Monday, 02 May 2016 20:36

Counting Zika Virus

Written by 
Published in Zika Diaries
Zika virus plaques on monolayers of African Green monkey (Vero) cells. Zika virus plaques on monolayers of African Green monkey (Vero) cells.

Last time on Zika Diaries, I recounted how we had obtained different strains of Zika virus. The next step was to make more of them. To do that we needed cells that can be infected with the virus, and a way to measure the amount of virus made.

Here’s another way to look at this problem: we have four small vials of different Zika viruses, each containing less than 1 milliliter of fluid. That won’t last very long. We need to make more Zika virus.

In general, viruses are fussy about what cells they reproduce in (you do know that viruses cannot reproduce without going inside a cell, right?). For example, we have been using HeLa cells for many years to produce stocks of poliovirus. But Zika virus does not multiply in HeLa cells! It’s necessary to find the right cell match for each virus.

A quick scan of the modest Zika virus literature revealed that Vero cells are the right cells for amplifying stocks of the virus. Vero is an immortal cell line produced years ago from kidneys of African Green monkeys. They are used for growing a number of different viruses. 

We took a small amount of each of our stocks of Zika virus, added it to Vero cells growing in a plastic dish, and in a few days the cells had all died. Virologists call this ‘cytopathic effect’. It’s a way of knowing the virus is replicating in cells.

Our stocks of Zika virus had just gone from 1 ml to 10 ml each. But how do we know how many virus particles we had? Put another way, what is the virus titer?

The number of virus particles in a stock can be measured in two general ways: by measuring the number of particles, or the number of infectious viruses. There is a difference between the two, because not all virus particles can infect a cell. In my lab we always measure the number of infectious viruses because that number tells us how many viruses we have that can infect cells.

My favorite assay for infectious virus particles is the plaque assay, an amazingly versatile and easy assay. Monolayers of cultured cells are incubated with a preparation of virus to allow adsorption to cells. The cells are then covered with nutrient medium containing agar, which forms a gel. When the infected cells release new progeny particles, the gel restricts the spread of viruses to neighboring uninfected cells. As a result, each infectious particle produces a circular zone of infected cells, or a plaque. Eventually the plaque becomes large enough to be seen with the naked eye. 

When we do a plaque assay, we can express the virus titer as plaque forming units per ml, or PFU/ml.

I love the plaque assay. It is simple and elegant. You can watch a movie of a microscopic plaque being formed in real time. And don’t forget to visit the Wall of Polio--a stack of plaque assay plates that I’ve built in my laboratory.

There were not many references for Zika virus plaque assays in the literature. In our laboratory we have several different types of plaque assays that we use to determine the titer of polioviruses and rhinoviruses. We decided to try one of those with Zika virus.

After 7 days incubation we stained the plaques and saw lovely holes in the cell monolayer--plaques--caused by Zika virus infection (photo). We had our plaque assay for Zika virus! We were able to determine that most of our Zika virus stocks contained about 10 million PFU/ml. We tried growing virus stocks in a mosquito cell line and obtained about 100 million PFU/ml.

We are happy to share our Zika virus plaque assay protocol with anyone who needs it. It’s interesting that several Zika virologists have complained that it takes too long--7 days. They do a variation of the plaque assay called a fluorescent focus assay, which takes only a day or two. In a fluorescent focus assay, the plaques are not visualized as a hole in the cell sheet, but by staining cells with a fluorescent antibody to identify virus-infected cells. It’s faster but more expensive. In science, everyone has their preferred methods.

Now we were ready to start doing experiments with Zika virus.

Next time: Zika virus in the brain.


See everything ASM is doing around Zika virus here.


Last modified on Monday, 23 May 2016 11:24
Vincent Racaniello

Vincent Racaniello, Ph.D. is Professor of Microbiology at Columbia University Medical Center. As principal investigator of his laboratory, he oversees the research that is carried out by Ph.D. students and postdoctoral fellows. He also teaches virology to graduate students, as well as medical, dental, and nursing students.

Vincent entered the world of social media in 2004 with virology blog, followed by This Week in Virology. Videocasts of lectures from his undergraduate virology course are on iTunes University and virology blog. You can find him on WikipediaTwitter, Facebook, and Instagram. His goal is to be Earth’s virology professor. In recognition of his contribution to microbiology education, he was awarded the Peter Wildy Prize for Microbiology Education by the Society for General Microbiology. His Wildy Lecture provides an overview of how he uses social media for science communication.