VIRO 3 | Research Methods for Studying Viruses

Virus cultivation initially involved _ bc _

• Infecting animals

• Viruses can only multiply inside living cells

When using animal models as research method for studying viruses, _ must be studied

natural course of infection (pathogenesis = how virus causes disease)

Why must pathogenesis (i.e., process of how a virus causes disease) be studied when using animal models as research method for studying viruses?

Without studying pathogenesis, the animal model may not be able to accurately reflect how virus causes disease bc key aspects, including which tissues are infected, how immune system responds, and what symptoms develop, may be missed or misinterpreted, leading to false assumptions in disease severity, transmission, & treatment effectiveness

From the 3 animal models (ferrets, mice, guinea pig), _ which share most human clinical features following an influenza virus infection, including _

• Ferrets

• Human clinical features following IV fnc gstd nw

  • Fever

  • Nasal secretions

  • Cough

  • Gastrointestinal complications

  • Serum abnormalities

    *shared with guinea pigs

  • Transmission to susceptible contacts

  • Distribution of sialic acid (SA) receptors in respiratory tract

    *shared with mice

  • Neurological complications

  • Weight loss/anorexia

Bc viruses can only multiple inside living cells, another way through which they can be cultivated would be by creating _

embryonated eggs, which is created by poking a small hole on surface of egg shell, inoculating an appropriate tissue with the virus, then sealing it with wax or gelatin

Explain 4 tissues commonly inoculated when cultivating viruses via embryonated eggs

• Chorioallantoic membrane hrp

  *Rich in capillaries and thus ideal for pock-foming viruses

  • Herpes simplex virus

  • Rous sarcoma virus

  • Poxvirus

• Amniotic im

  Suitable for isolating respiratory viruses

  • Influenza virus

  • Mumps virus

• Yolk sac

  Nutrient-rich, can support fastidious organisms

  • Herpes simplex virus

• Allantoic imna

  Used for high-yield virus production

  • Influenza virus

  • Mumps virus

  • Newcastle disease virus

  • Avian adenovirus

Explain significance of cultivating viruses through embryonated eggs

Influenza virus vaccines are produced through inoculation of embryonated eggs to date, specifically flu viral components, e.g., antigens

*Embryonated eggs at 10-12 days; 1st larger needle punching a hole in shell; 2nd smaller needle inject virus seed into allantoic cavity, followed by 2-3 days incubation

_ refers to the structural & functional changes occurring in host cell due to viral infection

Cytopathic effects

Give examples of cytopathic effects

plmb

• Pock marks or lesions on chorioallantoic membrane of egg

• Leaf lesions from infection with potato virus Y

• Mosaic & chlorosis in leaf lamina

• Bumps & ringspots in papaya infected with Papaya ringspot virus

T/F: Initially, observing cytopathic effects was a crude way to quantify virus titer because you can count the pock marks indicative of infectious virions in the solution

TRUE

When an egg is inoculated with virus, _ are observed as cytopathic effects indicative of viral infection

Pock marks or lesions

Except studying pathogenesis, the use of animal models for virus cultivation is generally being taken over by _

faster & cheaper molecular biology methods

Explain 3 choices of virus culture system

• Animal culture

  • Advantage

    • Natural course of infection

  • Disadvantages

    • Upkeep is expensive

    • Variations between individuals, even if inbred, require large numbers

    • Ethical considerations

• Organ culture (pieces of brain, trachea, gut)

  • Advantages

    • Natural infection (mimics site)

    • Differentiated cell types present

    • Fewer numbers needed

    • Less variation since 1 animal gives many organ cultures

  • Disadvantages

    • Unnatural since the culture is no longer subjected to homeostatic responses of their immune system

• Cell culture

  • Advantages

    • Can be cloned and thus variation between individuals is minimal

    • Good for biochemical studies bc environment can be quickly & exactly controlled

  • Disadvantages

    • Harder to study entire pathogenesis

  • 3 types of cell culture

    • Primary cells = derived from organ/tissue, differentiated but can only survive few passages

      • Cell senescence is bc of telomere shortening due to cell replication cycles

    • Cell lines = undifferentiated but are diploid & survive larger numbers of passages (~50)

      • Bc undifferentiated, they may not fully replicate specialized functions of original tissue

    • Continuous (permanent) cell line = dedifferentiated but immortal; infecting these with virus can create continuous cell line

What is the significance of the differentiation state of cells in a cell culture system?

• Differentiated cells in primary cells = closely mimic the structure & functions of original tissue but can only survive few passages

• Undifferentiated cells in cell line = have more proliferative capacity but could lose some specialized functions, making them less representative of in vivo tissue

• Dedifferentiated cells in continuous cell line = can divide indefinitely, making these ideal in large-scale / long-term experiments, but would often behave very differently from normal cells due to altered gene expression & abnormal karyotypes

TLDR: The more differentiated, the more representative of original tissue but the lower the survivability; the more dedifferentiated, the more the proliferative capacity but the farthest from being representative of original tissue

Explain examples of each type of cell culture

1. Primary human foreskin fibroblast

   1. Cells are derived from embryo / adult organism then cultured under growth medium

   2. Differentiated cells eventually stop dividing due to cell senescence or shortening of telomeres due to replication cycles

   3. After a few passages, differentiated cells (most representative of in vivo tissue) stop dividing

2. Established line of mouse fibroblast (3T3)

   1. Divide a little longer than primary cells but still have finite number of replications

   2. Undifferentiated = lost SOME specialized functions

3. Continuous cell line of human epithelial cells (HeLa)

   1. HeLa = first immortalized cell line

   2. Dedifferentiated cells that have undergone transformation via accumulation of mutations or viral infection, which gave them ability to divide indefinitely but also behave differently from original tissue

   3. Used for long-term experiments but do not mimic natural tissue behavior

How are these grown in the lab

• Cells form a monolayer at the bottom of culture dish, then immersed in growth medium

• Observed under inverted microscope (lens below, light source above) bc cells adhere and thus grow on bottom of plate

Explain extensively all considerations when setting up cell culture systems

• Difficulties

  • One of the difficulties would be avoiding bacterial & fungal contamination

  • Add antibacterials + antifungals

  • Add penicillin-streptomycin; BUT BE CAREFUL when adding bc this could overwhelm your animal cells and cause them to die

  • Apart from growth medium components, fetal bovine serum is also added to provide essential growth factors needed for cell division

• Source

  • Sources of primary cell cultures are usually either duck or mice embryo bc these are rapidly dividing cells

  • For duck embryo

    • Get fertilized egg, break it open, obtain embryo, then dissect for tissues you want to culture

    • e.g., connective tissue, heart cells, fibroblasts, liver, brain cells

    • On the correct medium, heart cells can still beat as long as provided with calcium in proteins

  • For mice embryo

    • Mucus plug in vaginal area indicates successful mating

    • You count days post-mating, then extract bead-like embryos and dissect them for tissues

  • Tissue obtained must be macerated & homogenized via

    • Physical means = blade

    • Enzymatic method = trypsin (proteolytic enzyme that digests extracellular matrix)

• Monolayer formation via contact inhibition, subculturing

  • Allow culture to adhere to bottom of flask

  • Eventually, this will grow into a monolayer then experience contact inhibition (when cells run into each other, they stop dividing)

  • Adhesion cell culture > primary cell culture

  • If you want to culture more cells, u can passage this after several days to form cell lines (undifferentiated & would lose SOME specialized function but could divide more than primary cells)

  • Eventually, after several passages, primary cells would no longer divide

  • OR they could undergo transformation via mutation accumulation / viral infection that allows them to become “immortalized” or divide infinitely but could likely become genetically unique from original tissue culture

• Incubation

  • Cell cultures are usually incubated in CO2 incubator with specific oxygen amount & humidity that must be maintained

• Glasswares

  • Culture disks or flasks are made of special plastics that allow cells to adhere to bottom

  • Ensure aseptic conditions; work in separate BSCs when working with diff types of cultures (bacteria/fungi/virus)

• Scaling up via suspension culture

  • However, adherent/monolayer culture are only small-scale cultures that can accommodate 20-50 mL

    • Adherent cultures can only accommodate small volume bc these are limited to available surface area and having small volume allows nutrients & O2 to reach cells more evenly & efficiently

  • To scale up to liters of culture, you can create suspension culture via trypsinization, which would detach adhering cells on bottom and allow them to float freely within medium

    • Suspension cultures can accommodate larger volumes bc cells are simply floating freely in medium that could be stirred by bioreactors, thus would have no concern with regards to even nutrient & O2 distribution

Explain how to scale up virus cultivation using adherent or suspension cells

• Adherent cells (cells grow attached to surfaces)

  • Wash them, give them fresh medium, then infect

  • Directly infect

• Suspension cells (freely floating)

  • Directly infect

  • Increase cell density, then infect

• Once virus is harvested, these are subjected to fip

  • Filtration

  • Inactivation

  • Purification

Explain cytopathic effects & adherent cells

• Cytopathic effects can also be observed in adherent cells

• Fetal tonsil fibroblast

  • Fibroblasts = connective tissue cells commonly used to make primary cell culture

  • (B) Fibro infected with adenovirus

  • (C) Fibro infected with HSV

  • Different viruses have different cytopathic effects

  • Not all viruses will produce cytopathic effects

  • Microscopically, you may not be able to confirm viral infection

• Vero cells

  • (B) Vero cells infected with poliovirus = cells are clumping together & no longer exhibit polygonal shape; not adhering to culture flask anymore

  • (C) Vero cells infected with herpesvirus = cells are merging and becoming multinucleated, forming a synctium

  • Synctium formation = glycoproteins on surface of infected cells binds receptors on neighboring cells, causing fusion

Explain tissue / organ culture

• Aside from cell culture, tissue/organ culture may also be used

• Tracheal organ culture is most commonly used for studying respiratory viruses

• You must keep the intact cell still with cilia on apical surfaces, basal cells

• When tissue culture is infect with rhinovirus, it becomes very disorganized

• But again this is limited by being unnatural since the culture is no longer subject to homeostatic responses of immune system; cannot study pathogenesis

Explain plaque assay

• Plaque assay is one way to observe cytopathic effects

  • Used to estimate virus titer; no. of plaques = no. of infectious virus particles in solution

  • Used to biologically clone a virus, i.e., isolate pure form from mixture of types

  • Only applicable for viruses that lyse their host cells

• Plaque formation

  • Plaque = hole in monolayer formed due to 1 cell being infected by a virus; viral replication lyses host cell

  • Adjacent cells become lysed, forming plaque

  • Plaque is like a colony; we assume that plaques are formed by the same virus infecting the cell

  • Number of plaques = depend on virus titer (number of virions present in initial solution)

• Multiplicity of infection = average no. of virions added per cell during infection

  • Not a natural trait of virus but a controllable quantifiable measure that depends on number of virions added; number of cells present

  • If MOI = 1, then 1 virion is added per cell during infection, BUT this does not necessarily mean that if u have 5 cells and if u add 5 virions, all will be infected

  • Instead, higher MOI = higher probability of all being infected; lower MOI = lower chances of all being infected (plaque formation)

• Serial dilution

  • e.g., Poliovirus inoculum, monkey kidney cell monolayer

  • Viral inoculum should be serially diluted; chose the most diluted or the dilution that will allow countable number of plaques

  • Inoculate diluted virus solution into monolayer culture, then allow virus to adhere

  • After some time, add semi-solid agarose solution because viruses move freely in liquid medium; thus, overlaying it with semi-solid agarose solution would prevent virus from diffusing all over the plate, preventing formed plaques from overlapping, and forming distinct countable plaques

• PFU/mL

  • Virus titer / original conc. = number of plaques/mL used (DF)

    • DF = fositive

  • PFU/mL because it is uncertain whether each plaque formed is formed by a single virion

Plaque assay problem

0.10 ml of a 10-6 dilution of the virus preparation yields 75 plaques. What is the original concentration of PFUs?

• PFU/mL = no. of plaques / mL (DF)

• = 75 / 0.10 mL (10⁶)

• = 7.5×10⁸ PFU/mL

Explain foci-forming units (FFU), quantal assays (LD50, ID50) as other ways to measure groups of infected cells

• Foci-forming units (FFU)

  • Pock marks can be counted as quantitative measure for infected cells

  • Sometimes, viruses don’t lyse/kill host cells, thus a group of infected cells called foci may be counted

  • e.g., Bovine coronavirus-infected cells express a viral envelope protein that binds to red blood cells

  • To quantify infected cells, u can immunostain virus with specific antibody conjugated to chromophore to count foci

• Quantal assays (endpoint dilution assays)

  • Sometimes, you’re not interested in inoculum but in LD50, ID50

  • LD50 = dilution with dose large enough to kill 50% of cells

  • ID50 = dilution with dose large enough to infect 50% of cells

  • Setup = 10-fold dilutions of virus inoculated into cell cultures grown in wells; 1 mL of virus suspension is inoculated into each well; after incubation, wells are marked with ± for CPE

  • Dose response curve = useful for determining dilutions needed to kill/infect a certain % of cells

    • Y = % of dead/infected cells

    • X = dilutions of virus suspension

Explain growth curve quantitative assay as another way to measure group of infected cells

• Bacteria = cell numbers over time

  • Optical density = limited bc there could still be dead cells in there

  • To count live cells, plate inoculum

• Viruses = abrupt

• One-step growth curve

  • Eclipse period = time until the viruses are assembled and thus detected

  • Latent period = time until viruses are released from infected cells

  • When cell is infected with virus,

    • undergoes eclipse phase = where no viruses are detected bc there is no virus assembly yet

    • even if virus has entered host cell & lysed it; lysate is used to infect other host cells > no assembly of virions yet

  • Log phase = occurs when virions have been assembled

  • Followed by stationary phase = release of all virions

• One-step vs. Two-step: which would have lower MOI?

  • One-step = higher MOI bc this means that it only took 1 burst for all cells to be infected, implying that there were sufficient virions to infect all cells

  • Two-step = lower MOI bc first burst released virions not enough to infect all cells, hence 2nd burst was needed to infect remaining viable cells

• Can be determined using plaque assay

  • However, eclipse phase cannot be detected intracellular & intracellularly

  • Virions are only detected intracellularly after assembly; while virions are detected only extracellularly after release from infected cells

Explain hemagglutination & ELISA as other ways to measure no. of infected cells

• Hemagglutination

  • Some viruses agglutinate RBCs bc they bind to proteins on RBC surface

  • If virus agglutinates RBCs, mixture spreads into a lattice at bottom of well (+)

  • If virus doesn’t, RBCs will settle as tight button at bottom of well (-)

  • Hemagglutination titer = reciprocal of highest dilution that shows positive agglutination results; as virus becomes more diluted, it loses the ability to agglutinate

    • Highest dilution = 1/128

    • Hemagglutination titer = 128

• Enzyme-linked Immunosorbent Assay (ELISA)

  • Enzyme-linked = conjugation of antibody with reporter enzyzmes

  • Immuno- = antibody-antigen interactions

  • -Sorbent = adsorption of antibody/antigen

  • Purpose

    • To detect viral antigens/host antibodies

    • Color change = binding / (+)

  • Setup components

    • Antigen/antibody is adsorped to plate

    • Blocking agent (skim milk) is added to prevent nonspecific binding

    • Primary antibody/antigen binds to target antibody/antigen

    • (Indirect) Secondary enzyme-linked antibody binds to conserved region of primary antibody

    • Add substrate > color change > (+)

  • Indirect ELISA = detects host antibodies

    • Coat plate with viral antigen

    • Add blocking agent then patient serum with antibodies

    • Target antibody binds to antigen

    • Secondary enzyme-linked antibody binds to conserved region of primary target antibody

    • Substrate > color change

    • Common for serological testing

  • Direct ELISA = viral antigens

    • Target antigens adsorp to plate

    • Enzyme-linked antibody binds to target antigen

    • Substrate > color change > (+)

  • Sandwich ELISA = low-level viral antigens

    • Coat plate with primary capture antibody

    • Target antigens bind to capture antibody

    • Secondary enzyme-linked antibody binds to different epitope of the same target antigen

    • Substrate > color change > (+)

  • Considerations

    • COVID-19 = lateral flow antigen test (ELISA principle)

    • HIV = direct virus detection is hard; detecting viral antibodies (indirect ELISA) is preferred

    • 2-antibody system = cost-efficient bc generic secondary metabolites can be reused; no need to label every primary antibody bc universal 2ndary antibody

Explain NAATs as viral detection techniques

Nucleic Acid Amplification Techniques (NAATs)

• PCR

  • Template = DNA

  • To amplify specifc DNA sequences using Taq polymerase, which has no proof-reading abilities and prioritizes speed > accuracy

  • High-fidelity amplification = PFU / PMX polymerase

    • Important if u plan to express DNA product later

• RT PCR

  • For viruses with RNA

  • Reverse transcriptase converts RNA to cDNA

  • DNA polymerase synthesize dsDNA

• High-Throughput Sequencing

  • Enables analysis of multiple samples at once from amplification to sequencing

  • Useful for vef

    • Virus population genomics (insights into how RNA viruses could have different sequences in an individual - quasispecies)

    • Evolutionary biology & outbreak tracing = u can look at outbreaks & figure out spatial relationship between outbreak & metagenomics (environment-derived genetic material)

    • Phylogenetic analysis = phylogenetic trees can show reinfection & lineage relationship of SARS-CoV-2

• Detecting viruses via metagenomic analysis

  • Analyzes environmental / biological samples without isolating individual viruses

  • Samples: Soil, oceanwater, human tissue, wastewater

  • Key techniques

    • DNA/RNA extraction

    • PCR / RT PCR

    • Sequencing

  • Identifies tvf

    • Taxonomic profiles = which viruses are present

    • Virulence factors = assess functional potential & pathogenic risk of microbial community

    • Functional genomics = what each gene does

• Real world example

  • During COVID-19, SARS-CoV-2 RNA was detected in wastewater

  • Sick individuals excreted viral particles into wastewater system

  • Wastewater was analyzed to ets

    • Estimate community viral load

    • Track prevalent strains

    • Support public health surveillance, contributing to early detection & monitoring emerging outbreaks before clinical testing is widespread

Explain viral diagnostics

• Targets for viral diagnostics

  • Viral proteins/antigens = detect via immunoassays using naturally occurring or recombinant antibodies

  • Viral genomes = NAATs

  • Antiviral antibodies = Indirect elisa; detects only exposure, not necessarily active infection

• Diagnostic test qualities

  • Sensitive = should detect even small infections

  • Specific = prevent false positives

  • Rapid = to provide quick results

• e.g., Lateral Flow Immunoassay (Rapid Antigen COVID-19 Test kit)

  • Nitrocellulose adsorbent membrane carries sample via capillary action

  • Sample pad = where u put sample

  • Conjugate (Primary antibody) pad = binds viral antigens present

  • Test line = immobilized labeled secondary antibody binds to different epitope of same antigen

    • (+) = red

  • Control line = to see if test works; red = valid if it binds unbound labelled primary antibodies

• Samples

  • Blood = detecting antibodies

  • Nasal swab = detecting antigens

• Antibody tests = exposure not active infection

• Antigen tests = active infection

• Gold Standard = RT-qPCR

  • Amplifies & quantifies viral RNA

  • Cycle threshold (Ct) = cycle number at which viral NA become detectable

  • Lower Ct = more virus particles present

Explain other methods of viral diagnostics (Pap smear, EM)

• CPE & Pap smear

  • Pap smear = used to detect cervical cancer caused by Human Papillomavirus (HPV)

  • In this method, epithelial cells (superficial, intermediate, parabasal, LSIL, HSIL) are observed under microscope

  • For HPV, CPE manifests as uncontrolled cell division, which may lead to tumor formation

• Electron microscopy (EM)

  • Uses electrons instead of light to form image

  • How it works

    • Beam of electron passes through magnets & is focused on specimen

    • Specimen scatters electrons & scattered electrons are detected by scanner

    • Computer then interprets these signals into high-res image

  • Transmission EM

    • Uses wide electron beam

    • Produces 2D image

    • Visualizes internal structures of virus

  • Scanning EM

    • Uses fine electron beam

    • Produces 3D image

    • Only shows surface structure of virus

  • Limitations

    • Requires sample fixation = samples need to be preserved & dehydrated

    • Biological samples don’t scatter electrons well; have to be coated with thin metallic layer (gold) to be visualized

Explain summarized Protocol for Isolating Influenza A Virus

1. Sample collection & processing

   1. Human surveillance (swabs & liquids)

   2. Field surveillance (animal tissues)

   3. Experimental source

2. Virus culture

   1. Embryonated chicken eggs

   2. Mammalian cell culture

3. Identification of Influenza A virus

   1. Classical = HA = uses RBCs to detect presence of viral hemagglutinin

   2. Molecular = RT-PCR = amplifies viral RNA