viral diagnostics

Question 1

Gold standard for virus detection

Answer 1

virus isolation in cell cultures

Question 2

describe traditional tube cultures

Answer 2

Uses a round bottom screw cap tube. Cell monolayer adheres to one side of the tube and are inoculated with clinical specimin. They are incubated then examined by light microscopy for CPE

Question 3

describe Cytopathic effects

Answer 3

cell vacuolization, rounding up of cells, cell fusion and cell lysis

Question 4

Hemadsorption test

Answer 4

alternative approach fro detecting viruses that produce CPE slowly, or not at all. Cell culture medium is removed from traditional tube culture and replaced with solution of erythrocytes. Then incubated and examined by light microscopy. Hemadsorbing viruses will cause the RBCs to adhere in clumps to the cell monolayer.

Question 5

Shell vial cultures

Answer 5

Cell monolayers are grown on a round glass coverslip in a small shell vial. Then, cell growth medium is removed and clinical specimen is added to cells to inoculate. The entire vial is centrifuged at low spped for 1 hr to enhance virus attachment to cells and virus penetration of cells. The vials are then incubated and detected later by staining coverslips with virus-specific monoclonal antibodies. Detected by fluorescent microscopy

Question 6

Advantages and disadvantages of virus cultivation

Answer 6

advantage: isolate wide variety of viruses, provides virus isolate for further studies. Shell vials do not require development of CPE. Disadvantages: long incubation times, some viruses don’t proliferate in cell culture, need for technical expertise to evaluate for CPE

Question 7

Describe PCR for virus detection

Answer 7

Most commonly used method for rapid virus diagnosis. RNA or DNA is isolated from patient > reverse transcription of RNA to form cDNA > DNA or cDNA amplified by PCR using primers for virus pathogen. PCR is qualitative (agarose gels) or quantitative (to determine viral loads). Can also determine sequence of patients sample.

Question 8

List viruses where viral load is clinically relevant

Answer 8

HIV, HCV, CMV

Question 9

multiplex PCRs

Answer 9

can detect multiple different viruses in one assay. Ie HSV-1 and HSV-1

Question 10

PCR advantages/ disadvantages

Answer 10

advantages: high sensitivity and specifity, short turnaround time, useful for viruses that cant be cultured. Disadvantages: lack of standardized protocols or FDA approved kits, technical expertise required, cost of equipment and reagents

Question 11

rapid immunoassays

Answer 11

FDA approved kits for rapid detection of influenza A and B, RSV. rapid (10-15 minutes). A nasal or throat swab is mixed with buffer to extract antigens. Test strip embedded with antibodies against flu, RSV, etc placed in buffer. Color develops if virus protein is in the sample

Question 12

Rapid immunoassays advantages and disadvantages

Answer 12

High specifity, minimal technical expertise required. Poor sensitivity (false negatives are common)

Question 13

Western blot

Answer 13

Uses antibodies to recognize viral proteins separated in an SDS polyacrylamide gel. Can be used to detectanti-viral antibodies or actual viral proteins in patient sera

Question 14

Direct immunofluorescence

Answer 14

Used to detect virus infected cells. Cells are placed on glass slide and monoclonal antibodies against the viruses are used. Quite labor intensive, but fast and definitive

Question 15

serology

Answer 15

Since there are many normal viral flora, it is important obtain serum from patient during acute infection, then several weeks later during covalescence. A positive test requires at least a four fold increase in circulating antibody titer against the virus btw acute and convalescent serum specimens.

Question 16

ELISA

Answer 16

Viral proteins are attached to a solid phase, then incubated with patients serum to allow antibodies from pts serum to attach. Then an anzyme linked reagent is added which binds to IgG or IgM from pts serum. Amount of antibody is quantitated by intensity of color reaction. sensitive

Question 17

Classic method used to determine viral size

Answer 17

Tissue culture medium with virus is filtered through 300nm, 100nm and 50nm filters. Then the filtrate is inoculated onto tissue culture and observed for evidence of viral replication

Question 18

Classic method to determine type of nucleic acid in virus

Answer 18

virus is inoculated into one normal tissue culture medium and into the same medium containing BrDU which inhibits replication of DNA containing viruses but not RNA containing

Question 19

classic method to determine presence of lipid containing viral envelope

Answer 19

virus is treated with chloroform or ether, which solubulizes the envelope and inactivates the virus, but does not affect infectivity of non enveloped viruses

Question 20

Virus neutralization test

Answer 20

Antisera against different strains of a virus are mixed with patients virus in tissue culture and incubated. Evidence of virus replication indicates that the virus was not neutralized by the antiserum and therefore not the same as the reference virus.

Question 21

Hemagglutination assay

Answer 21

Many viruses can bind RBCs and form a lattice. Dilutions of virus are mixd with defined quantity of RBCs in 96-well plate. Unabsorbed RBCs fall to the bottom of the well and form a sharp dot. Agglutinated RBCs form a lattice that coats the entire well

Question 22

Hemagglutination inhibition

Answer 22

If a virus is identified by hemagglutination, treatment of the virus with anti-viral antibody can prevent hemagglutination. In this assay, dilutions of serum are incubated with virus and red blood cells.

Question 23

How can you identify a wild type virus vs live attenuated virus from vaccine

Answer 23

Live attenuated viruses in vaccines contain known marker mutations that make them different from the virulent wild type. PCR and DNA sequencing can be used to determine which variety a patient has. This can be important for polio, because the oral polio vaccine virus virtually always mutates to become virulent, so it can be passed on to contacts of the vacinee (though the vacinee rarely gets sick)