Bio 220: Lecture 21 - Cultivation Of Viruses (Continued)

Front 1

Quantification of viral populations

Back 1

1. Electron Microscopy
2. LD (Lethal Dose) 50 or ID (Infectious Dose) 50
3. Pock Method
4. Plaque Method
5. Tissue Culture Method
6. HA titer (hemagglutination end point or titer assays)

Front 2

electron microscopy

Back 2

one can count the particles in a viral preparation. This is time consuming, costly and laborious. Furthermore, one cannot differentiate between those particles which are infectious (virion) vs. noninfectious by just looking at them. However, it does provide an absolute count.

Front 3

LD (Lethal Dose) 50 or ID (Infectious Dose) 50 methods

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scientists get a relative idea of the potency of a viral preparation. The LD 50 method finds the concentration of a viral preparation at which 50% of the test animals will die. The ID 50 method finds the concentration of a viral preparation at which 50% of the test animals are infected. In comparing two viral preparations, create a series of dilutions of both preparations and inject these into the test animals. At which dilution will 50% of the test animals die (LD 50). One can assess the viral concentrations of the two preparations relative to each other based upon which dilution from the two preparations was the LD 50 dilution. If one preparation required more diluting to observe the LD 50 effect, than this was the preparation with more viral quantity.

Front 4

Pock method

Back 4

pocks are observed as areas of increased growth due to viral replication on embryonic chicken membranes. The chorioallantoic membrane is commonly used. The pocks are observed as “pits” or “bumps” upon the membrane. Counting the number of pocks on two separate chorioallantoic membranes following the inoculation of two viral preparations, allows for the assessment of these viral concentrations. This quantification is relative and only virions provide results.

Front 5

Plaque method

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pour plates are created from agar that is mixed with bacteriophage and host bacteria. The host bacterial cells involved with viral replication are destroyed to reveal areas of clearing in the plates. These clearings are called plaques. Two separate viral preparations are used to create two separate pour plates. Upon examination of the two plates, one can count the number of plaques (clearings) following bacterial host cell growth and viral replication. The plate with more plaques is the plate with more viral particles in the original suspension. This quantification method is relative and only virions produce results.

Front 6

Tissue culture method

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tissue cells are grown in monolayer. Viral particles are introduced. Foci of infection can be observed where the tissue cells are damaged or destroyed by the viruses. This pattern of cell destruction is referred to as the cytopathic effect (CPE). Two separate viral preparations can replicate in separate monolayer tissue cultures. The more CPE’s in a culture, the greater the viral concentration. This quantification method is relative and only virions produce results.

Front 7

HA titer (hemagglutination end point or titer assays)

Back 7

some viruses can attach to erythrocytes and form bridges between them. This causes clumping or agglutination of the RBC’s. The influenza virus has this capability. One can check the viral concentration of two separate preparations by creating a series of dilutions of the two preparations and then inoculating these dilutions into RBC’s. The highest dilution the shows agglutination is referred to as the end point. Examine the end points from the two series of inoculations. If the end point established from one of the preparations required an additional dilution, than that preparation is more concentrated. This quantification method provides relative values.

Front 8

What are the Virus-host relationships

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1. Lytic State
2. Lysogenic State
3. Viral Release without Lysis

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Lytic State

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virus kills and lyses host cell.

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Lysogenic State

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viral infection without host cell lysis. In this situation, there is no release of viral
particles. Virus is in a quiet or in a latent state within the host cell.

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Viral Release without lysis

Back 11

viral particles are extruded out of the cell without lysis. The host cell is
therefore not lysed and killed. This provides the virus with a method for obtaining an envelope.

Front 12

In some cases, viruses can go from a ___ to a ___ or from a ___ to a ___.

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In some cases, viruses can go from a lytic cycle to a lysogenic cycle or from a lysogenic cycle to a lytic cycle.
When one is showing the classical lesions from a Herpes simplex 1 infection (cold sores or fever blisters), the virus is in a lytic cycle and the host cells are being lysed. The virus can then retreat into a lysogenic cycle or phase and remains dormant or latent. UV light, stress or temperature variations can activate the virus into its lytic phase.


Front 13

The viral DNA from a bacteriophage can integrate into the bacterial host cells chromosome and replicate with the host cell DNA during ....

Back 13

The viral DNA from a bacteriophage can integrate into the bacterial host cells chromosome and replicate with the host cell DNA during transverse binary fission without active infection.
This inserted phage DNA is known as the prophage or temperate phage or lysogenic phage. The phage remains latent in a state of lysogeny. The host bacterial cell is known as a lysogenic cell. UV light or chemicals or some other spontaneous event can cause the prophage to pop out of the host chromosome and start the lytic cycle. A temperate (lysogenic) phage is therefore capable of both a lysogenic cycle and a lytic cycle within its bacterial host.