Virology Class1

8/3/2019 Virology Class1

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Virology


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Virologyisthestudyof

-Virusesandvirus-likeagents

-Structure -Classifica9on

-Evolu9on

-Reproduc9on,thediseasestheycause

-Thetechniquestoisolateandculturethem

-Theiruseinresearchandtherapy.

WhatisVirology?


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VirologicalMethods


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Biosafety classified laboratoryMicrobiological safety cabinet

Sterilization & disinfection

Protective clothing for workersVaccination

Knowledge & discretion

Safety in handling viruses


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WHO classification: BioSafety Level 1 to 4 for

Risk Groups 1 to 4 microorganisms

BSL 1 laboratories

In the interest of safety, floors should be slip resistant, seamless, be impermeable to liquids and resistant to

most, if not all chemicals that are normally used in laboratories. Each laboratory should have a hand basinand disposable paper towels provided.

BSL 2 laboratories

Ventilation systems designed to prevent the distribution of infectious airborne particles were developed

based on the "Clean-to-Dirty" airflow principle, where more air is extracted from the rooms where hazardousmaterials are handled than from any other area. Close-fitting doors are used and thereby resulting in a

pressure gradient so that air always flows from clean to potentially contaminated areas ie. from corridors to

laboratories and not in the opposite direction. The air extracted from contaminated areas may be ducted

directly to the atmosphere. It is important that adequate lighting is provided.

BSL 3 laboratories

The object of level 3 laboratories is to confine, or contain the organisms so that only a minimum number of

people are exposed to them. Access to Level 3 laboratories should be strictly limited and controlled and the

doors should be locked when the rooms are not in use. Microbiological safety cabinets are essential features

of these laboratories. An incubator room could open directly from a Level 3 laboratory.

BSL 4 laboratoriesWork with Hazard Group 4 agents is usually severely restricted in most countries by government decree. The

laboratory should be isolated or physically separated from other parts of the same building so that access is

difficult. It should be airtight and access is through airlocks. The ventilation system should be completelycontrolled so that air flows via air locks into the laboratory. All effluent air is passed through double banks of

HEPA filters before discharge to the atmosphere. A double-ended autoclave is essential to ensure that

nothing passes outside the room without being sterilized.


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Sterilization & disinfection

Moist heat (autoclaving 120*C x 20 minutes) ordry heat (oven, 180*C for 60

minutes) are effective against all viruses - lesser degrees of heat may inactivatemany viruses (e.g. simple boiling) but may not reliably inactivate resistant viruses

especially if times of exposure are short.

Chemicals: halogens, especially chlorine ashypochlorite are effective againstviruses but corrosive on instruments where activated gluteraldehyde ("Cidex") ispreferred.

Detergents and lipid solvents inactivate readily the enveloped viruses which needan intact envelope for effective cell adsorption. Phenolic disinfectants damage

proteins and thus inactivate bacteria but do not affect nucleic acids. Phenolics are

not recommended for viral disinfection.

UV light


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Diagnos9cMethodsinVirology

1. DirectExamina9on2.IndirectExamina9on(VirusIsola9on)

3.Serology


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DirectExamina9onDirect examination methods are often also called rapid diagnosticmethods because they can usually give a result either within thesame or the next day

1. Electron Microscopy morphology of virus particles

immune electron microscopy

2. Light Microscopy histological appearance

3. Viral Genome Detection hybridization with specific

nucleic acid probes

polymerase chain reaction (PCR)


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IndirectExamina.on

1. Cell Culture cytopathic effect (CPE)haemabsorption

immunofluorescence

2. Eggs pocks on CAM

haemagglutination

3. Animals disease or death

Cell cultures, eggs, and animals may be used for isolation. Howevereggsand animals are difficult to handle and most viral diagnostic laboratoriesdepend on cell culture only


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DirectExamina9on


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Electron Microscopy

Viruses may be detected in the following specimens.

Faeces Rotavirus, Adenovirus

Norwalk like viruses

Astrovirus, Calicivirus

Vesicle Fluid HSV (Herpes simplex virus)

VZV (Varicella zoster virus)

Skin scrapings Papillomavirus,

Virus particles are detected and identified on the basis of

morphology.


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Negative staining

With heavy metal

Rotavirus


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EMdetec.onofCoronavirus


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RabiesVirus


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TMV (TEM 207,480x)


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Electronmicrographs

Shape and Location Specific ???


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ProblemswithElectronMicroscopy

Expensive equipment

Expensive maintenance

Require experienced observer

Sensitivity often low

Limited use, good if virus shape andlocation are specific.


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Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune

electron microscopy.

Immune electron microscopy (IEM) electron microscopy ofspecimens labeled with antibodies that have been conjugated

with gold. The gold makes the antibody labels electron-dense.


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Viral Genome Detection

Methods based on the detection of viral genome are alsocommonly 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 isindeed 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 willincrease rapidly in the near future.


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Dot-blot, Southern blot, in-situ hydridization are examples ofclassical techniques. They depend on the use of specific

DNA/RNA probes for hybridization.

The specificity of the reaction depends on the conditionsused for hybridization. However, the sensitivity of thesetechniques is not betterthan conventional viral diagnostic

methods.

However, since they are usually more tedious andexpensive than conventional techniques, they never foundwidespread acceptance.

ClassicalMolecularTechniques


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PolymeraseChainReac.on

PCRallows the in vitro amplification ofspecific target DNA sequencesby a factor of 106 and is thus an extremely sensitive technique.

It is based on an enzymatic reaction involving the use of syntheticoligonucleotides 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 thetemplate DNA (at 94oC), annealing of primers to their complementary

sequences (50oC), and primer extension (72oC) 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.


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PolymeraseChainReac.on(cont)

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 operatorskillrequired 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.

Cytomegalovirus (CMV)


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Schematic of PCR

Each cycle doubles the copy number of the target


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OtherNewerMolecularTechniques

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

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

amplification.

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

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

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

DNA chip is another promising technology where it would be possible to detect alarge number of viruses, their pathogenic potential, and their drug sensitivity at

the same time.


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IndirectExamina9on

(VirusIsola9on)


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EmbryonatedEggvirusinocula9on

Before the development ofcell culture, many viruses

were propagated in embryonated chicken eggs

Today this method is most commonly used for growth

ofinfluenza virus.

The excellent yield of virus from chicken eggs has led

to their widespread use in research laboratories andforvaccine production.

In fact the vast majority ofinfluenza vaccines are

produced in chicken eggs.


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For propagation of

influenza virus, pathogen-free eggs are used 11-12days after fertilization.

The egg is placed in frontof a light source to locatea non-veined area of the

allantoic cavity just below

the air sac.


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After all the eggs have been nicked and drilled, they are

inoculated with virus using a tuberculin syringe

The virus is placed in the allantoic cavity, which is filled with

allantoic fluid.

The holes in the shell are sealed with melted paraffin, and the

eggs are placed at 37 degrees C for 48 hours.


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The shell membrane and chorioallantoic membrane

are pierced with a pipette which is then used to

remove the allantoic fluid about 10 ml per egg.

Sufficient virus may be produced in one or two eggs

(depending on the viral strain) to produce one 15

microgram dose of vaccine.


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Tostudyviruseswhichcannotbepropagatedinvitro,egHBV Tostudythepathogenesisofvirusinfec.onsEgCoxsackieviruses Totestvaccinesafety,egoralPoliovirusvaccineNevertheless,theyareincreasinglybeingdiscardedbecause:

Breeding&maintenanceofanimalsinfectedwithvirusesisexpensive Wholeanimalsarecomplexsystems,inwhichitissome.mesdifficulttointerpret Resultsobtainedarenotalwaysreproducible,duetohostvaria.on Unnecessaryorwastefuluseofexperimentalanimalsismorallyrepugnant Theyarerapidlybeingovertakenbycellculture&molecularbiology


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VirusIsola.on

Cell Culturesare most widely used for virus isolation, there are 3

types of cell cultures:

1. Primary cells - Monkey Kidney These cells can only be

passaged once or twice.

2. Semi-continuous cells - Human embryonic, kidney and skin

fibroblasts, may be passaged up to 50 times

3. Continuous cells - HeLa, Immortalized cell line, may be passaged

indefinitely.

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 ofviruses supported is often limited.


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Tissuecultureprepara9on:

Fromthedesired,ssuethefollowingstepsarefollowed:

Minceinto1mmfragments.

Incubatewithproteoly9cenzyme(trypsin)todispersethecells.

Addgrowthmediatomakeacellsuspension.

Incubateinsta9onaryflasksortubes,cellsseleonthedependentsurfaceandgrowintoconfluentmonolayer.

Re-dispersemonolayercellsandincreasenumberofculturesforcellculturepassage.


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Identification of growing virus

The presence of growing virus is usually detected by:

1. Cytopathic Effect (CPE) - such as the ballooning of

cells, may be specific or non-specific e.g. Herpes

Simplex Virus (HSV) and Cytomegalovirus (CMV)produces a specific CPE, whereas enteroviruses do not.

Cytopathic = characterized by pathological changes in

cells.


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CytopathicEffect

Cytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning ofcells.

( CPE = a virus is growing )


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Many viruses (eg influenza, parainfluenza, measles and mumps

virus and some picornaviruses) contain surface glycoproteins knownas haemagglutinins (HA). These are capable of binding red blood

cells. As these viruses replicate in cell culture, HA molecules appear

on the cell surface. If red blood cells of the appropriate species areadded to the cell culture tube in which the virus is replicating, they will

adhere to the cell sheet

a phenomenon known as haemadsorption

2. Haemadsorption - cells acquire the ability to

stick to mammalian red blood cells.

Haemadsorption is mainly used for the detection of

influenza and parainfluenzaviruses.

Identification of growing virus (Cont.)


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Haemadsorp9on

Haemadsorption of red blood cells onto the surface of a cell

sheet infected by mumps virus


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Haemadsorp9on

Macrophages infected by the

African Swine Fever virus


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Serumneutraliza9ontest

It is suitable for the detection and quantification of

Virus-specific antibodies in serum

The SNT is sensitive and specific, compared to the

ELISA

Cell Culture

Viral Particle Virus SpecificAntibody


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Serum neutralization test

1. A specific amount of virus is added to progressively diluted serum probes.2. Susceptible cells are added to the virus-antibody mixture.

3. The neutralization effect is made visible (cytopathic effect or

immunostaining).


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No Cytopathic Effect

Cells Survival

Cytopathic Effect at difference level

Cells Death


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Limita9onofcellculture

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.

Aged cells Requires equipment and operator skill. Ongoing costs even without doing any tests. Many viruses will not grow in cell culture e.g. Hepatitis

B, Diarrhoeal viruses, parvovirus, papillomavirus.


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VirusesIsolatedbyCellCulture


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RapidCultureTechniques

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 plasticbottle.

Following inoculation, the bottle then is spun at a low speed forone 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 presenceof CMV early antigens by immunofluorescence.


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DEAFFtestforCMV

Detection ofEarly Antigen Fluorescent Foci

Cytomegalovirus (CMV)


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Serology


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Serological

Antigen - antibody reactions studied under laboratory conditions are

known as serological reactions, so named because they commonlyinvolve serum from a patient.

Serology (the testing for antibodies) is used to determine antibody positivity

A large variety of serological tests are available eg.

complement-fixation (CFT)

haeagglutination-inhibition (HAI)enzyme-linked immunoassay (EIA)

radioimmunoassay (RIA)

particle agglutinationimmunofluorescence

western blot


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The complement fixation test is an immunological medical test that can

be used to detect the presence of either specific antibody or specificantigen in a patient's serum.

ComplementFixa.onTest

The complement fixation assay can be used to look for the presenceof i) specific antibody or ii) specific antigen in a patient's serum. Thetest utilizes sheep red blood cells (SRBC), anti-SRBC antibody and

complement, along with specific antigen (if looking for antibody inserum) or specific antibody (if looking for antigen in serum). If

antibody (or antigen) is present in the patient's serum, then the

complement is completely utilized and SRBC lysis is minimal.

However, if the antibody (or antigen) is not present in the patient'sserum, then the complement binds anti-SRBC antibody and lysis ofthe SRBCs ensues.


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ComplementFixa.onTest


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Principle of Immunoassays

ANTIGEN

SAMPLE ANTIBODY

ANTI-HUMAN IMMUNOGLOBULIN WITH

DETECTOR


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ELISAforHIVan.body

Microplate ELISA for HIV antibody: coloured wells indicate reactivity


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WesternBlot

HIV-1 Western Blot

Lane1: Positive Control Lane 2: Negative Control Sample A: Negative Sample B: Indeterminate Sample C: Positive


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WesternBlot

Expensive$80-100technicallymoredifficultvisualinterpreta9onlackstandardisa9on


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UsefulnessofSerologicalResults

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 theappearance of antibodies such as respiratory. 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 yearsafter seroconversion e.g. HIV and rabies. In the case of these viruses, the

mere presence of antibody is sufficient to make a definitive diagnosis.


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ProblemswithSerology

Long period of time required for diagnosis for paired acute andconvalescent sera.

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

immunocompromised patients often give a reduced or absent humoralimmune response.

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

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Virology Class1