Analysis of Virus Particles in Media by Dynamic Light Scattering

Introduction

Traditional visual methods of observing virus particles in solution give a snapshot of a very small sampling volume, while the technique of particle analysis using dynamic light scattering yields an ensemble average of particles present in the solutions. Virus particles incubated in the laboratory present a challenge, as they must be grown within cells in a media containing albumin and other small proteins, such as those present in Fetal Bovine Serum (FBS) or Minimum Essential Media (MEM) solutions. When virus particles are released from the cells, the cell fragments are large and can be separated by centrifugation, but the smaller proteins of the media cannot be removed. Careful selection of the distribution parameters from a dynamic light scattering experiment allows the size distributions of virus particles to be clearly observed in the presence much smaller proteins that constitute the media.

Background

Fish Viruses

There are a number of fish viruses that have the potential to wreak significant economic damage on both recreational and commercially important fish populations. (For more information, see the USGS Western Fisheries Research Center Website). Four virus types are studied here with representative images of family virus types are shown below.

1. CTV (Cutthroat Trout Virus – an emerging virus), a small round virus 25-35nm in diameter of the Hepeviridae family; (below left)
2. IHNV (Infectious Hematopoietic Necrosis Virus of the family Rhabodoviridae is a bullet shaped virus with dimensions ~ 70nm wide x 170 nm long) that infects Rainbow Trout and Sockeye Salmon (Second from left).
3. ISAV (Infectious Salmon Anemia Virus) is mostly present is farmed Atlantic salmon populations and has a very high mortality rate. It is of the Orthomyxoviridae family, and virions are generally between 50-120nm diameter (second from right).
4. LMBV (Largemouth Bass Virus) is of the family Iridoviridae and is an Icosahedral shaped particle ~150nm diameter and is present in the largemouth bass populations in the Southeast US (below right).

We wanted a quick method of assessing the presence of virus particles in solution under known incubation conditions and chose DLS as the method to explore.

Dynamic Light Scattering

The Autocorrelation Function

Experimental

1. Samples were kindly provided by the USGS Western Fisheries Research Center in Seattle WA. Virus particles were grown in Chinook Salmon embryo cells in Minimum Essential Medium containing fetal bovine serum (FBS) and virus particles were expressed into the media. Cells and cell fragments were separated by centrifugation. Cell media with viral particles were kept refrigerated at 5 °C until measurements were made.
2. Dynamic Light Scattering (DLS) measurements were made on a Brookhaven Instruments ZetaPALS instrument equipped with a BI-MAS particle sizing capability.
3. Samples were allowed to equilibrate to 25 °C in the sample compartment for at least 5 minutes and measured in polystyrene cuvettes at 25 °C. DLS autocorrelation data was reduced using the NNLS deconvolution algorithm. The reduced particle size data was evaluated by both intensity and number distribution, and the results exported to an exported into an Excel format and graphed in Origin 8.0.

Distribution Curves
NNLS distributions can be weighted by number, surface area, volume, or the measured intensity. The weighting by intensity greatly emphasizes the largest particles because of the power relationship in diameter, where number weighting emphasizes the smallest particles.

Summary and Conclusions
The results clearly show the presence of virus particles with the expected average dimensions when observed by an intensity weighted distribution, but these particles are absent in the number distribution. This shows the virus particles are present, but in relatively small numbers compared to the proteins and other small molecules in cell growth media. There does appears to be some other large particles present, possibly other cell fragments or expressed molecules (RNA, proteins) that would not be expected to be removed in the centrifugation step due to similar size ranges to the virus particles. Proper control samples should allow the differentiation of virus particles from free cell fragments.