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

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 June 18, 2014

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.

What is a DIP? Conventional DIP counting An alternative method Conclusions Influenza A viral replication cycle & model τ E τ I length of eclipse phase length of infectious phase c τ C virus clearance rate length of co-infection window p V (virus) STANDARD BRANCH virus production rate Virus ... ... ... E 1 E 2 E ic E ic+1 E nE I 1 I nI β Target Eclipse Eclipse Eclipse Eclipse Eclipse Infectious Infectious Dead infection c DIP DIP clearance rate β ... rate D (DIP) p DIP production rate Virus ... ... ... A CE 1 CE 2 CE ic CE ic+1 CE nE CI 1 CI nI β Abortive Co-inf. eclipse Co-inf. eclipse Co-inf. eclipse Co-inf. eclipse Co-inf. eclipse Dead Co-inf. infectious Co-inf. infectious Co-infection window CO-INFECTION BRANCH DIPs are indistinguishable from virus. So, how have DIPs been counted?

What is a DIP? Conventional DIP counting An alternative method Conclusions Existing method to count DIPs Infect with sample + diluted UV'd sample (pure DIP). f cell receive one or more virus = 1-e - N V + + + # cells N D N D ẟ - f cell receive no DIP = e - # cells # cells (N D + N V ) (0 + 0) (N D + N V ) (ẟN D + 0) (N D + N V ) (N D + 0) Pure DIP dose (sample - virus) f cells receiving = f cells still producing virus (ẟ) cells receiving one or more 0.2 0.4 0.6 0.8 1 virus but no DIP N V N D N D ẟ - = (1-e - )e - # cells # cells # cells So we have, N D f cells receiving virus but no DIP ẟ e - # cells relative virus = f cells receiving virus in absence of sample yield N V N D N D ẟ - = (1-e - )e - # cells # cells # cells = N V N D - = (1-e - )e # cells # cells N D Bellett & Cooper (1959) ẟ = e - # cells Bellett & Cooper (B&C) calculation uses Poisson distribution parameterized by N V =# virus or N D =# DIP . # cells # cells

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. V Infecting with 4 virus/cell + 8 DIP/cell 0 10 D D V D Relative virus yield -1 10 V V D -2 10 ? co-inf window 0 h (none) depends on co-inf window 1.1 h co-inf window 2.2 h co-infection -3 10 co-inf window 3.3 h window co-inf window 4.4 h co-inf window 5.5 h So order co-inf window 6.6 h (full) -4 10 D+V ≠ V+D 0 0.2 0.4 0.6 0.8 1 DIP dose ( δ ) B&C valid for intermediate co-infection windows when order of events equally likely, and there are no newly produced DIPs.

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. Infecting with 4 virus/cell + 8 DIP/cell Infecting with 4 virus/cell + 8 DIP/cell Virus-only infected cell fraction Virus-only infeceted cell fraction 0 Eclipse cells Infectious cells 0 10 10 Eclipse cells (not yet virus-producing) -1 Infectious cells (virus-producing) -1 10 10 co-inf window 0 h -2 -2 co-inf window 0.2 h 10 10 co-inf window 0.44 h co-inf window 0.66 h co-inf window 0.88 h -3 -3 10 10 co-inf window 1.1 h B&C co-inf window 6.6 h -4 -4 10 10 -5 -5 10 10 0 1 2 3 4 5 6 0 6 12 18 24 Time [h] Co-infection window [h] 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.

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. 10 12 10 10 Infectious virus (PFU/mL) Total particles (RNA/mL) 9 11 10 10 8 10 10 10 7 10 9 6 10 10 5 8 10 10 4 7 10 10 3 10 6 10 2 10 5 1 10 10 -5 virus/cell + ?/4x10 -5 DIP/cell 10 -5 virus/cell + ?/4x10 -5 DIP/cell 10 4 virus/cell + ? DIP/cell 4 virus/cell + ? DIP/cell 4 0 10 48 96 120 10 0 24 48 72 96 120 0 24 72 Time [h] Time [h] Our method proposes to use the drop in virus to estimate DIPs.

What is a DIP? Conventional DIP counting An alternative method Conclusions Adding DIP to achieve observed virus drop Given a virus drop of 10 4 , work backwards to find how much DIP was present in sample. Infecting with 4 virus/cell + adding DIP/cell Infecting with 4 virus/cell + DIP/cell (below) -4 to find virus drop of 1.9x10 ideal B&C co-infection window (~3 h) 10 10 4 10 Infectious virus [pfu/mL] 9 10 Estimated DIP/cell 8 3 10 10 7 10 2 6 10 10 DIP-free 2 DIP/cell 5 10 4 DIP/cell 1 6 DIP/cell 10 4 8 DIP/cell 10 10 DIP/cell >20 DIP/cell 3 10 0 10 0 12 24 0 1 2 3 4 5 6 Co-infection window [h] Time [h] 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.

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 actual DIP/cell -1 actual DIP/cell Infecting with 4 virus/cell + 8 or 10 Infecting with 4 virus/cell + 8 or 10 actual DIP/cell (high) actual DIP/cell (high) 1 1 10 10 Estimated DIP/cell Estimated DIP/cell 0 0 10 10 actual DIP/cell (low) actual DIP/cell (low) -1 -1 10 10 -2 -2 10 10 B&C B&C Our method Our method -3 -3 10 10 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Co-infection window [h] Co-infection window [h]

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. ours 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.