Method and means for determining the replication rate of a viral population

1. A method for determining a replication rate of a viral population, said method comprising the steps of:

a) diluting the viral population into at least 2 different dilutions;

b) providing cells into wells, wherein the amount of wells is the amount of the different dilutions from step a) in duplicate, triplicate or any other multiple;

c) infecting the multiple wells of cells with each dilution of the viral population so as to promote replication of said viral population, wherein said cells or viral population comprise a phenotypic marker, whose signal is proportional to the logarithm of the viral population count;

d) measuring in a suitable device for each well the signal expressed by the phenotypic marker for at least 2 different time points;

e) calculating a weight (w) for each group of signals of the wells with the same dilution at each time point, wherein each group of signals of the wells with the same dilution at each time point is a dilution multiple set, whereby said weight is a monotone decreasing function of the standard deviation of the signals;

f) plotting the logarithm of the signal data obtained from step d), in function of the time, and disregarding the signal data which is outside the linear region;

g) extrapolating each remaining signal data by adding said each remaining signal data to the logarithm of each dilution factor at which the viral population was diluted; and

h) calculating the replication rate by performing a linear regression on the extrapolated remaining signal data and their corresponding time points; wherein the replication rate of the viral population (RR) is calculated according to the formula:

RR

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wherein the replication of a viral population refers to the completion of an entire viral life cycle, “w” is the weight for each signal data set, “x” is a time point, “y” is each signal data for a given time point multiplied by the dilution factor, and “n” is the total number of signal data.

2. The method according to claim 1 wherein in step e) the weight (w) is calculated according to the formula:

w= 1/( SD+F ),

wherein “SD” is the standard deviation in the log domain for each dilution multiple set, and “F” is a constant.

3. The method according to any one of claims 1 - 2 wherein before step g), each dilution multiple set within which those extreme values are more than 1-log apart from each other, is disregarded.

4. The method according to claim 1 wherein the signal expressed by the phenotypic marker is measured at 5 time points, and 3 linear regressions are performed on each three consecutive signal data obtained from step g) and their corresponding time points, whereby the replication rate is the greatest slope obtained.

5. The method according to claim 1 wherein the signal expressed by the phenotypic marker is measured at 6 time points, and 4 linear regressions are performed on each three consecutive signal data obtained from step g) and their corresponding time points, whereby the replication rate is the greatest slope obtained.

6. The method according to claim 1 wherein the replication rate is expressed as the factor by which the viral population grows by calculating the inverse logarithm of the obtained greatest slope.

7. The method according to claim 1 wherein the replication rate is expressed as an increase or a drop in viral population count between 3 time points.

8. The method according to claim 1 wherein the replication rate is calculated for a given viral population and for a reference viral population, and the replication rate of said given viral population is divided by the replication rate of the reference viral population, and the replication rate of said given viral population is expressed in a percentage relative to the replication rate of the reference viral population.

9. The method according to claim 1 wherein the phenotypic marker is a reporter gene inserted in the cells.

10. The method according to claim 1 wherein the viral population consists of human immunodeficiency virus (HIV).

11. The method according to claim 1 further comprising assessing the efficiency of a subject's therapy or for evaluating or optimizing a therapy.

12. A computer apparatus or computer-based system comprising a memory for storing computer executable program instructions, and a processor operative to execute steps e)-h) of the method of claim 1 in response to the computer executable program instructions.