Virology

What is Virology ?
Virology is the study of - Viruses and virus-like agents - Structure - Classica9on - Evolu9on - Reproduc9on, the diseases they cause - The techniques to isolate and culture them - Their use in research and therapy.

Virological Methods

Safety in handling viruses

Biosafety classified laboratory Microbiological safety cabinet Sterilization & disinfection Protective clothing for workers Vaccination Knowledge & discretion

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 basin and 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 hazardous materials 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 laboratories Work 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 completely controlled 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.

Sterilization & disinfection

Moist heat (autoclaving 120*C x 20 minutes) or dry heat (oven, 180*C for 60 minutes) are effective against all viruses - lesser degrees of heat may inactivate many viruses (e.g. simple boiling) but may not reliably inactivate resistant viruses especially if times of exposure are short. Chemicals: halogens, especially chlorine as hypochlorite are effective against viruses but corrosive on instruments where activated gluteraldehyde ("Cidex") is preferred. Detergents and lipid solvents inactivate readily the enveloped viruses which need an 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

Diagnos9c Methods in Virology

1. Direct Examina9on 2. Indirect Examina9on (Virus Isola9on) 3. Serology

Direct Examina9on
Direct examination methods are often also called rapid diagnostic methods because they can usually give a result either within the same or the next day

1. Electron Microscopy 2. Light Microscopy 3. Viral Genome Detection

morphology of virus particles immune electron microscopy histological appearance hybridization with specific nucleic acid probes polymerase chain reaction (PCR)

Indirect Examina.on
Cell cultures, eggs, and animals may be used for isolation. However eggs and animals are difficult to handle and most viral diagnostic laboratories depend on cell culture only

1. Cell Culture

cytopathic effect (CPE) haemabsorption immunofluorescence pocks on CAM haemagglutination disease or death

2. Eggs

3. Animals

Serology

Direct Examina9on

Electron Microscopy
Virus particles are detected and identified on the basis of morphology.

Viruses may be detected in the following specimens. Faeces Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus HSV (Herpes simplex virus) VZV (Varicella zoster virus) Papillomavirus,

Vesicle Fluid

Skin scrapings

Negative staining With heavy metal

Rotavirus

EM detec.on of Corona virus

Rabies Virus

TMV (TEM 207,480x)

Electronmicrographs

Shape and Location Specific ???

Problems with Electron Microscopy

Expensive equipment Expensive maintenance Require experienced observer Sensitivity often low Limited use, good if virus shape and location are specific.

Immune Electron Microscopy

The sensitivity and specificity of EM may be enhanced by immune electron microscopy.

Immune electron microscopy (IEM) electron microscopy of specimens labeled with antibodies that have been conjugated with gold. The gold makes the antibody labels electron-dense.

Viral Genome Detection

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 Reac.on

PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 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 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.

Polymerase Chain Reac.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 operator skill required 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)

Schematic of PCR

Each cycle doubles the copy number of the target

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.

Indirect Examina9on (Virus Isola9on)

Embryonated Egg virus inocula9on

Before the development of cell culture, many viruses were propagated in embryonated chicken eggs Today this method is most commonly used for growth of influenza virus. The excellent yield of virus from chicken eggs has led to their widespread use in research laboratories and for vaccine production. In fact the vast majority of influenza vaccines are produced in chicken eggs.

For propagation of influenza virus, pathogenfree eggs are used 11-12 days after fertilization.

The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac.

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.

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.

To study viruses which cannot be propagated in vitro, e.g. HBV To study the pathogenesis of virus infec.ons. E.g. Coxsackieviruses To test vaccine safety, e.g. oral Poliovirus vaccine.

Nevertheless, they are increasingly being discarded because: Breeding & maintenance of animals infected with viruses is expensive Whole animals are complex systems, in which it is some.mes dicult to interpret Results obtained are not always reproducible, due to host varia.on Unnecessary or wasteful use of experimental animals is morally repugnant They are rapidly being overtaken by cell culture & molecular biology

Virus Isola.on
Cell Cultures are 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 of viruses supported is often limited.

Tissue culture prepara9on:

From the desired ,ssue the following steps are followed: Mince into 1mm fragments. Incubate with proteoly9c enzyme (trypsin) to disperse the cells. Add growth media to make a cell suspension. Incubate in sta9onary asks or tubes, cells seTle on the dependent surface and grow into conuent monolayer. Re-disperse monolayer cells and increase number of cultures for cell culture passage.

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.

Cytopathic Eect

( CPE = a virus is growing )

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

Identification of growing virus (Cont.)

2. Haemadsorption - cells acquire the ability to stick to mammalian red blood cells. Haemadsorption is mainly used for the detection of influenza and parainfluenzaviruses.
Many viruses (eg influenza, parainfluenza, measles and mumps virus and some picornaviruses) contain surface glycoproteins known as 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 are added to the cell culture tube in which the virus is replicating, they will adhere to the cell sheet a phenomenon known as haemadsorption

Haemadsorp9on

Haemadsorption of red blood cells onto the surface of a cell sheet infected by mumps virus

Haemadsorp9on

Macrophages infected by the African Swine Fever virus

Serum neutraliza9on test

It is suitable for the detection and quantification of Virus-specific antibodies in serum The SNT is sensitive and specific, compared to the ELISA

Viral Particle

Virus Specific Antibody

Cell Culture

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).

No Cytopathic Effect Cells Survival

Cytopathic Effect at difference level Cells Death

Limita9on of 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. 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.

Viruses Isolated by Cell Culture

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

Detection of Early Antigen Fluorescent Foci

Cytomegalovirus (CMV)

Serology

Serological
Antigen - antibody reactions studied under laboratory conditions are known as serological reactions, so named because they commonly involve 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 agglutination immunofluorescence western blot

Complement Fixa.on Test

The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum.

The complement fixation assay can be used to look for the presence of i) specific antibody or ii) specific antigen in a patient's serum. The test utilizes sheep red blood cells (SRBC), anti-SRBC antibody and complement, along with specific antigen (if looking for antibody in serum) 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's serum, then the complement binds anti-SRBC antibody and lysis of the SRBCs ensues.

Complement Fixa.on Test

Principle of Immunoassays
ANTI-HUMAN IMMUNOGLOBULIN WITH DETECTOR

SAMPLE ANTIBODY ANTIGEN

ELISA for HIV an.body

Microplate ELISA for HIV antibody: coloured wells indicate reactivity

Western Blot
HIV-1 Western Blot

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

Western Blot
Expensive $ 80 - 100 technically more dicult visual interpreta9on lack standardisa9on

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