Laboratory diagnosis of viral infection

Question 1

Virus isolation/culture

3 types

_____
_____
_____

Answer 1

Cell culture
Egg inoculation
Animal inoculation

Question 2

Egg inoculation

________ are among the most useful form of living animal tissue

Used for the ____ of viruses or _____ viruses

Used for _________ cultivation in the production of viral vaccines

Answer 2

Embryonated eggs

isolation; titrating

large quantity

Question 3

Animal inoculation

In 1909, Landsteiner & Popper used ______ to isolate Polio virus.

_____,______,_______,_______ are now commonly used

Answer 3

monkeys

Rabbit, Guinea pig, mouse, rat

Question 4

Earliest method for cultivation of viruses causing human disease is by ______

Answer 4

Animal inoculation

Question 5

Animal inoculation

The growth of virus in inoculated animals may be indicated by _____,_____ or _______

Answer 5

death, disease or visible lesions.

Question 6

Disadvantages of animal inoculation are that they may ______ with viral growth and the animals often _________

Answer 6

interfere

harbour latent viruses.

Question 7

Chorioallantoic membrane inoculation is used for which viruses

Answer 7

Herpes simplex
Pox
Rous sarcoma

Question 8

Amniotic inoculation is used for which viruses

Answer 8

Influenza
Mumps

Question 9

Yolk sac inoculation is used for which viruses

Answer 9

Herpes simplex

Question 10

Allantoic inoculation is used for which viruses

Answer 10

Influenza
Mumps
Newcastle disease virus
Avian adenovirus

Question 11

Cell culture

Primary cell culture

It consists of ____ cells, freshly taken from body & cultured.

They are capable of _____ growth in culture & cannot be maintained in _____

Eg. Monkey, kidney, human embryonic kidney, human amnion & chick embryo cell culture.

Answer 11

normal; limited; serial culture.

Question 12

Cell culture

Primary cell culture

They are useful for __________ and for ________

Answer 12

isolation of viruses & for vaccine production.

Question 13

_______ is best cell culture systems available

Why?

Answer 13

Primary cell culture

since they support the widest range of viruses

Question 14

Primary cell culture is quite cheap

In Primary cell culture it is often difficult to obtain a reliable supply

T/F

Answer 14

F, they are very expensive

T

Question 15

Cell culture

Diploid cell culture

These are used to ensure ___________

After ____ serial passages, they undergo ________.
Eg. Human _______

Answer 15

a continuous supply of cell line.

50; senescence

fibroblasts

Question 16

Diploid cell culture are useful in viral vaccine production.

T/F

Answer 16

T

Question 17

Cell culture

Continuous cell culture

These are single type usually derived from _____ cells that are capable of __________________________________.

Eg. _____ for rabies vaccine, ___ , HEp-2, LLC-MK2

Answer 17

cancer

continuous serial cultivation indefinitely

Verocell; Hela

Question 18

Continuous cell culture are not useful for vaccine production.

T/F

Answer 18

F

They are

Question 19

________ cell culture is most easy to handle

Answer 19

Continuous

Question 20

the range of viruses supported by continuous cell culture is limitless

T/F

Answer 20

F

often limited

Question 21

Mention the types of cell cultures

Answer 21

Primary
Diploid
Continuous

Question 22

Cell cultures

Growing virus may produce

1. ________
2. ________

Answer 22

Cytopathic Effect (CPE)

Haemadsorption

Question 23

Cell cultures

Growing virus may produce

1. Cytopathic Effect (CPE) - such as the ______ of cells or ______ formation, may be specific or non-specific.
2. Haemadsorption - cells acquire the ability to _______________

Answer 23

ballooning; syncytia

stick to mammalian red blood cells

Question 24

Cell cultures

Confirmation of the identity of the virus may be carried out using ______,__________, or __________ tests.

Answer 24

neutralization, haemadsorption-inhibition or immunofluorescence

Question 25

Syncytium formation is seen in cell culture caused by ____ ,and _____ virus

Answer 25

RSV measles

Question 26

Problems with cell culture _____ period (up to _____ ) required for result. Often very poor ________ ,which depends on a large extent on the _________. Susceptible to ___________.

Answer 26

Long; 4 weeks sensitivity; condition of the specimen. bacterial contamination

Question 27

Problems with cell culture Susceptible to ________ which may be present in the specimen. Some viruses will not grow in cell culture e.g. ________,________,________,________

Answer 27

toxic substances Hepatitis B, Diarrhoeal viruses, parvovirus, papillomavirus.

Question 28

Light microscopy Detecting the ____________ in smear & tissue.

Answer 28

viral inclusion body

Question 29

Light microscopy Inclusion bodies are ________ of stainable substances, usually proteins. Can either be : _______ inclusions or ________ inclusions

Answer 29

dense aggregates Intra nuclear Intra cytoplasmic

Question 30

Electron microscopy Virus particles are detected and identified on the basis of ______ ______ virus particles per ml required for visualization ______-______ magnification normally used.

Answer 30

morphology 10^6 50,000 - 60,000

Question 31

Electron microscopy Use Vesicle Fluid for ______ or ____ Skin scrapings for _____,_____,____ Faeces for ____,____,_______,______ ,______

Answer 31

HSV VZV papillomavirus, orf molluscum contagiosum Rotavirus, Adenovirus Norwalk like viruses Astrovirus, Calicivirus

Question 32

Molecular methods ________ _________ reaction _______ reaction __________ based amplification ________ mediated amplification

Answer 32

Hybridisation Polymerase chain Ligase chain Nucleic acid sequence Transcription

Question 33

Polymerase chain reaction or PCR is a simple laboratory technique to obtain ______ of _____ fragments even from samples containing only ____ quantities of DNA or RNA.

Answer 33

multiple copies specific DNA minute

Question 34

Essential components of PCR ______ ________ ________ ______ cations Deoxynucleoside triphosphates (dNTPs) ________ solution

Answer 34

Template DNA Primers Thermostable DNA polymerase enzyme Divalent Buffer

Question 35

Essential components of PCR Template DNA that contains the ______ of interest to be amplified during the PCR.

Answer 35

target sequence

Question 36

Essential components of PCR Primers- A pair of synthetic ________ (______ and ____ primers) that are complementary to the ___’ ends of each of the two strands of target DNA.

Answer 36

oligonucleotides forward and reverse 3

Question 37

Essential components of PCR Thermostable DNA polymerase enzyme like ____ polymerase (originally isolated from ————————- ) is a vital ingredient of a PCR to _____ the _____________________ of DNA.

Answer 37

Taq thermophilic bacterium Thermus aquaticus catalyse; template-dependent synthesis

Question 38

Essential components of PCR Divalent cations, usually ____ are required in (minute or optimum?) concentration for the activity of most __________ as well as for several other steps in PCR.

Answer 38

Mg 2+ optimum thermostable DNA polymerases

Question 39

Essential components of PCR Deoxynucleoside triphosphates (dNTPs)- ________ amounts of each dNTP (dATP, dCTP, dGTP, dTTP), which are ______ used by the DNA polymerase enzyme to synthesize a new strand of DNA.

Answer 39

Equimolar building blocks

Question 40

Essential components of PCR Buffer solution to maintain ____________ for optimum activity and ______ of the _____

Answer 40

suitable ionic environment stability DNA polymerase.

Question 41

STEPS IN PCR ______ ———— ________

Answer 41

Denaturation Annealing Extension

Question 42

STEPS IN PCR Denaturation: The first step of a PCR where the sample is ____ to _____ or _____ the two strands of the DNA (>_____oC).

Answer 42

heated separate or denature 90

Question 43

STEPS IN PCR Annealing: the reaction temperature is ______ (usually ___-__ 0C below the Tm of _____ )to allow the ————- to bind to the single strands of the template DNA.

Answer 43

lowered 3-5; primer oligonucleotide primers

Question 44

STEPS IN PCR Extension: the temperature is ______, typically to ____°C, allowing specific enzymes to synthesize a new DNA strand complementary to the DNA template.

Answer 44

Raised 72

Question 45

STEPS IN PCR Typically about ___-___ cycles of PCR are performed depending upon the _________ used, the __________ and the ______________ for post-PCR processing.

Answer 45

25 to 45; type of PCR ; amount of initial template DNA ; number of amplicon copies desired

Question 46

STEPS IN PCR The PCR is commonly performed in a reaction volume of ____ μl in small reaction tubes (___-___ ml volumes) in a _______ that heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction.

Answer 46

10–200; 0.2–0.5 thermocycler

Question 47

Immunological assay Antigen detection •_________ assay (ELISA) •_____________ •___________/_________ assay

Answer 47

Enzyme linked immunosorbent Immunoflourescence Immunochromatographic /lateral flow

Question 48

Immunological assay Antibody detection and quantification •_____ •_____________ test •__________ test

Answer 48

ELISA Complement fixation Immunoflourescence Rapid

Question 49

Post PCR analysis Agarose gel electrophoresis. PCR products can be visualized by staining the gel with _______ such as ______ which binds to DNA and intercalates between the stacked bases. Confirmation of size of the DNA product is done by ______________________

Answer 49

fluorescent dye ethidium bromide comparing the size with DNA ladder.

Question 50

The appearance of discrete band of the correct size may be indicative of a successful PCR amplification. T/F

Answer 50

T

Question 51

POST PCR analysis ______ electrophoresis ______ the PCR product __________ with a specific _______

Answer 51

Agarose gel Sequencing Hybridization; oligonucleotide probe