Virology Lab Technique Training

Virology Lab Technique Training

Basic Virological Techniques

18th November-23rd November

This past week I participated in a basic virology lab techniques course hosted by the Virology Research Laboratory at Manipal. Virology is a branch of science that deals with the study of viruses. Each day we learned about different techniques used in virology through lecture and hands on time in the lab. This week long course was one of my favorite experiences that I have had while in India. Since learning about scientific research on communicable diseases in South Africa, I have been inspired to investigate the field of virus research. I signed up for this course not sure what to expect but was blown away by the course material, which provided me with insight into a another whole area of academia. I had a blast working in the lab and look forward to doing more lab work in the future.
Although this blog post is a bit dense I wanted to provide you all with an idea of what we covered during the conference. I would be glad to talk with anyone that is interested about the specific techniques that we learned or the results that we obtained. I am still learning and hope that having the opportunity to talk about this experience on a scientific level will help me to understand it more myself! I have been told that teaching others is a great way to test your knowledge and better understand a concept.

Our schedule was as follows:

Monday

Introduction and Laboratory approach in Diagnostic Virology
Syndromic Approach in Diagnostic Virology and Sample Collection
Specimen Collection, Packaging, processing

Tuesday

Cell Culture
Virus Isolation

Wednesday

Haemagglutination Assay
Haemagglutination Inhibition Assay

Thursday

Quantification of Virus- TCID50
Neutralization Assay

Friday

Conventional PCR
Real time/ multiplex PCR

Saturday

Immunofluorescence Assay

In addition, there were a number of other techniques that I learned throughout the week including: how to preform a two-fold dilution, isolate RNA, navigate a level two lab, make liquids of a desired concentration and do a Plaque Assay. 

This image includes some of the instruments that we used on a daily basis in the lab. The bottles or flasks are used for cell culture, the trays with larger wells are used for techniques that involve staining such as a plaque assay which is used to  preform virus inoculation, the smaller tray in the back in a 96 well microtiter plate like the one we used for our hemagglutination assays, and the small tube on the right is a sample swab. 

Monday

On Monday we learned about the basic approaches to laboratory diagnostic virology, how to do syndromic diagnosis of diseases, and the processes for collection, packaging and transportation of samples. Diagnostic virology is used to increase awareness of viral diseases and is a major factor in: technological advances and modern management of infections. It is focused on the research of many public health issues such as AIDS, immunosuppressive therapy, and anti-virals.  There are a number of important factors that affect lab diagnosis of viral diseases including; specificity of the sample, transportation methods, selection of the appropriate assays, knowledge of disease kinetics, details of virus isolation, and clinical details. The basic approaches in laboratory diagnosis include microscopy (light and electron microscopy), antigen detection via. immunofluorescence assay or other assays, antibody detection through a neutralization assay or a haemagglutination inhibition assay and nucleic acid detection through PCR techniques.

Syndromic Diagnostic Approach in Virology:

Syndromic diagnosis is used by health care providers in order to quickly identify and treat a consistent group of symptoms that are easily recognizable. Treatment for such syndromic diseases is targeted towards the main organisms responsible for causing the syndrome. The syndromic treatment approach is preferred because it focuses on multiple symptoms at once, emphasizes direct doctor- patient interaction, and treatment is immediately initiated at the primary healthcare level. When we visited the public health facilities this semester we viewed the various guideline and protocol cards used by doctors to do syndromic diagnosis.

Major Clinical Syndromes-
1. Fever
2. Influenze like illness
3. Dengue like illness
4. fever and arthralgia (joint pain)
5. fever and rash (measles like)
6. Fever and parotitis (infection of the salivary glands)
7. Acute encephalitis syndrome
8. Adult respiratory distress syndrome
9. Acute jaundice syndrome
10. Acute diarrhea syndrome
11. Acute hemorrhagic fever syndrome

Specimen Collection, Packaging and Transportation

The specimen collection method is determined based on the syndrome that a patient exhibits.
When choosing a collection method there are a number of things to consider including; duration of viral shedding, type of virus, systemic involvement, the kinetics of the disease, and the age or immune system of the patient. Specimen collection techniques include respiratory samples, dermal samples, blood, CSF, urine, stool, saliva, conjunctival swab, and bone marrow. For the purpose of this course we focused on throat and nose swabbing. We were able to  practicing taking samples and packaging them for transport.

Tuesday

Tuesday we learned how to make a cell culture and perform isolation of a virus.

This is a video on cell culture.

Viruses are obligate intracellular parasites and require living cells to replicate. An essential step in propagating them in the laboratory is provision of cells in some way. The most widely used method is cell culture. Cell cultures are fairly easy to maintain and can have a wide host range. Cells in culture may be grown as a monolayer, a single layer of cells growing on a surface or in suspension in which cells multiply while suspended in a liquid media. Suspended cultures are used primarily for growing large volumes of viruses whereas monolayers are used widely in diagnostic virology for viral isolation. Though three types namely primary, diploid and continuous cell cultures are available, continuous cell lines are maintained commonly for the ease of maintenance and are most commonly used for experiments. Each cell line is chosen for a particular assay based on its specific qualities and the goal of the assay.
Cells grow best in the laboratory if the culture environment resembles the conditions experienced by the cells in vivo. Many types of synthetic media have been developed and modified to meet the needs of certain types of cells. Cell lines can be propagated or maintained dependent on the type of medium they are kept in. In virology, cell lines are chosen for their ability to act as hosts to specific viruses. For example, the Vero cell line that we used for many of our experiments was from the African green monkey kidney. We chose this cell because of its success in vitro.
Virus isolation can be done via animal inoculation, embryonated egg inoculation or cell cultures (the most common, safe, and ethically sound method and the method that we used).  Cell cultures are a convenient in-vitro method of virus isolation. There are three types of cell lines used in cell cultures; primary cell lines, diploid cell lines and continuous cell lines. In order to preform virus isolation one needs a tissue culture flask with confluent monolayer, a clinical sample with a suspected virus, maintenance media (solution with the necessary components for cell growth and division or life), and the necessary laboratory equipment (microscope, flasks, micro pipet, etc.).
In the lab we created a subculture of Vero cells. Subcultures are created when cells are taken from the maintained cell line and placed in growth medium in a new vial. After we had created a subculture we learned how to isolate a virus via cell culture inoculation. This means that we took a sample that was thought to contain a pathogen and added it to a cell line in a medium conducive to maintenace of cells. We then observed the cells over a number of days in order to determine whehter the virus had infected the cells. Cells that displayed the cytophathic effect; characteristic cellular changes such as ballooning and aggregation of cells, were suspected to have been infected by the virus and could be used for further studies.

Brain Break! (Don't worry we were using precautionary methods and inactive viruses) 

Wednesday 

On Wednesday we learned how to perform a hemagglutination assay and a hemagglutination inhibition assay.

Hemagglutination Assay (aka. virus titration)

The hemagglutination assay is a method used to quantify a virus in a sample or cell. Many viruses have a surface protein known as a hemagglutinin protein (HA) that binds to specific receptors such as those found on the plasma membrane of red blood cells. When red blood cells are mixed with the viruses containing HA protein (in the appropriate ratio) the virus bridges the red blood cells and changes their normal settling pattern creating a dense latex as opposed to a diffused monolayer. This processes is called hemagglutination. Hemagglutination assays are used after virus isolation and inoculation of a designated virus in order to determine the viral load.
Steps in a Hemagglutination Assay:

1. Prepare red blood cells  (RBS) and virus suspension
2. Add Phosphate buffer saline (PBS,  keeps the pH stable and prevents the cells from lysing) to all designated wells of a 96 well microtiter plate
3. Add the virus suspension to well 1
4. Preform a two-fold serial dilution, decreasing the concentration from left to right using the suspension in well 1 as initial source
5. Add fixed concentration and volume of RBS suspension to each well
6. Incubate the wells to promote binding of virus and RBS

Results:
The highest dilution of virus that causes complete hemagglutination is the endpoint of titration. One unit of HA is contained in the endpoint dilution of HA titration and is inversely equal to the total concentration of virus that was in the original sample. (aka. the endpoint is the highest dilution of virus that will still react with the red blood cells causing a latex to form, it is at this point that only one unit of HA protein is in the well therefore the concentration of the original sample is equal to the the number of times the dilution). A sample with an endpoint of 32 means that the original sample was diluted 32 times before there was only one HA unit and therefore originally the HA concentration was 32.

Hemagglutination Inhibition Assay:

If a person is infected by a virus bearing the HA protein, ant-HA antibodies may appear in his serum (sample collected may be blood, sputum, or other). These antibodies can block hemagglutination by binding to their target antigen and lead to the hemagglutination inhibition phenomenon. As a result no glutination will occur. Antibodies bind to the virus blocking binding sites on the viral surface and preventing the agglutination of the red blood cels. This assay is used to determine presence of antibodies in a patient serum by using an unknown serum and known virus or to determine the type or strain of virus by using a typing serum with known antibodies instead of a patient serum and an unknown virus isolated from a patient sample. In this case, the formation of the button or hallo is a negative result indicating that the serum did not have the target antibodies present. If the antigen antibody pair is a match, the red blood cells will not bind with the antigen and there will be no glutination.

This image is of the 96 well microtiter plate. If you look closely you can see the evidence of hemagglutination in the mirror; the red dots all the way to the right of the mirror image are "buttons" or congregates of red blood cells. The cells to the left are the more concentrated samples providing evidence that as the concentration of antibodies decreases the blood cells begin to bind with the antigens resulting in hemagglutination.  

Serial two-fold titration: a useful technique often used in diagnostic virology. Two fold titrations are used in order to manipulate the concentration of a sample (the antigen or antibody concentration).

Thursday 

Thursday we learned to preform a virus neutralization assay. We also learned how to quantify a virus using TCID50.
Neutralization of a virus is defined as loss of infectivity through reaction of virus with specific antibodies. Neutralization assay is primarily performed in order to quantify the concentration of antibody present in a sample serum. In vitro testing of virus neutralization is preformed by incubating the known virus concentration with the sera containing antibodies. Residual viral activity is detected by its ability to subsequently infect cell cultures or other host systems. Antibody concentration is equal to the reciprocal of highest dilution of serum that shown 50% or greater reduction in cytopathic effect (50% or less neutralization). The results of this study are viewed using specific staining techniques in which the the cells that have been infected by the virus are stained and the cells that have not been infected are washed away. This process is used to identify viruses, differentiate between closely related viruses, and determine specific antibody response following infection/ vaccination.
Tissue culture infective dose 50 (TCID50) is a viral quantification technique. It determines the titer of infectious virus present in a viral stock until neutralization occurs. The TCID50 technique determines the highest dilution of virus that infects 50% of cell cultures as evidenced by the cytopathic effect. The results of the TCID50 titration (infection ratio, cumulative # of infected and not infected cells, and percent infected) are entered into an equation that determines the quantity of viral load in the original sample.

This image is of a 96 well microtiter plate used in TCID50. The wells all the way to the right and left are cell controls. They are stained dark purple because they were not infected by the virus. Their purpose is to insure that the cells were viable and correctly fixed without any experimental variables. Wells 1, 2, and 3 do not have any cells because the cells that were present were infected by the virus. Infected cells are not fixed with the addition of ethanol and can be literally washed away by water whereas infected cells rows 5-10 are fixed and are therefore stained. Row 4 is evidence of the success of the TCID50 titer, some of the cells have been fixed and stained( were not infected) while others were washed away (infected by the virus). It is up to the experimenter to determine the % of infected cells in each well. Remember, the TCID50 assay determines quantity of a virus based on the viruses ability to infect > 50% of cells at different dilutions. 

This day was particularly challenging as I had to recall calculous from Mrs. Niller's math class in 11th grade (not my brightest moments). It was a lot of learned but I thoroughly enjoyed myself!

Friday

On Friday we did Conventional Polymerase Chain Reaction (PCR) and Real-time PCR. Polymerase chain reaction is a technique used to amplify a target gene sequence in vitro using the cells "built in" mechanism for replication of DNA. Conventional PCR is used for disease diagnosis, molecular epidemiology by genotype (hereditary DNA diseases), drug resistance studies, phylogeny analysis, DNA fingerprinting and studies of genetic variability.

A gel electrophoresis technique is used to view the results of conventional PCR. The amplified target sequence is loaded into an agarose gel and an electrical current is run through the gel propelling the negatively charged DNA away from the anion and towards the cation. Larger segments of DNA take longer to navigate through the agarose gel while small segments of DNA easily move through the well resulting in a separation of DNA segments by size. This technique is used to determine the success of PCR by identifying the target sample of known length. 

In comparison, real-time PCR is a specialized technique that allows PCR reactions to be visualized "in real time" during each cycle by using fluorescent dyes or probes. The advantages of real-time PCR include the ability to monitor the progress of the PCR reaction as it occurs, quantification of target sequence present based on the nucleic acid concentration, the specificity and sensitivity of the test, and the speed of procurement of results. The amount of target sequence during amplification is quantified by using fluorescent dyes which increase in a logarithmic pattern until the threshold concentration (point of maximum amplification) is reached. The concentration of virus is found by determining where the threshold and log phase intersect. This point, known as the Ct value, is inversely related to the amount of target sequence present.

Saturday 

On the final day of our course we learned how to preform an immunofluorescence assay. Immunofluorescence facilitates the rapid detection of viral antigen in clinical specimens and viral isolates. It is useful for the detection of respiratory viruses such as herpes sinplex virus, cytomegalovirus, and varicella zoster virus. This technique is based on the phenomenon of photo-excitation and subsequent emission of light molecules produced by fluorochromes or fluorophores (light absorbing and emitting molecules). Antibodies can be molecularly attached to fluorochrome molecules for experimental purposes. In immunofluorescence, antibodies are chemically conjugated to fluorescent dyes. Fluorochrome-conjugated antibodies against specific viruses can detect their protein antigens in clinical specimen and infected cells.  This concept is used in order to view antigen- fluorochrome-conjugated antibody complexes under UV lights. When molecules such as fluorochromes absorb light their electrons are excited to a higher energy state, as the electrons fall back to their original energy state they emit energy in the form of light. A high energy wavelength of light will be absorbed causing excitation of the electrons, the return of the electron to its original energy state emits excess energy in the form of a lower energy wavelength of light.  Immunofluorescence can be used to identify the presence and quantity of virus in a sample as the antigen(virus) will be bound to fluorochrome-conjugated antibodies and can be viewed using microscopy.

The end!

If you are interested in learning more about the lab that I was working in you can visit the departments webpage: http://www.manipal.edu/institutions/universitydepartments/departmentof%20virusresearch/pages/drgarunkumar.aspx