In a recent study of influenza virus reassortment in ferrets, the authors used polymerase chain reaction (PCR) to search for viruses with RNA segments from the 2009 pandemic H1N1 strain and seasonal H1N1 and H3N2 strains. I thought you might like to see how I did a similar experiment in 1979 – a very different era for laboratory techniques.
For my Ph.D. thesis project, I wanted to isolate reassortants of two influenza B virus strains, B/Lee and B/Maryland. The goal was to obtain viruses with a genome consisting of one RNA segment from one parent, and 7 RNA segments from the other parent. These viruses would then be used to identify the protein product of each viral RNA.
To isolate these reassortants, I co-infected cells in culture with both viruses, allowed them to replicate, and then harvested the newly synthesized viruses. I then did a plaque assay with the viruses produced by the co-infected cells, and isolated individual clones by plaque purification. I prepared virus stocks from each plaque-purified clone, and then asked if any of these viruses were reassortants.
In 1979, we identified viral reassortants by a tedious method. Each virus to be examined was used to infect cultured cells in the presence of radioactive phosphate. The viruses were purified by centrifugation, and the viral RNA was extracted, concentrated, and fractionated by gel electrophoresis. The gel was dried and exposed to X-ray film. Because the viruses were propagated in the presence of radioactive phosphate, which was incorporated into the viral RNAs, it was possible to visualize each viral segment as a band on the film. The X-ray is shown above.
The RNAs of three different viruses are included: B/Lee on the left, B/Maryland in the middle, and a plaque-purified virus called R3. You can see that each lane contains 8 RNAs, as expected. It is also evident that the migration pattern of the RNAs is different for B/Lee and B/Maryland. This property allowed us to determine that the plaque purified virus R3 is a reassortant that inherits RNA 2 from B/Lee, and the remaining 7 RNAs from B/Maryland. Just what I wanted!
In fact, in just one infection, I isolated all the different reassortant viruses that I needed. I remember showing the gel to my thesis advisor: he told me I didn’t deserve such luck.
The whole procedure – from infecting cells in the presence of radioactive phosphate, to producing the X-ray, took about a week. And to get all the right viral reassortants required a great deal of luck.
Today, the process of identifying influenza viral reassortants is far simpler and faster. The process begins in a similar way – co-infect cells (or animals) with two different viruses. Once the infection is complete, a small sample of the cell culture medium is taken, heated to disrupt the virions, and the viral RNA is converted to DNA using reverse transcriptase. The DNA is amplified by PCR, in eight separate reactions, using primer pairs specific for the individuals segments. The products are then fractionated by gel electrophoresis, as shown here.
Total time to identify reassortants by PCR – less than a day. That’s progress.
In a few years, we’ll skip the gel electrophoresis and simply determine the sequence of the RNAs using a small, inexpensive machine that will be on most laboratory benches. And who knows what will be next? That’s one of the beauties of science: it is driven forward by technological innovation.
Racaniello VR, & Palese P (1979). Influenza B virus genome: assignment of viral polypeptides to RNA segments. Journal of virology, 29 (1), 361-73 PMID: 430594