Virus Detection and Diagnosis

1. immunological methods
2. molecular methods
3. hemagglutination methods

What are the 3 main virus diagnostic methods?

immunological methods

This type of virus diagnostic method detects viral proteins (antigens).

molecular methods

This type of virus diagnostic method detects viral nucleic acids.

hemmaglutination

This type of virus diagnostic method is often used for some enveloped viruses like influenza; RBCs clump together in the presence of viral particles as a result of interaction between HA proteins of viruses and RBC, leading to lattice formation.

1. direct
2. indirect
3. capture (sandwich)

What are the 3 common formats of enzyme-linked immunosorbent assay (ELISA) under immunological methods?

enzyme-linked immunosorbent assay (ELISA)

This immunological method is the most frequently used diagnostic tool for virus detection; uses a solid phase enzyme immunoassay to detect the presence of an antigen in a liquid sample.

True.

ELISA can be formatted to detect antigens as well as antibodies.

T or F?

immunoflyorescence assay (IFA)

This immunological method is a microscopic method that can detect and visualize the viral proteins expressed in cells via antigen-antibody reactions.

secondary antibody (specific to the Fc fragment of the IgG molecule of the primary antibody)

In IFA, which antibody is fluorophore-labeled and attaches to another antibody with the target protein?

paraformaldehyde

In IFA, cells are fixed by?

Triton X-100

In IFA, cells are permeabilized by a detergent such as?

patient sera

In IFA, a specific antibody termed is applied on the surface so that the antibody recognizes the viral antigen.

immunoblotting

This immunological method is also known as the Western blot; can readily detect proteins transferred onto a solid support (e.g., nitrocellulose membrane).

SDS-PAGE

What is the technique used before engaging into Western blotting to ensure that the proteins are resolved?

primary antibody: for antigen

secondary antibody: linked to another enzyme (AP or HRP) that allows for visualization

In immunoblotting, which antibody (primary, secondary) is linked to another enzyme (AP or HRP) that allows for visualization? Which is specific for an antigen?

enhanced chemiluminescence (ECL)

In the last step of immunoblotting, the protein bands on the membrane are visualized by .

polymerase chain reaction (PCR)

This molecular method for viral diagnostics allows the diagnosis of the viral infection at very low titer (<106 particle per mL); relies on thermal cycling, consisting of cycles of heating and cooling of the reaction for DNA melting and enzymatic amplification of the DNA.

real-time PCR

This molecular method for viral diagnostics is capable of monitoring PCR products as it is being amplified; main feature is a specially designed oligonucleotide probe that has a fluorophore attached to one end, while a quencher is attached at the other end.

hemagglutinin (HA)

In hemmaglutination, this is an enveloped glycoprotein of some enveloped viruses which imparts the property of viruses that can adsorb to RBCs.

False. Highest dilution.

In hemagglutination, the lowest dilution at which clumping is observed is regarded as the HA titer of the sample.

T or F?

1 HA = 10^4 particles per mL

In hemagglutination, estimate the virus titer in a sample by miltiplying the dilution fold wherein 1 HA is equal to how many particles per mL?

cell lines

This is the most convenient laboratory virus cultivation method; usually immortalized cells (coming from cancer cells) that have acquired the ability to proliferate indefinitely.

primary cells

These cells are isolated from normal human/animal cells; filtered, centrifuged, and transferred to a plate for cultivation and culture by adding culture medium with 10% fetal bovine serum (FBS).

subculturing, definite multiple passages, and immortalization

How can primary cells become immortalized cell lines (indefinite multiple passages)?

embryonated eggs

This is considered to be the earliest method for the cultivation of some viruses prior to the use of tissue culture technology; utilized for the propagation of influenza virus, for vaccine production in particular.

True.

For a given virus, both natural and nonnatural hosts are used as experimental animals which are indispensable for antiviral drug development and vaccine development.

T of F?

humans

This animal model is the only natural host for some human pathogenic viruses such as HIV.

mice

This animal model is the foremost choice for human influenza virus.

primates (chimpanzee)

This animal model is the foremost choice for HIV.

plaque assay

This virological quantification assay is a standard method that has been long used to determine thr virus titer (infectious dose).

end-point dilution assay

This virological quantification assay is the method of choice for a subset of viruses that do not form plaques but cause cytopathic effect (CPE) in the host cells which are viewed under the microscope.

1. plaque assay
2. end-point dilution assay
3. hemagglutination assay

What are the 3 virological quantification assays?

PCR and RT-PCR

What are the 2 molecular quantification assays for viruses?

1. plaque isolation (biological)
2. centrifugation (biochemical/physical)

What are the 2 ways to purify viruses?

plaque isolation

This biological method to purify viruses involves a qualitative isolation of genetically identical virus particles.

True.

Plaque isolation as a biological method to purify viruses is only available for plaque-forming viruses.

T or F?

density-gradient centrifugation (specific for virus particle isolation)

This method to purify viruses is a separation technique that separates objects from specifmens by centrifugal force.

PCR-mediated mutagenesis

This process enables us to generate all kinds if mutations on the replicon plasmid, thanks to molecular virology and recombinant DNA technology.