyngeal aspirate + + Urine, saliva Lesion sample e.g. vesicle fluid, skin scrapping Eye swab Pericardial fluid + Autopsy After use, swabs should be broken into a small bottle containing 2 ml of virus transport medium.

Swabs should be sent to the laboratory as soon as possible without freezing. Faeces, CSF, biopsy or autopsy specimens should be put into a dry sterile container.

Molecular Methods

 Methods based on the detection of viral genome are also commonly known as molecular methods. It is often said that molecular methods is the future direction of viral diagnosis.

 However in practice, although the use of these methods is indeed increasing, the role played by molecular methods in a routine diagnostic virus laboratory is still small compared to conventional methods.

 It is certain though that the role of molecular methods will increase rapidly in the near future.

Classical Molecular Techniques

 Dot-blot, Southern blot, in-situ hydridization are examples of classical techniques. They depend on the use of specific DNA/RNA probes for hybridization.

 The specificity of the reaction depends on the conditions used for hybridization. However, the sensitivity of these techniques is not better than conventional viral diagnostic methods.

 However, since they are usually more tedious and expensive than conventional techniques, they never found widespread acceptance.

Polymerase Chain Reaction (1)

     PCR allows the in vitro amplification of specific target DNA sequences by a factor of 10 6 and is thus an extremely sensitive technique.

It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest.

These oligonucleotides act as primers for the thermostable Taq polymerase.

Repeated cycles (usually 25 to 40) of denaturation of the template DNA (at 94 o C), annealing of primers to their complementary sequences (50 o C), and primer extension (72 o C) result in the exponential production of the specific target fragment.

Further sensitivity and specificity may be obtained by the nested PCR.

Detection and identification of the PCR product is usually carried out by agarose gel electrophoresis, hybridization with a specific oligonucleotide probe, restriction enzyme analysis, or DNA sequencing.

Polymerase Chain Reaction (2)

   Advantages of PCR:    Extremely high sensitivity, may detect down to one viral genome per sample volume Easy to set up Fast turnaround time Disadvantages of PCR   Extremely liable to contamination High degree of operator skill required   Not easy to set up a quantitative assay.

A positive result may be difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

These problems are being addressed by the arrival of commercial closed systems such as the Roche Cobas Amplicor which requires minimum handling. The use of synthetic internal competitive targets in these commercial assays has facilitated the accurate quantification of results. However, these assays are very expensive.

Schematic of PCR

Each cycle doubles the copy number of the target 92

Other Newer Molecular Techniques

      Branched DNA is essentially a sensitive hydridization technique which involves linear amplification. Whereas exponential amplification occurs in PCR.

Therefore, the sensitivity of bDNA lies between classical amplification techniques and PCR. Other Newer molecular techniques depend on some form of amplification.

Commercial proprietary techniques such as LCR, NASBA, TMA depend on exponential amplification of the signal or the target.

Therefore, these techniques are as susceptible to contamination as PCR and share the same advantages and disadvantages.

PCR and related techniques are bound to play an increasingly important role in the diagnosis of viral infections.

DNA chip is another promising technology where it would be possible to detect a large number of viruses, their pathogenic potential, and their drug sensitivity at the same time.

Comparison between PCR and other nucleic acid Amplification Techniques

Method Target Amplification Signal Amplification Thermocycling Sensitivity Commercial Examples PCR LCR NASBA

Exponential No Exponential

TMA

Exponential

Qß-Replicase

Branched DNA

No No Exponential No No Exponential Linear Yes Yes No No No No High High High High High Medium Roche Amplicor Abbot LCX Organon Teknika Genprobe None Chiron Quantiplex 94