This work is supported in part NIH grants R01 A1064826-01, U19 AI062627-01, NO1-HHSN26600500032C (to S.M.). We thank our laboratory members for discussion and technical help, Tina Heil for the critical review of the manuscript. We would like to thank David Bina, Milwaukee Health Department Laboratory for virus culture and PCR.

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We have nothing to disclose.

The recent epidemic outbreak of avian influenza in Southeast Asia and a global fear of the swine flu pandemic impose enormous concerns to public health and safety. Critical focus has been dedicated in generating vaccines for existing and newer strains of influenza virus. Availability of rapid high-throughput technologies for precise detection and accurate viral titer calculations will tremendously improve treatment. The most commonly used techniques employed to calculate virus titer include plaque forming and influenza hemagglutination assays. The plaque forming assay was first developed to estimate bacteriophage titers and was adopted by Renato Dulbecco to calculate animal viral titers 4. To perform this assay, 10-fold dilutions of influenza stock or animal specimens such as BAL fluid are prepared and 0.1 ml aliquots are inoculated onto the monolayer of MDCK cells in 6-well plates. The virus is allowed to adsorb onto the MDCK cells followed by the addition of a nutrient medium and agarose. During the incubation, the original infected cells release viral progeny that spread only to the neighboring cells. The cytopathogenic effect of the influenza viral infection produces a circular zone of infected cells called a plaque. Each plaque is presumably formed after infection of a single cell with a single virus particle followed by replication of virus. Contrast between the living cells and the plaques can be enhanced by dyes such as crystal violet. A major drawback of this assay is lack of reproducibility and huge variations within experiments. Another assay used to determine the influenza viral titer is the hemagglutination assay. The hemagglutinin protein present on the surface of the influenza virus efficiently binds to N-acetylneuraminic acid-containing proteins on mammalian and avian red blood cells 1. This interaction between the influenza virus and the erythrocytes leads to agglutination in the form of a lattice. The level of agglutination is used to estimate the titer of the influenza virus in test specimens. Erythrocyte agglutination assay is used only to determine the titration of a known virus and cannot be used for strain identification purposes. Since this assay does not differentiate between the live and the dead viruses, it is difficult to obtain a precise calculation of viral titers.
Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,6. The new method described here also has clinical and laboratory diagnostic potential. Influenza detection kits Directigen FLU-A (BD Biosciences, San Jose, CA) and BinaxNow (Binax, Inc., Scarborough, ME) use immunochromatography assay to detect viral antigen such as nucleoprotein 7. Using these kits, detection of virus can be achieved within 15 min; however, all negative samples have to be further screened by the cell culture method. Various studies have also reported low sensitivity of these kits, particularly, when infection was mild or low 8. In our method, we consistently detect as low as 100 PFU of virus. This suggests our technique is useful in detecting H1N1 virus in clinical samples, even when the infection is mild. A fluorescence-based method to detect viral antigens has been reported, where either nasopharyngeal aspirate specimens were directly analyzed or were inoculated onto a monolayer of susceptible cells for virus multiplication and subsequent analyses 9. However, these methods are limited for diagnostic purposes and are not suitable for virus titer calculations. Q-PCR helps to identify the presence of viral RNA and does not estimate the infectivity of live viruses. Compared to these assays, IR dye-based assay system will help in the detection and viral titer enumeration in clinical and laboratory samples. In case of influenza outbreak, it is critical to diagnose thousands of samples in a short time period. Our method based on a 384-well plate and IR scanner can scan 6-plates at a time (&gt;2,000 samples), therefore offering a greater advantage over other direct fluorescent methods. Feasibility of rapid processing of thousands of clinical samples could reduce test variability, length of assay time and costs. The use of an M2-specific antibody offers the advantage of being independent of strain differences in H and N proteins. The M2-protein is relatively conserved in most influenza strains infecting humans. If antibodies specific to circulating stains are combined with our system one can measure both the titer of virus and identify the strain. However, since M2 is the target for the Amantadine-like drugs, mutants can arise that might change the binding site for the mAb used here. This represents a potential limitation for this system in individuals who are being treated by these drugs.
Multiple assays are used to enumerate influenza titers in test samples. To calculate virus titer, 50% tissue culture infectious dose (TCID50) and egg allantoic fluid-based assays are widely used 1,10. However, requirements of multiple days make these methods less reliable. Further, in these methods calculations based on the 50% change in the number of plaques provide theoretical but not accurate viral titrations. Plaque forming assays can be performed within 96 hours; however, lack of reproducibility is a major concern in viral titer calculations. The present technique described here has several distinct advantages. First, it is a highly reproducible technique with consistent outcomes. Second, viral titer calculations are based on simple quantification of fluorescence intensities that are compared against defined standard curves. Third, two or more primary antibodies can be used to diagnose multiple serotypes in a given sample. Also, IR dye-conjugated antibodies have minimal autofluorescence and have been used for cell imaging 11. UV or visual radiation (300-600 nM) results in high cellular autofluorescence due to the presence of high quantum yielding endogenous fluorophores. These include aromatic amino acids, lipo-pigments, pyridinic nucleotides (NADPH), elastin, collagen and flavin coenzymes. This intrinsic property of cells makes it difficult to utilize these sources of radiation as probes to quantify the expression of specific proteins. In contrast, near-IR dyes excite and emit between 670-1000 nm. Many host cellular molecules and polyaromatic hydrocarbons do not excite at this wavelength and due to reduced light scattering, emit extremely low background fluorescence. Further, the window between the background and specific detection is vastly improved due to a high extinction coefficient of the near-IR fluorophores. Photostability, superior signal-to-noise ratio, is extremely relevant in the quantification of viral titers. Use of microfluidic chambers will automate the screening process through robotic controls, and could allow us to test highly contagious clinical samples with ease. Thus, the utilization of near-IR dyes in these methods is ideal and highly reliable to enumerate viral titers in tissue samples.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Bovine serum albumin</td>
 <td>Atlanta Biologicals</td>
 <td>S11750</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PR8 virus</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>From Dr Thomas M Moran</td>
 </tr>
 <tr>
 <td>Goat anti-mouse IRDye@800</td>
 <td>LI-COR biosciences</td>
 <td>926-32210</td>
 <td>1:200 dilution</td>
 </tr>
 <tr>
 <td>LI-COR Odyssey IR scanner</td>
 <td>LI-COR-biosciences</td>
 <td>9201-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>384-wells flat bottom plate</td>
 <td>Nalge Nunc international</td>
 <td>164730</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96-wells flat bottom plate</td>
 <td>Nalge Nunc international</td>
 <td>165305</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DMEM medium</td>
 <td>Invitrogen</td>
 <td>11965126</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RPMI medium</td>
 <td>Invitrogen</td>
 <td>11875135</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>Invitrogen</td>
 <td>25080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-glutamine 100x</td>
 <td>Invitrogen</td>
 <td>25030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypson-EDTA</td>
 <td>Invitrogen</td>
 <td>R001100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Pyruvate</td>
 <td>Invitrogen</td>
 <td>11360070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Anti-M-2 antibody</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>From Dr Thomas M Moran</td>
 </tr>
 <tr>
 <td>Anti-NP mAb</td>
 <td>CDC</td>
 <td>V2S2208</td>
 <td>Obtained with an MTA</td>
 </tr>
 </tbody>
</table>

high-throughput detection method for influenza virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

Watch this Scientific Journal Video about High-throughput Detection Method for Influenza Virus at JoVE.com

High-throughput Detection Method for Influenza Virus

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

high-throughput-detection-method-for-influenza-virus

Research

JoVE Journal

Immunology and Infection

25.9K 조회수.  Blood Research Institute. 이 방법은 높은 감도에 감염된 생쥐들의 bronchioalveolar 세척 (BAL) 유체에서 H1N1의 탐지를위한 적외선 염료 기반 이미징 시스템의 사용을 설명합니다. 이 방법론은 96에서 수행할 수 있습니다 - 또는 판 384 자, 시험 물질 