BTE4610 Chapter 7: Laboratory Diagnosis of Viral Diseases and Working with Viruses in the Research Laboratory

Virus Culture

• cultivation of viruses in a laboratory

• viruses need a host system—can be grown in tissue/cell cultures (preferred method), embryonated/fertilized eggs (harder to control and can introduce contaminants), animals (highly regulated nowadays), plant cells, amoeba/protozoa, etc)

• therefore, culture is absolutely necessary to be able to study a virus 

Laminar flow hoods

• contains HEPA (high efficiency particulate air) filter, which removes the vast majority of particles 0.3 microns or higher from the air

• combined with design of the hood and UV light, helps prevent contamination of samples

One-step growth experiments 

• step 1—infect monolayers of tissue culture cells and allow infection to proceed in a CO2 incubator 

• step 2—monitor experiments via inverted microscope (should be red due to pH indicator; color changes to yellow when pH drops due to organic acids) 

• step 3—collect infected cell lysates at various time points after infection

  • virus replicates in the cell, but is released, so you have to look at both inside the cell (genome and viral proteins) and extracellular media (secreted viruses) 

• step 4—perform serial dilutions on infected cell lysates and do plaque assays 

• step 5—stain and record plaque assay results; analyze/quantify viral proteins and nucleic acids at different points in time 

  • in general: replication = increased nucleic acids; translation = increased proteins; maturation = increased intracellular viral particles; release = increased extracellular viral particles 

  • simplified profile; the different stages do overlap (ex: RdRp must be translated before the genome can be replicated)

Plaque assay

• type of quantitative assay

• prepare serial dilutions of virus (usually tenfold)

• plate dilutions on susceptible cells

• after attachment, overlay cells with semi-solid medium to restrict diffusion of virus particles so that virus only spreads to neighboring cells → restricted spread = localized destruction of cell monolayer, visible as clear plaques

• count plaques x dilution = virus concentration (account for units and whatnot)

• can study virus by performing a plaque purification

Cytopathic effects (CPEs)

• morphological changes in infected cells; can be observed with inverted light microscopes 

• rounding/detachment from plastic flask (loss of adherence)

• shrinkage

• cell lysis/death

• increased refractility

• syncytia/fusion (presence of giant cells/multinuclear cells)

• aggregation

Biosafety level

• laboratory ranking depending on how equipped/prepared a laboratory is to contain pathogens of various risks

• BSL-1: minimum containment, for work with well-characterized, non-pathogenic agents; no particular containment equipment or design necessary 

• BSL-2: moderate hazards; restricted lab access, basic hoods and equipment; most molec and cell bio labs belong in this category 

• (A)-BSL-3: serious or potentially lethal disease causing agents; much stricter safety features (PPE, sealed labs/ventilation systems, etc) (A is for animal labs) 

• BSL-4: maximum containment, for work with extremely dangerous and exotic agents; crazy safety features

Plaque purification

• method for producing clonal virus stocks

• separate virus from host cell by filtering/centrifugation (obviously, depends on having a cell line which can act as host); usually done 3x to remove contaminants 

• observe virus with microscope (EM)—note morphological characteristics such as naked vs enveloped, size, etc 

• sequence to identify and compare the virus to existing viruses/strains (fragment, PCR, sequencing) (reverse transcription from RNA → DNA is necessary if it is an RNA virus) 

• if the nucleic acid sequence is novel, you must do a proteome analysis to confirm it is a virus (presence of capsid sequences, etc) 

• explore virus—virulence check; inject for animal studies, etc

End-point dilution assay

• another quantitative assay which measures the infectivity of a virus 

• TCID₅₀ = 50% tissue culture infective dose = amount of virus necessary to produce a CPE in 50% of inoculated tissue culture cells

• LD₅₀ = 50% lethal dose 

• PD₅₀ = 50% paralysis dose

• ex: do an assay with 10 wells (or animals) for each virus dilution, and see at which dilution 5 dead/CPE wells/animals are produced 

Particle-to-PFU ratio

• another quantitative assay that measures the infectivity of a virus 

• is the ratio of the number of virus particles in a sample to the number of infectious particles in the sample (PFU = plaque forming unit = infectious particle)

• not all viruses are infectious—might lose infectivity due to exposure to detergents, chemicals, heat, etc

• ~1 for many bacteriophages (1 particle per plaque forming unit)

• is pretty high for many animal viruses

  • damaged particles or empty/genome-free virus particles

  • mutations in viral genome

  • complex infection cycle—failure at any one step rpevents completion of viral life cycle

  • non-ideal host/cell line

Fluorescence forming unit (FFU)

• another method for quantifying virus titers

• viral proteins expressed in the infected cell can be visualized immunologically

• dsRNA (which is unusual in host cells) can also be used as an antigen; increases/accumulates in the infected cell as viral replication progresses

• uses a specific antibody to recognize the specific viral antigen + a secondary fluor (or gold)-conjugated antibody against the primary antibody (indirect immunostaining—cheaper and better results than direct immunostaining)

• genome antigen → qRT-PCR or Northern/Southern blot)

• protein antigen → western blot or ELISA 

• cell-based assay; used to detect viruses that do not form a plaque 

Hemagglutination assay (HA titer)

• another method for quantifying viruses which interact with red blood cells

• does not give any measure of viral infectivity

• HA (hemagglutinin) proteins are responsible for viral entry and cause aggregation of the red blood cells → carbohydrates on the cell surface will interact when viruses infect the cells, leading to aggregation, which eventually leads to formation of a “bullseye” in the culture (all the RBCs stuck together) -—> is the endpoint of the dilution  

• can be used to quantify influenza viruses (but does not indicate infectivity because the influenza cannot replicate due to lack of nucleus; only indicates attachment) 

ASSURED diagnostic criteria (WHO)

• affordable

• sensitive

• specific

• user-friendly (simple to perform in a few steps with minimal training) 

• robust and rapid results (less than 30 minutes) 

• equipment free 

• deliverable to those who need the test (logistically—stable at high temperatures, able to withstand transportation, etc) 

Diagnosis in clinical settings

• over 60% of all infectious disease cases seen by a physician are viral, most of which are RNA viruses)

• quality of patient specimen and transport to laboratory is important for diagnosis and treatment

• five general approaches for laboratory diagnosis of viral infections

  • microscopy 

  • culture (only method to conclusively determine infectivity) 

  • nucleic acid detection (genome identification) 

  • detection of viral antigens 

  • antibody detection (against nucleic acid or proteins) 

• other methods of detecting viral components—antisense nucleic acid which can recognize a target sequence (viral genome); RNA/DNA with tertiary structure that can bind to target components, etc

Direct virus detection (microscopy)

• electron microscopy (EM) or immuno-EM (visualizes gold nanoparticles conjugated to antibodies bound to viruses) visualizes virus particles directly in the specimen

  • pros: rapid (15 minutes - 3 hours); works for viruses that cannot be cultured; suitable for “unknowns”

  • cons: requires expensive EM and highly skilled technician + a separate microscope room; not sensitive and specific

• cryo-EM—don’t have to dry the virus particke; just freeze in solution and look at the structure (allows higher resolution/fidelity visualization of the surface)

• indirect immunofluorescent staining/microscopy of infected cells can allow rapid diagnosis of infections such as epstein-barr virus due to specific characteristics of the virus

  • pros: quick (1-3 hours), semiquantitative, good sensitivity 

Centrifugation culture (shell vial technique)

• used in clinical labs for rapid and sensitive diagnosis to test infectivity 

• speeds up amount of time to detect CPE 

• glass vial with tissue culture monolayer in shell vial → inoculation with specimen/virus → centrifuge to enhance infection of monolayer (key step) → incubate → stain with anti-viral fluorescent monoclonal antibodies → mount coverslip on slide → read with fluorescent microscope 

• physical centrifugation increases attachment to host 

• to detect a specific virus, must use a specific antibody 

• pros of culture: indicates that there is infectious virus present in sample; can be used to study and quantify viruses

• cons of culture: slow (results in days to weeks); labor intensive; requires skill and experience both to prepare cells and read CPEs 

Nucleic acid detection/genome identification

• rapid, sensitive, and specific detection of viral genome—PCR (DNA) and RT-PCR (RNA) 

• however, does not tell if samples have LIVE, INFECTIVE viruses 

• can be used to detect viruses that cannot be grown in culture 

• can be used to manage patients (e.g. monitoring viral load in response to antiviral drugs)

• obtain specimen from patient → RNA isolation (bind, wash, elute) → (RT)-PCR amplification (taq DNA polymerase, forward and reverse primer, dNTPs, Mg2+ cofactor, etc) → detection (classic method—gel electrophoresis; modern method—real-time PCR melt curves)

• pros: highly sensitive; can be “same-day” results; can be automated for large numbers 

• cons: may be oversensitive; expensive; requires good technique to avoid cross-contamination 

Real-time PCR (TaqMan-probe method) (qPCR)

• quenching dye disrupts the observable signal from the reporter dye when it is within a short distance 

• TaqMan probe (quenching dye, reporter dye, complementary sequence to target) is located somewhere between the forward and reverse primer 

• Taq polymerase digests the phosphodiester bond linking the reporter dye to the probe (5’ to 3’ exonuclease function) during PCR

• released reporter dye, now away from the quenching dye, can now be excited and observed; the sooner this signal is amplified (closer to the left), the higher the virus titer (more particles) 

• CT value = cycles to threshold; lower = more virus (less cycles needed to reach threshold levels of fluorescence)

Western blot/immunoblotting

• antigen samples → separate proteins via gel → blotting (transfer proteins from gel to nitrocellulose sheet by putting it and then a number of paper towels on the gel, which aspirate the blotting fluid toward them by capillary action, transferring the proteins with it)  → immunostaining of blot → visualization

• enzyme-conjugated antibody → color development

• radiolabeled antibody → autoradiography

• fluorophore-tagged antibody → image analysis

Immune response to viral infections

• IgM (1st exposure): will see within 10 days; covalently linked pentamer (10 antigen recognition sites); Shorter half life than IgG

• IgG (2nd exposure): will see within 2 weeks; lifespan is much longer than other immunoglobulins; monomer; creates a very strong and quick response upon second exposure (height and magnitude of antibody levels in serum)

  • this is why many vaccines (such as the COVID one) have a second/booster shot

• detecting the amount of each allows you to determine what stage of infection (roughly) you’re in

antigen

• a foreign substance that stimulates an immune response in the form of antibody formation or a cell-mediated response

• bacteria and viruses are common sources of antigens

Antibody

• three-lobed globulin molecule found in the blood or other body fluids that can be produced by the presence of an antigen

• has a destructive influence on the antigen

• heavy chain and light chain 

• Fab = fragment antigen-binding region; variable sequence allows it to recognize diverse antigens 

• Fc = fragment crystallizable region → if you remove Fab, the remaining Fc readily forms a crystal; constant seuence 

Enzyme-linked immunosorbent assay (ELISA)

• used to detect viral (or other) antigens or antibodies 

• can be sandwich or indirect 

Sandwich ELISA

• scaffold/solid support has a conjugated capture antibody which binds to the target antigen, and then a second antibody which has some indicator (fluorescent dye, radiolabel, or an enzyme such as alkaline phosphatase which can generate a color or light signal) 

• sample is added to solid support → wash → secondary antibody specific to particular viral antigen → wash → detection 

• if the viral antigen is present in the patient’s sample, then the patient has been infected with that specific virus (is for detection of antigen)

• pros: rapid (1-3 hours); can be read by a machine; very sensitive; no special skills needed, many commercial kits available, can process large numbers of samples 

• cons: requires specific antisera; problems with false positives and borderline results 

Indirect ELISA

• solid support has the viral antigen (or other antigen of interest) conjugated to it; if corresponding antibody (IgG) is present in the patient’s sample, it binds

• → secondary antibody is added; if the primary antibody is present in the patient sample, it binds its constant region and is not washed away → visualization 

• can also detect cytokines (inflammatory response) 

• pros: can be automated and read by a machine; presence of IgM indicates recent infection

• cons: not applicable to all viruses; interpretation may be difficult; patient response can take up to 10 days to develop 

Protein arrays

• chips probed with blood sample (viral antigen or antibody) from a patient

• is just a more advanced/powerful sandwich ELISA, but it’s quite expensive so it’s not really practical to use

DNA microarrays/chip uses 

• can be used to identify novel viruses via sequence homology-based first round screening

• can be used to rapidly diagnose/identify things such as bioterror agents

• can be used to identify specific viral genes or related sets of genes together 

• can be used to monitor virus titer in patients 

• can be used for vaccine quality control by detecting unwanted/contaminated pathogens (both viral and microbrial) in vaccines 

DNA Microarray creation

• preparation of target: amplify genome of target through PCR → spot on the glass chip/microarray (each well corresponding to different genes)

• preparation of probe: isolate RNA from samples → cDNA → label isolated nucleic acid (red for control, green for infected cell) → apply sample on chip → laser scanning to generate heatmap 

• if well 1 is red, that indicates that the gene encoding the oligonucleotide in the well is highly expressed in the control and not expressed much in the infected cells (green) → indicates differential expression of that gene under different conditions 

• depending on mRNA abundance (gene expression), more or less cDNA will be generated, which will generate different colors

• yellow = half/half; both the infected and noninfected cell express the target gene equally 

• not really used nowadays 

One-step growth curves

• used to study a single replication cycle of viruses

• developed by Delbruck to study E.coli T4 bacteriophage

• infection at a high multiplicity of infection (MOI) to ensure simultaneous infection of every cell

• plaque assays for quantification of intracellular and extracellular virus titers

Multiplicity of infection (MOI)

• MOI =1 → have to apply 100 virus particles to 100 cells to ensure simultaneous infection (to study virus propagation kinetics)

• is the ratio of infectious agents to infection targets (i.e. host cells) 

• is influenced by several factors, including the infectivity of the virus, environmental conditions (pH, temperature, etc) and duration of exposure to the virus 

Bacterial growth

• proceeds in a series of phases—lag, log, stationary, and death

• curve shaped like a bucket hat kinda

Viral growth curve

• attachment + penetration: virus titer decreases as viruses enter the cells and disassemble/inject genome/etc → no longer infectious

  • attachment, penetration, and uncoating are ideal to target viral infections (in particular, attachment, since antibodies can be made against the virus’s membrane proteins, receptor proteins, etc → can inject antibodies against the vaccine, which can be helpful especially for those whose bodies cannot generate antibodies well, like the elderly) 

• eclipse: flat, very low as viruses replicate and transcribe genomes

• maturation: increasing linear growth for cell-associated viruses as translation occurs and viruses assemble; remains flat for cell-free viruses (intracellular virus particles form first)

• release: increasing linear growth for cell-free viruses (extracellular virus particles)