Virology

Near-infrared (NIR) Western blotting and the In-Cell Western™ Assay are valuable tools for virology researchers seeking quantifiable assays for their studies. Results from the In-Cell Western have been shown to correlate well with traditional methods used in virology, such as the standard plaque assay (SPA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry (FCM), and enzyme-linked immunosorbent assay (ELISA).

Virus Infectivity Characterization

When investigating viruses, it is critical to characterize viral infectivity.

Traditionally, infectivity of viruses that exhibit cytopathic effect (CPE) has been characterized using assays such as SPA. While effective, these methods can be tedious, time-consuming, and the results are subject to human error.

The In-Cell Western Assay, a quantitative immunofluorescence assay performed using microwell plates, combines the specificity of Western blotting with the replicability and throughput of an ELISA. The result is a high-quality and reliable means to perform many assays, including viral titrations on both plaque forming and non-plaque forming viruses, antiviral molecule screens, and more.¹

The In-Cell Western Assay is effective for quantifying virus titrations of both cytopathic and non-cytopathic viruses.² The “Reliable Quantification of Virus Using Infrared Digital Imaging” webinar provides an in-depth look at the many advantages of the In-Cell Western Assay for viral titration and other assays.

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Performing HSV-1 Titration Using the In-Cell Western Assay Imaged on an Odyssey^® Imager

Example Study: “A Novel Method to Titrate Herpes Simplex Virus-1 (HSV-1) Using Laser-Based Scanning of Near-Infrared Fluorophores Conjugated Antibodies.” Research conducted by Fabiani, M., et. al., Frontiers in Microbiology.³

The SPA can be used to evaluate virus infectivity and replication, but it is prone to operator subjectivity and is labor intensive. In this study, viral titer of Herpes simplex virus-1 (HSV-1) was determined in parallel using NIR immunodetection of viral proteins in an In-Cell Western Assay and using SPA.

The In-Cell Western Assay and SPA produced nearly superimposable results, but the In-Cell Western Assay was faster, detecting HSV-1 24 hours post infection versus 48-72 hours post infection for SPA, enabled many samples to be measured in parallel, and was found to be more suitable for titrations that produced plaques too numerous to count using SPA.

HSV-1 Titration with ICW — **Figure 1. The In-Cell Western Assay is a suitable technique to detect HSV-1 infection in cells.** Vero cells were seeded in 96-well plates and infected with HSV-1 in 10-fold serial dilutions. A) Cells were fixed and immunostained 24 hours post-infection with anti-gB antibody followed by IRDye^® 800CW Goat anti-Mouse Secondary Antibody. Plates were imaged using an Odyssey Imager with a representative image shown. B) A representative image of SPA performed using the same HSV-1 dilutions used for the In-Cell Western Assay. C) Standard curve for the mean value of fluorescence intensity detected for each point.

Mechanism of Action Characterization

Understanding the mechanism of action (MoA) of a virus is key to developing a plan for effective treatment options. If the MoA can be determined, then a means of preventing infection or mitigating disease symptom severity might be developed.

Researchers often use Western blotting to learn more about the MoA of a virus.^{4, 5 , 6} The In-Cell Western Assay can also be used to identify aberrent signalling pathways, detect apoptosis, or screen therapeutic agents. Both assays can be performed with exceptional quality and consistency using an Odyssey imaging system, such as the Odyssey M.

Evaluating Cell Transfection using Near-Infrared Western Blotting on an Odyssey Imager

Example Study: “Positive Selection of a Serine Residue in Bat IRF3 Confers Enhanced Antiviral Protection.” Research conducted by Banerjee, A., et. al., iScience.⁴

Bat species harboring viruses often do not demonstrate signs of disease because of a robust antiviral response at low levels of infection. According to a study by Banerjee, A., et al., a serine residue in interferon regulatory factor 3 (IRF3) confers enhanced antiviral protection in bats.

Replacing a leucine residue with a serine residue in the human IRF3 resulted in a significant increase in the antiviral response of human cells. Conversely, replacing a serine residue with a leucine residue in the bat IRF3 led to decreased antiviral protection in the bat cells.

Figure 2: Graph — **Figure 2. Human IRF3 deleted cells transfected with plasmids expressing IRF3-serine-185 demonstrated increased antiviral response when compared to the wild type. A)** Human IRF3 deleted fibroblast cells were transfected with varying concentrations of plasmids expressing wild type (IRF3-WT) or altered IRF3 (IRF3-S185). Then green fluorescent protein expressing vesicular stomatitis virus (VSV-GFP) was used to infect the cells. Cells were then mock treated or treated with poly(I:C) and VSV-GFP levels were quantified. B) Transfection was evaluated using Western blot to determine if increasing concentrations of plasmid containing IRF3-WT or IRF3-S185 led to increased expression of respective IRF3. Detection was performed using IRDye Secondary Antibodies, images were acquired using an Odyssey Imager, and results were quantified using Image Studio™ Software.

**Figure 2. Human IRF3 deleted cells transfected with plasmids expressing IRF3-serine-185 demonstrated increased antiviral response when compared to the wild type. A)** Human IRF3 deleted fibroblast cells were transfected with varying concentrations of plasmids expressing wild type (IRF3-WT) or altered IRF3 (IRF3-S185). Then green fluorescent protein expressing vesicular stomatitis virus (VSV-GFP) was used to infect the cells. Cells were then mock treated or treated with poly(I:C) and VSV-GFP levels were quantified. B) Transfection was evaluated using Western blot to determine if increasing concentrations of plasmid containing IRF3-WT or IRF3-S185 led to increased expression of respective IRF3. Detection was performed using IRDye Secondary Antibodies, images were acquired using an Odyssey Imager, and results were quantified using Image Studio™ Software.

“This automated technique provides not only rapid detection of viral infection status but can also be optimized to detect changes in host-cell protein expression during JCPyV challenge.”
DuShane⁷

Screening Antiviral Therapeutics and Vaccines

Antiviral therapeutics or vaccines that have shown promise in preclinical studies may then move on to clinical trials. During clinical trials, potential antiviral therapeutics or vaccines will have their efficacy in humans evaluated.

NIR Western blotting and In-Cell Western Assays performed using Odyssey Imaging Systems have played a critical role in assessing the efficacy of both antiviral therapeutics and vaccines.¹

“The ICW assay was also applied to assess Nab (neutralizing antibodies) titers in patients and vaccine recipients… Our results indicate that the use of the ICW assay to evaluate non-CPE Hantaan virus titer demonstrates a significant improvement over current infectivity approaches and a novel technique to screen antiviral molecules and detect NAb efficacies.”
Ma¹

Comparing the In-Cell Western Assay to Common Virology Research Methods

Detecting infectious virus titers commonly rely on visible CPEs; however, viruses such as the Hantaviruses do not produce CPE in cell culture. Instead, a study by Ma, H.W., et. al. sought to quantify virus replication by monitoring viral protein expression and neutralizing antibody (NAb) titers with Hantaan virus (HTNV)—the prototype hantavirus—using an In-Cell Western Assay.¹

The study compared the In-Cell Western Assay with three common methods: qRT-PCR, FCM, and ELISA. It determined that the In-Cell Western Assay was as equally sensitive as qRT-PCR and ELISA and more sensitive than FCM. These results suggest that an In-Cell Western Assay is a highly accurate method for assessing HTNV and NAb titers, detecting NP expression and production, and evaluating HTNV replication.

**Figure 3. Comparing the detection of nucleoprotein (NP) production between qRT-PCR and an In-Cell Western Assay and NP expression between FCM and an In-Cell Western Assay. A)** To first determine the optimal detection points for an In-Cell Western Assay, human umbilical vascular endothelial cells (HUVACs) and A549 cells were infected with HTNV then harvested at five time points. HUVAC viral loads were detected at 1 day post-infection (dpi) and the viral loads of both cell types increased from 1 to 3 dpi then plateaued at 4 dpi. Images were acquired using the Odyssey imaging system. B) To compare the detection of NP production between qRT-PCR and an In-Cell Western Assay, a second plate of HUVECs and A549 cells were infected. The viral loads increased from 1 to 4 dpi. Both assays therefore had consistent data. C) To compare the detection of NP expression between FCM and an In-Cell Western Assay, a third plate of HUVECs were infected at an MOI of 0.1. FCM detected NP expression at 2 dpi, compared to the detection of NP expression at 1 dpi using an In-Cell Western Assay.

**Figure 4. Comparing the detection of HTNV titers between an ELISA and an In-Cell Western Assay. A)** A549 cells for the In-Cell Western Assay were grown in 96-well microplates then infected (positive control (PC)) or mock infected (negative control (NC)) with serially diluted HTNV at an MOI of 0.1. Cells were stained with IRDye 800CW Goat anti-Mouse IgG Secondary Antibody and IRDye 680 Goat anti-Rabbit IgG Secondary Antibody. Images were acquired at 2 dpi using the Odyssey imaging system. B) E6 cells for the ELISA were infected or mock infected under the same conditions then collected at 10 dpi. The HTNV titer values calculated for the C) In-Cell Western Assay and the D) ELISA were both 1.25 x 105 TCID50/mL.

“For viral titer assessment, the TCID50 detected by the ICW assay was more convenient and rapid than the ELISA-based TCID50 assay.”
Ma¹

**Figure 5. Comparing the detection of NAb titers of monoclonal antibodies 3D8 and 3G1 between an ELISA and an In-Cell Western Assay.** A549 cells for the In-Cell Western Assay were grown in microplates then stained with IRDye 800CW Goat anti-Mouse IgG Secondary Antibody and IRDye 680 Goat anti-Rabbit IgG Secondary Antibody. Images were acquired on the Odyssey imaging system. The NAb titer values measured for the In-Cell Western Assay at 2 dpi were A) 1/1334 (0.0075μg/μl) for 3D8 and B) 1/2240 (0.0045μg/μl) for 3G1. The NAb titer values measured for the ELISA were C) 1/1585 (0.0063μg/μl) for 3D8 and D) 1/2240 (0.0045μg/μl) for 3G1.

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