virology lec.5 lab diagnosis of viral diseases

Why are definitive lab tests for viral diseases developed?

• patient management

• Availability of some antivirals

• Rapid advancements in drug therapies requires proper diagnosis

• Screening blood supply from donors

• Tracking novel viral stains

• Initiating disease-specific control measures

• Surveillance

Based on Koch’s 4 postulates for proving causation in bacterial disease (1880s)

• specific microbe causes a disease

Challenges with adapting for viruses

Asymptomatic carrier state with a subclinical infections (HIV, Hepatitis C virus)

River’s 6 criteria for viruses

• identification of the etiological agent 1930s

• Isolated viruses from spinal fluid of the patients

1. Isolate virus from diseased hosts

2. Cultivation of virus in host cells

3. Proof of filterability

4. Production of a comparable disease when the cultivated virus is used to infect experimental animals

5. Reisolation of the same virus from the infected experimental animal

6. Detection of a specific immune response to the virus

Fredericks and Relman (1990s)

Used biotechnology and PCR to offer updated casusation guidelines

Factors influencing lab outcome

• type and quality of specimen

• Transport condition and time

Five approaches for lab diagnosis of viral infections

1. Microscopy

2. Viral antigen detection

3. Culture

4. Nucleic acid detection

5. Antibody detection

Microscopy

Light microscopy

• used to observe intracellular inclusions

• Immunihistochemistry (IHC)

Electron microscopy

• used to observe individual virus particles

• Immunoelectron microscopy- concentrate the number of virus particles in a specimen, virus-antibody complexes are concentrated by centrifugation then stained

Viral antigen detection

• enzyme-linked immunosorbent assay (ELISA)

Inexpensive

Technically easy to perform

Rapid turnaround

Culture

Cell cultures used for virus isolation and identification

• monitored by cytopathic effects (CPEs)

Centrifugation culture (shell vial technique)

• rapid diagnosis

• Detects viral antigens before CPEs are present

Nucleic acid detection

Nucleic acid- amplification tests (NAATs):

detect viral nucelic acids

• PCR (DNA virus) and RT-PCR (RNA virus) technology

• NASBA and TMA are non-PCR methods

Diagnosis

Management of patient

• Ex: HIV, hepatitis C patients

• Monitor viral load - virus escaping the treatment

Antibody detection

Presence of antibodies are indirect measure of viral infection

• patient serum contains antibodies

• Recent viral infection: IgM

• Re-infection with same virus: IgG

Methods used

• indirect immunofluorescent assays (IFA)

• ELISA

• Western blot (separation of viral proteins through an acrylamide gel)

DNA microarrays (DNA chip)

Applied to cancer biology and drug and therapy development

Applied in clinical virology

• diagnostic

• Detects agents of bioterror

• Detect presence or absence of viral pathogenicity genes

• Patient management

• Vaccine quality control

• Study of host gene responses to viral infection

Protein arrays

Arrays of antibodies instead of DNA probes are immobilized on the chip

PCR-based strategies

• used to measure and monitor viral load

- PCR

- RT-PCR

• compare viral load measurement after treatment to baseline measurement

• Plaque reduction assays

- gold standard for measuring effects of antiviral drugs

- toxicity assays performed

Working with viruses in the research lab

• viruses need a “host” system

• Viruses can be grown in:

Animals

Embryonated eggs

Tissue (cell) cultures (preferred method)

• optimal growth conditions vary greatly

Vertical flow laminar hood

• Air filtered through high-efficiency particulate air (HEPA) filter

• Removes 99.97% of particles ≥0.3 μm

Cytopathic effects (CPEs)

Visual changes in host from viral infections:

• Formation of inclusion bodies

• Rounding of the cells

• Shrinkage

• Increased refractility

• Fusion/syncytia formation

• Aggregation

• Loss of adherence

• Cell lysis/death

CPEs occur as a result of

• Entry into host cell that is susceptible and permissive

• Inhibition of cellular transcription or stimulation of cellular RNA

polymerase activity

• Virus interactions with RNA processing pathways

• Virus interactions with ribosomes

• Host responses to viral infection

Common methods used to study viruses in the research lab

• plaque assays

• Tissue cultures infectious dose (TCID)

• Neutralization, hemagglutination, and hemagglutination- inhibition assays

• Transformation (focus) assays

• Interference assays

Plaque assays

Quantitative assay measuring number of viruses in a prepared virus stock.

• if know infection is virus, use plaque assays.

• Not in clinical lab

• After centrifuge use water not solid

Tissue culture infectious dose (TCID)50

Endpoint dilution assay

• quantify virus

Neutralization, hemagglutination, and hemagglutination

inhibition assays- detect or quantify virus and strain-specific neutralizing antibodies

• Part virus with antibody and see if neutralized

• Scientists take samples from ppl infected, put omicron, no affect

Transformation (focus) assays

Determine immortalization of cells in culture

Ex: Tumor viruses

• cells transform and cause cancer

Interference assays

Detect viruses that do not cause visual CPEs

How do virus mutat?

Infect population, change thru ppl, and evolve

PCR-based methods uses

• Discover emerging or reemerging viruses, Nucleic acid

sequencing

• Study virus replication in vitro

• Restriction fragment length polymorphism (RFLP)

• Real-time PCR

• Combined with fluorescence resonance energy transfer

(FRET) in biomedical research

Lab safety

Labs classified by biosafety level (BSL)

• BSL-1 (minimum containment)

• BSL-2

• BSL-3

• BSL-4 (maximum containment)

• Protective equipment varies by BSL

Which is typically the first test used to screen for HIV infection?

ELISA

Which of the following would be the most useful in detecting a newly emerging virus that cannot be cultivated in cell cultures?

Nucleic acid-amplification tests

Loss of adherence and increased cell refractility are examples of cytopathic effects observed in virally infected cells. True or false?

True cuz change in cell structure

Differentiate between PCR and RT-PCR, specifically with regard to how they are used to identify viruses.

PCR is used for DNA virus such as Herpes. RT-PCR is used for RNA virus such as SARS CoV2 , it changes the RNA to cDNA to read the virus. 

Detail the advantages and disadvantages of current methods used to diagnose viral infections.

Describe a method that could be used to detect an emerging virus in blood drawn from a patient even though the virus was unable to replicate in cell cultures or animals in the laboratory.

ELISA

Explain why guidelines for designated biosafety levels were created for technicians and researchers in laboratories.

Different labs have different levels of risk and having a guideline for each one helps keeps everyone safe and not exposed to infection.