Counting defective interfering particles: Easy as 1, 2, 3, . . . ? - - PowerPoint PPT Presentation

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Counting defective interfering particles: Easy as 1, 2, 3, . . . ? - - PowerPoint PPT Presentation

Oct 06, 2023 201 likes •310 views

What is a DIP? Conventional DIP counting An alternative method Conclusions Counting defective interfering particles: Easy as 1, 2, 3, . . . ? Laura Liao 1 1 Department of (Viro) Physics, Ryerson University Supervisor: Prof. Catherine Beauchemin

slide-1
SLIDE 1

What is a DIP? Conventional DIP counting An alternative method Conclusions

Counting defective interfering particles: Easy as 1, 2, 3, . . . ?

Laura Liao1

1Department of (Viro) Physics, Ryerson University

Supervisor: Prof. Catherine Beauchemin

June 18, 2014

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SLIDE 2

What is a DIP? Conventional DIP counting An alternative method Conclusions

Defective interfering particles (DIPs)

Defective interfering particles (DIPs) are improperly formed virus. DIPs impact the outcome of virus experiments. Therefore, it is important to count them.

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SLIDE 3

What is a DIP? Conventional DIP counting An alternative method Conclusions

Influenza A viral replication cycle & model

β β β

Co-infection window

Co-inf. eclipse

CE1

p p

virus production rate DIP production rate

V (virus)

c c

virus clearance rate DIP clearance rate CE2

Co-inf. eclipse

CO-INFECTION BRANCH STANDARD BRANCH infection rate

τE

length of eclipse phase

τI

length of infectious phase

τC

length of co-infection window DIP Virus

Target E1 Eclipse E2 Eclipse Eic+1 Eclipse EnE Eclipse I1 Infectious InI Infectious CEnE

Co-inf. eclipse Co-inf. eclipse

CEic+1

... ...

Dead Dead A Abortive Eic Eclipse

Co-inf. eclipse

CEic D (DIP) Virus

Co-inf. infectious

CI1

Co-inf. infectious

CInI

... ... ... ... ...

DIPs are indistinguishable from virus. So, how have DIPs been counted?

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SLIDE 4

What is a DIP? Conventional DIP counting An alternative method Conclusions

Existing method to count DIPs

Bellett & Cooper (1959)

(ẟ)

fcell receive one or more virus = 1-e- NV

# cells

fcell receive no DIP = e-

# cells

ẟ

fcells receiving = fcells still producing virus

cells receiving

  • ne or more

virus but no DIP

= (1-e- )e-

# cells

ẟ

# cells

So we have,

relative virus yield

= fcells receiving virus but no DIP fcells receiving virus in absence of sample = (1-e- )e-

# cells

ẟ

# cells

= = e-

# cells

ẟ

e-

# cells

ẟ

Pure DIP dose (sample - virus) 0.2 0.4 0.6 0.8 1 = (1-e- )e

# cells

+ + + Infect with sample + diluted UV'd sample (pure DIP).

(ND + NV) (0 + 0) (ẟND + 0) (ND + NV) (ND + NV) (ND + 0) ND

# cells

ND

  • # cells

ND

  • ND

NV

# cells

ND

  • # cells

ND

  • ND

NV NV ND ND

Bellett & Cooper (B&C) calculation uses Poisson distribution parameterized by NV =# virus

# cells

  • r ND=# DIP

# cells

.

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SLIDE 5

What is a DIP? Conventional DIP counting An alternative method Conclusions

B&C does not account for order of infection events

B&C assay curves (predictions) impacted by co-inf window.

D D V V D V V D

?

depends on co-infection window

So order D+V ≠ V+D

0.2 0.4 0.6 0.8 1 DIP dose (δ)

10

  • 4

10

  • 3

10

  • 2

10

  • 1

10

Relative virus yield

co-inf window 0 h (none) co-inf window 1.1 h co-inf window 2.2 h co-inf window 3.3 h co-inf window 4.4 h co-inf window 5.5 h co-inf window 6.6 h (full)

Infecting with 4 virus/cell + 8 DIP/cell

B&C valid for intermediate co-infection windows when order of events equally likely, and there are no newly produced DIPs.

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SLIDE 6

What is a DIP? Conventional DIP counting An alternative method Conclusions

B&C valid for intermediate co-infection windows

Compare frac virus producers for varying windows to B&C calc.

6 12 18 24

Time [h]

10

  • 5

10

  • 4

10

  • 3

10

  • 2

10

  • 1

10

Virus-only infected cell fraction

co-inf window 0 h co-inf window 0.2 h co-inf window 0.44 h co-inf window 0.66 h co-inf window 0.88 h co-inf window 1.1 h co-inf window 6.6 h

Eclipse cells Infectious cells Infecting with 4 virus/cell + 8 DIP/cell

1 2 3 4 5 6

Co-infection window [h]

10

  • 5

10

  • 4

10

  • 3

10

  • 2

10

  • 1

10

Virus-only infeceted cell fraction

Eclipse cells (not yet virus-producing) Infectious cells (virus-producing)

B&C Infecting with 4 virus/cell + 8 DIP/cell

B&C valid for co-infection window between 1.5 h and 3.5 h. The biological co-infection window is between 1 h and 3 h. So, B&C is suited for influenza A DIP counting.

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SLIDE 7

What is a DIP? Conventional DIP counting An alternative method Conclusions

Our method

Infect with sample (conc. and diluted). Equivalent total particles produced, but loss of infectious virus due to DIPs.

24 48 72 96 120 Infectious virus (PFU/mL) Time [h] 10 10

1

10

2

10

3

10

4

10

5

10

6

10

7

10

8

10

9

10

10

4 virus/cell + ? DIP/cell 10-5 virus/cell + ?/4x10-5 DIP/cell

Total particles (RNA/mL)

4 virus/cell + ? DIP/cell 10-5 virus/cell + ?/4x10-5 DIP/cell

24 48 72 96 120 Time [h] 10

4

10

5

10

6

10

7

10

8

10

9

10

10

10

12

10

11

Our method proposes to use the drop in virus to estimate DIPs.

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SLIDE 8

What is a DIP? Conventional DIP counting An alternative method Conclusions

Adding DIP to achieve observed virus drop

Given a virus drop of 104, work backwards to find how much DIP was present in sample.

12 24 Time [h] 10

3

10

4

10

5

10

6

10

7

10

8

10

9

10

10

Infectious virus [pfu/mL]

DIP-free 2 DIP/cell 4 DIP/cell 6 DIP/cell 8 DIP/cell 10 DIP/cell >20 DIP/cell

Infecting with 4 virus/cell + DIP/cell (below)

ideal B&C co-infection window (~3 h)

1 2 3 4 5 6 Co-infection window [h] 10 10

1

10

2

10

3

10

4

Estimated DIP/cell Infecting with 4 virus/cell + adding DIP/cell to find virus drop of 1.9x10

  • 4

Our method estimates 8 DIP/cell that begin the infection, or 2 DIPs/virus present in the sample. Estimates consistent for all co-infection windows > 1 h.

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SLIDE 9

What is a DIP? Conventional DIP counting An alternative method Conclusions

Us vs. them

Infect with 4 virus/cell + 8 DIP/cell, varying co-inf window. Get virus drop as a fn of window. Fix window to 3 h, as in B&C (left); ask “How much DIP to add to achieve virus drop?”. If co-inf window unknown, ours does just as well as B&C. If known, fix co-inf window to actual co-inf window (right). Ours performs better than B&C for long co-inf windows.

1 2 3 4 5 6

Co-infection window [h]

10

  • 3

10

  • 2

10

  • 1

10 10

1

Estimated DIP/cell

B&C Our method Infecting with 4 virus/cell + 8 or 10

  • 1 actual DIP/cell

actual DIP/cell (high) actual DIP/cell (low)

1 2 3 4 5 6

Co-infection window [h]

10

  • 3

10

  • 2

10

  • 1

10 10

1

Estimated DIP/cell

B&C Our method Infecting with 4 virus/cell + 8 or 10

  • 1 actual DIP/cell

actual DIP/cell (high) actual DIP/cell (low)

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SLIDE 10

What is a DIP? Conventional DIP counting An alternative method Conclusions

Conclusions on DIP counting

Have two methods (ours and B&C): if co-inf window not known, do as well as B&C. if co-inf window known, ours does better for long windows.

  • urs does not use UV’d DIPs.

Validation — use both to count a sample: disagree revise and test assumptions on DIP biology (exciting!). agree evaluate how both methods fare with uncertainty in data.