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VIRUS

Update: 2017-01-15 14:35      View:

Instrument configuration

 

The Flow NanoAnalyzer is equipped with a 532 nm CW laser, and the detection channel is side scatter.
 


1.     Label-Free Detection of Single Viruses

 

Introduction

Viruses are by far the most abundant biological entities on our planet. Whereas animal viruses are notoriously responsible for a large number of fatal diseases, plant viral pathogens cause significant losses in crop yields worldwide each year. On the other hand, the high transfection efficiency of viral gene-therapy vectors and the monodisperse structures that have a precise shape and size make viral particles powerful drug delivery vehicles and versatile nanotechnological building blocks. Therefore, high-resolution and high-throughput analysis of single viral particles is in high demand for virology research, disease diagnosis and treatment, and biotechnology and nanotechnology applications.
However, due to the small size (mostly ranges from 20 to 200 nm in diameter) and simple structure of viruses, conventional flow cytometry, which focuses on particles larger than 200 nm, is not the best option for virus detection. There is a great necessity for the development of advanced approaches enabling rapid, label-free and accurate detection of viruses at single particle level. The development of Flow NanoAnalyzer opens a new avenue for virus characterization.
 

Results

 

 
Figure 1. Label-free detection of levivirus MS2 by Flow NanoAnalyzer.


Discussion


The virus used in this experiment is levivirus MS2, which is one kind of non-enveloped, spherical virion about 27 nm in diameter, and the genome is monopartite, linear ssRNA (+) about 3.5 kb in size. The signal-to-noise (S/N) ratio, which is calculated as the average burst height of all the nanoparticles detected in 1 min divided by the standard deviation of the background signal (noise), is 11 for the MS2 viruses, indicating that the Flow NanoAnalyzer provides exceptional sensitivity in discerning MS2 viruses against the background noise. This sensitivity could meet the detection demand of most virus nanoparticles in nature.

 

2.     Effect of the Refractive Index on the Light Scattering Detection


Introduction

Based on the theory of Rayleigh scattering, the scattering cross section of a nanoparticle is not only dominated by the particle size but also influenced by the refractive index contrast, which is the ratio of the refractive index of the particle over that of the medium. A silica nanoparticle scatters 3.8-fold less light than a polystyrene nanoparticle of the same size at a wavelength of 532 nm and with refractive indices of 1.46 and 1.59, respectively. The exact refractive index of the virus is not known, but it can be assumed to be 1.45 or 1.46, which is similar to that of proteins and DNA. The effect of the refractive index on the light scattering detection of single viruses is examined with the bacteriophage T7, a bacterial virus with an icosahedral capsid head of ~60 nm diameter, along with silica nanospheres and polystyrene nanobeads of a comparable size.


Results


 

 
Figure 2. Light scattering analysis of single nanoparticles with similar diameters.


Discussion

1.     The SS signals of three kinds of nanoparticles are well above the background, and nice SS burst area histograms are derived.
2.     Compared with the highly monodisperse silica nanospheres of 60 ± 2 nm diameter, the naturally occurring bacteriophage T7 is more uniform with a very narrow distribution profile of light scattering intensity
3.     The comparable burst height/burst area for the T7 virions and 60-nm silica nanospheres confirms that the virus and silica nanoparticles exhibit comparable refractive indices. The approximately 6-fold higher scattering intensity observed for the polystyrene nanobeads of 63-nm diameter than that of 60-nm silica nanospheres agrees with the theoretical prediction of 5.1-fold.
 
 
 
 

3.     Discrimination and Size Measurement of Viruses in a Mixture


Introduction

Nano-sized biological agents and pathogens such as viruses are known to be responsible for a wide variety of human disease such as flu, AIDS and herpes, and have been used as biowarfare agents. Viral size plays a substantial role in transmission dynamics, disease outbreaks and outcomes. Rapid and accurate detection and characterization of single viral particles has become increasingly important for virology research, disease diagnosis and treatment, and biotechnology applications. Transmission electron microscopy (TEM) has historically been the method of choice for determining the size and morphology of single viruses. However, the tedious process for sample preparation and image analysis, along with the high cost, prevent its use on a routine basis. A standard calibration curve is constructed with monodisperse silica nanospheres of five different diameters ranging from 43 to 113 nm. The SS burst areas of silica nanoparticles are plotted as a function of the diameters determined by TEM, and the SS burst area of every single virus is converted to the corresponding particle size.
 

 

Results

 

 

 

 
Figure 3. Differentiation and size measurement of different virus types in a mixture.


Table 1. Accuracy and precision comparison among the Flow NanoAnalyzer, DLS, and TEM for virus size measurement.
 
EPD: The equivalent particle diameter of a sphere with a volume equivalent to the volume of a virus.

 


Discussion

1.      The SS burst area distribution histogram in Figure 1(b) indicates that four types of viruses can be resolved with baseline reparation.
2.      Employing silica nanoparticles with five different sizes to calibrate a standard curve, rapid and accurate virus size measurements with a resolution comparable to that of TEM is achieved. Particularly, with a throughput up to 10,000 particles per minute, a statistically reliable size distribution profile can be obtained in 2-3 minutes.
3.      Compared with the hydrodynamic diameters measured by DLS, the optical diameters measured by Flow NanoAnalyzer agrees well with the equivalent particle diameters derived from their structural dimensions measured by TEM. From the perspective of measurement precision, this approach is much higher than that of the DLS.
 
 
 
 

Angew. Chem. Int. Ed. 2016, 55, 10239-10243.

 


 

4.     Purity Assessment and Dynamic Monitoring of the Viral Genome Release Process


Introduction

Quality control is indispensable to ensure the adequate purity of virus products in the biopharmaceutical industry. Purity assessment of virus products is indispensable in many biotechnology applications. As we can see, many particles can coexist in the lysates of the host cells infected with a wild type phage, including cell debris, DNA-free proheads, empty capsids (after DNA ejection), and mature virions. In the meanwhile, the natural process of viruses delivering their genes into the hosts for self-replication has inspired the design of virus-like particles to deliver therapeutic or imaging agents. Monitoring the viral genome release process will allow for deeper insights into the mechanisms of the cargo release. The Flow NanoAnalyzer is applied to analyze the composition of the viral sample and to monitor the viral genome release process following treatment with NaClO4.
 


Results

 

 

 
 
Figure 4. Analysis of crude samples for bacteriophage T7 from different batches.

 
Figure 5. Analysis of NaClO4-triggered DNA ejection process of bacteriophage T7.


Discussion

1.      A crude extract of bacteriophage T7 obtained via polyethylene glycol precipitation is analyzed on the Flow NanoAnalyzer, the peak of lower scattering intensity is assumed to consist of cell debris, proheads, and empty capsids, whereas the peak of higher intensity can be ascribed to the mature virions.
2.      Simply by detecting the scattering signal of individual particles, the Flow NanoAnalyzer can easily distinguish the empty capsid of T7 from mature T7 virions.
3.      The effect of the NaClO4 concentration on the production of the T7 capsid is observed, and the dynamic process of DNA release is monitored.
4.      Broad applications in the analysis of virus products during both the manufacturing process and storage can be envisioned.
5.      The differentiation of capsids before DNA packaging (proheads, ~1100 counts) and after DNA release (empty capsids, ~600 counts), indicates that the Flow NanoAnalyzer could be an efficient tool for virology study.
 
 
 
 

Angew. Chem. Int. Ed. 2016, 55, 10239-10243.