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For more than two decades, Dr. Jason Roberts has been routinely observing underworld microscopes and observing the behaviors of some of nature’s deadliest nanometer-sized insurgents.
Roberts, a senior scientist at the Victoria Infectious Diseases Reference Laboratory in Melbourne, is also a virologist consultant to the Australian government’s polio eradication program.
In early February, he was one of the first Australians to observe the virus, which, a few days later, would receive the official name of SARS-Cov-2.
And as an accomplished scientific illustrator, he’s also adept at using powerful electron microscopes (EMs) to capture images of viruses and then use them to recreate intricate three-dimensional models.
Although I didn’t know it at the time, I was photographing a virus that would become the mother of all disruptive pathogens, a biological wrecking ball that has become an existential threat to our way of life.
“You are looking at the enemy,” says Roberts, recalling those first encounters. “It is invisible to everyone else in the world.
“But for us in virology, especially in MS, it is not invisible. We see it. We know how it looks.”
Observing the virus is knowing its shape and structure. Armed with that knowledge, scientists can begin to decipher their genomic mission, a vital first step in understanding how to neutralize this invisible enemy.
Genetic code snippet
As with all viruses, SARS-CoV-2 exists in a limbo somewhere between the animate and the inanimate.
Like a seed, a virus will only kick in if it finds the right kind of conditions. Otherwise, it will perish.
A virus at this stage is known as a virion. Since they cannot replicate on their own, virions must find a suitable host to survive.
“It really is just a short snippet of genetic code,” explains Roberts. “[But] it’s surprising that a little bit of code can wreak havoc. “
Roughly spherical with a strip of projections called pointy proteins or peplomers, a SARS-CoV-2 virus particle generally measures around 100 nanometers in diameter. That is approximately 10,000 times smaller than a grain of salt.
Each virus is equipped with an outer shell, or viral envelope, designed to penetrate a cell and deliver its load of genetic material to the host.
Photo:
Jason Roberts
Roberts describes that charge as “spring loaded, like a cat in the box.” When he finds the right target, he “just pings and delivers it.”
“Once the code enters a cell, it hijacks the [cell’s] Replication machinery. Now it can reproduce; it is capable of evolving; is able to metabolize energy. “
And there is no escape from them. Our biosphere is full of viruses.
A 2011 article in the research journal Nature Reviews Microbiology calculated that there were approximately ten million viruses on earth, that’s 1×1031 or a number with 1 followed by 31 zeros.
Despite the bad reputation they get, not all viruses are bad for us.
Roberts says that the human race could not survive without some of them, such as those found in our guts, that help keep our digestive system in balance.
“Viruses,” he says, “are the oil that lubricates the gears of evolution.”
‘Really pretty scary’
Roberts’ laboratory is part of the Peter Doherty Institute for Infection and Immunity. That’s the same organization that in late January became the first laboratory outside of China to successfully grow a sample of the SARS-CoV-2 virus.
They used a sample taken from one of the first people in Australia to test COVID-19 in Australia on Friday, January 24.
A few days later, Roberts and Dr. Andrew Leis, an electron microscopist and viral expert at the Bio21 Institute for Molecular Science and Biotechnology at the University of Melbourne, placed samples under the lens of a transmission electron microscope (TEM).
These were the first blurry images they produced. They were not published or distributed due to the quality of the image.
Roberts vividly remembers that first impression and the mood in the lab that day.
“Initially, there was excitement … and then it was like this fear: This is really scary.”
The sample showed the presence of many virus particles, much more than they expected to see.
“We had this moment of clarity and the kind of urgency you need to get to what needs to be done, get results, collaborate …”
Around January 31, the Doherty Institute had also delivered a small vial containing a sample of the virus that it had grown to CSIRO’s Australian Center for Disease Preparation (ACDP) in Geelong.
Members of the ACDP dangerous pathogens team took the sample in the safest part of the facility where they grew the virus from the original culture.
ACDP is one of the few facilities that includes a certified Physical Containment Level 4 (PC4) laboratory area, the place where dangerous pathogens are to be grown, tested and stored.
The facility has been built on a box-in-box principle.
Sections are air-blocked, ensuring that physical containment is doubled or tripled, should one fail.
The safe area is surrounded by a 30 cm thick concrete wall and is kept at a lower air pressure than the outside world, to keep any infectious agent in the air inside.
ACDP is also where Sandy Crameri has worked for 30 years as a specialized electron microscope for viruses. She is also one of the most experienced.
Photo:
Sandy Crameri
Over the years, you’ve seen and imagined the worst of the worst: pathogens like SARS, MERS, HIV, Hendra, Ebola, and H1N1, the virus that triggered the 2009 swine flu pandemic.
Around February 10, Crameri remembers receiving her first sample to examine.
“When I first looked at peplomers, which are crown-shaped things, I thought, ‘wow, they’re really obvious,'” she says.
“Then I invited my colleagues … to come take a look because everyone was interested in applauding him when we got here.”
Crameri quickly flipped her first image over, a monochrome version of this image below, which after coloring, which took a day or so, was published by CSIRO.
His image of a SARS-CoV-2 virion was taken with an EM transmission, which shoots electrons through the sample to reveal the structure, producing a two-dimensional image.
It was prepared using what is called the negative staining method, which improves the contrast of the sample. It is among the oldest and most widely used EM imaging techniques and produces relatively quick results.
Instead of using visible light, electron microscopes shoot an accelerated beam of electrons at the sample. Since electrons have a much shorter wavelength, they can offer higher magnification and better resolution, producing much more detailed images.
All electron microscope images are grayscale with coloration added later.
While color is used in research to highlight certain characteristics, Crameri says it was also added with the goal of doing something that will grab the attention of the public.
“I didn’t want to choose red, for example, because red is a kind of alarmist color,” he says, explaining his choices. “So I asked my colleague what color she liked and she said burnt orange.”
The orange burned and Crameri chose the complementary color, which was blue, to color the background, allowing the virion to come out more.
The image is similar to one of a series of images produced by Roberts, assisted by Andrew Leis on February 7.
It shows a single virion, with its distinctive spike proteins clearly visible. This is also a negative staining image captured in an EM transmission.
“If you look at the first images that came out, they were bad enough, to be honest,” he says. “It is through fine tuning the growth of the virus and the adjustments that we managed to get some really amazing particles.”
Roberts chose to color the shades of yellow-orange with a dark background to reflect the danger posed. The background is what he calls the peacock effect: a purple, blue and green gradient that reflects the plumage of the bird.
Limited diagnostic value
Another type of virus imaging is one produced using a scanning electron microscope (SEM), which is not equipment used by Crameri or Roberts.
As the name suggests, it scans the surface of an object instead of looking at it. And it provides a much more contoured effect and, after applying colors, texture.
The scanning EMs were used to take images like the following produced by the US National Institute of Allergy and Infectious Diseases. USA (NIAID) for its Integrated Research Center.
Each of the yellow dots in the image below is a coronavirus particle and corresponds to the images of the individual virus particles shown above that were captured by Sandy Crameri and Jason Roberts.
This image shows a cell taken from a patient sample (in blue) heavily infected with SARS-CoV-2 virus particles.
Roberts says the scanning MS has limited diagnostic value when investigating a new virus.
“While the NIAID images are visually interesting, they are not high enough in resolution to provide real structural information about the virus,” he says.
Scanning EM micrographs of SARS-Cov-2 virions sprouting from a cell.
NIAID, spearheaded by Dr. Anthony Fauci, one of the top advisers to the President of the United States, Donald Trump, during the current coronavirus crisis, has produced a series of startling EMs to scan for the SARS-CoV-2 virus.
A new frontier in microscopy.
Roberts is also using a process known as cryogenic electron microscopy, or cryo-EM for short, an advanced molecular imaging technique.
The 2017 Nobel Prize in Chemistry was awarded to three scientists for their work in developing cryo-EM, which, according to the quote, had “taken biochemistry into a new era.”
Although the technique was developed in the years between 1970 and 1990, it is only in more recent times that the technology has been brought up to date, allowing images to be transformed into sophisticated 3D models.
What the experts say about the coronavirus:
The process involves rapidly freezing the virus in a thin layer of ice approximately 1,000 thick in human hair, essentially capturing the specimen in suspended animation.
Roberts produced this image, which shows a single virion suspended in that ice cap, which appears as the darkest background area.
With cryo-EM images, it is also possible to combine thousands of these images, all taken at slightly different angles, to form a three-dimensional image or tomogram.
Tomography is the T on CT image, sometimes known as computed tomography, which produces a cross-sectional image of the anatomy and is a form of diagnostic imaging used by physicians.
The only difference is that EM uses electrons instead of X-rays.
Roberts says cryo-EM imaging is a recent technique and it is the process his team is currently using to build their models.
“It will give you a very good idea of how the virus is designed and what its general structure is.”
This image, below, is an example of a tomogram taken by Roberts on April 17.
It shows nuclei of virus particles, red in color, enclosed in their yellow viral envelopes.
The green and purple parts are part of the host cell, which comes from an animal kidney.
One of the virus particles can be seen leaving the cell in a process known as budding, which is a form of replication.
This is a short 3D effect video of the tomogram showing the same portion of infected cell (as the image above).
It was produced by stitching together 126 individual two-dimensional EM images taken at different angles.
The thousands of collected EM images become parts of a puzzle, allowing scientists to reconstruct the virus’s atomic structure.
That will help explain your tactics: your strategy for getting in and out of the cell, how it reproduces, and what your weaknesses are.
Researchers can use existing drugs or develop new drugs to try to interrupt those viral processes.
The key is in those atoms, says Roberts.
“Once you understand the atomic structure of a virus, you have a pretty good idea of how to knock it down.”
Credits
- Reports: Stephen Hutcheon
- Design and production: Alex Palmer
- Images courtesy of Jason Roberts / VIDRL – Doherty Institute (with technical assistance from Andrew Leis / Bio21 Institute), Sandy Crameri / CSIRO and NIAID-RML.
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