# US 2007/0111296 A1 · Methods and Devices for Quantitative Viral Assays

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- Number: US 2007/0111296 A1
- Document ID: US20070111296A1
- Status: Published application
- Kind: A1
- Application number: 11/560,714
- Filed: 2006-11-16
- Published: 2007-05-17
- Earliest claimed: 2005-11-16
- Assignees: Wisconsin Alumni Research Foundation
- Inventors: John Yin; Ying Shu
- Web page: https://patentgrep.com/patent/US20070111296A1

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 15/86: Viral vectors
- C12N 2760/16051: Methods of production or purification of viral material
- C12N 2760/20211: Vesiculovirus, e.g. vesicular stomatitis Indiana virus
- C12N 2770/36251: Methods of production or purification of viral material
- C12N 2795/14151: Methods of production or purification of viral material
- C12Q 1/00: Measuring or testing processes involving enzymes, nucleic acids or microorganisms (measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34); Compositions therefor; Processes of preparing such compositions
- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method for quantifying infectious particles of a virus in a sample comprises providing a layer of host cells of the virus, contacting the layer of host cells with a preparation of the sample, and culturing the cells under conditions wherein the cells are submerged in a thin layer of liquid culture medium, and wherein the virus infects host cells and releases its progeny from said infected host cells, imposing a flow of the liquid medium, wherein the spread of the viral progeny to uninfected host cells is enhanced, culturing the cells under conditions to allow further virus infection and viral gene expression, wherein infected host cells develop an observable indication of viral gene expression, and determining the number of infected host cells, whereby the number of infectious particles of the virus in the sample is quantified. The method may be used for measuring viral growth rate or for screening for antiviral compounds. Also provided are microfluidic devices suitable for the inventive method.

## Claims

### Claim 1

A method for quantifying infectious particles of a virus in a sample, the method comprising:
1) providing a layer of host cells of the virus,
2) contacting the layer of host cells with a preparation of the sample, and culturing the cells under conditions wherein the cells are submerged in a thin layer of liquid culture medium, and wherein the virus infects host cells and releases its progeny from said infected host cells,
3) imposing a flow of the liquid medium, wherein the spread of the viral progeny to uninfected host cells is enhanced,
4) culturing the cells under conditions to allow further virus infection and viral gene expression, wherein infected host cells develop an observable indication of viral gene expression, and
5) determining the number of infected host cells, whereby the number of infectious particles of the virus in the sample is quantified.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the observable indication is a plaque.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the plague is comet-shaped.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the host cells are stained with a suitable dye to show viral infection or viral gene expression or a host reaction to the virus infection.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the observable indication is digitally imaged and computer processed.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the layer of host cell is formed on the surface of a cultural medium or a cultural plate.

### Claim 7 (depends on 1)

The method according to claim 1, wherein a microfluidic device is used,
wherein the microfluidic device comprising a first and a second end reservoir connected by an enclosed channel, wherein the channel has an enclosed bottom surface suitable for host cell adherence and growth, and the end reservoirs are accessible for liquid loading or removal, and
wherein the layer of host cells is formed on the bottom of surface of the microfluidic device, and wherein the liquid culture medium is controlled to flow from one reservoir to the other reservoir.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the controlled flow of the liquid culture medium is effected by a differential in pressure of the liquid medium in the first and second reservoirs.

### Claim 9 (depends on 7)

The method according to claim 7, wherein the liquid culture medium comprises inert particles such that diffusion of viral particles in the liquid culture medium is enhanced.

### Claim 10 (depends on 9)

The method according to claim 9, wherein capture by host cells of the viral particles is enhanced.

### Claim 11

A method for measuring growth rate of a virus, the method comprising:
1) providing a layer of host cells of the virus,
2) contacting the layer of host cells with a preparation of the sample, and culturing the cells under conditions wherein the cells are submerged in a thin layer of liquid culture medium, and wherein the virus infects host cells and releases its progeny from said infected host cells,
3) imposing a flow of the liquid medium, wherein the spread of the viral progeny to uninfected host cells is enhanced,
4) culturing the cells under conditions to allow further virus infection and viral gene expression, wherein infected host cells develop an observable indication of viral gene expression, and
5) measuring the observable indication in relation to time.

### Claim 12 (depends on 11)

A method for determining antiviral activity of a compound against a virus, comprising:
1) measuring the growth rate of the virus according to claim 11 in the presence and in the absence of a candidate compound, and
2) comparing the growth rate of the virus in the presence to the growth rate in the absence of the candidate compound,
wherein a decrease in the viral growth rate in the presence of the candidate compound indicates that the candidate compound has antiviral activity against the virus.

### Claim 13

A microfluidic device comprising a first and a second end reservoir connected by an enclosed channel, wherein the channel has an enclosed bottom surface suitable for host cell adherence and growth, and the end reservoirs are accessible for liquid loading or removal.

### Claim 14 (depends on 13)

The microfluidic device according to claim 13, wherein the end reservoirs are suitable for storing different amounts of liquid.

### Claim 15 (depends on 14)

The microfluidic device according to claim 14, wherein the enclosed channel and the end reservoirs are filled with a liquid, and the end reservoirs contain the liquid in different volumes, and a flow occurs in the channel due to the evaporation of liquids in the end reservoirs.

### Claim 16 (depends on 15)

The microfluidic device according to claim 15, wherein the end reservoir at one end of the channel is a source reservoir, and the reservoir at the other end of the channel is a sink reservoir, wherein the source reservoir contains an excess amount of liquid in comparison to the sink reservoir, and wherein evaporation from the sink reservoir causes flow of the liquid in the channel towards the sink reservoir.

### Claim 17 (depends on 14)

The microfluidic device according to claim 14, comprising a plurality of microfluidic channels.

### Claim 18 (depends on 17)

The microfluidic device according to claim 17, wherein the plurality of microfluidic channels are linked by at least one cross channel.

### Claim 19 (depends on 17)

The microfluidic device according to claim 17, wherein at least one of the plurality of microfluidic channels is Y shaped.