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Clinical Laboratories and Pathology Groups

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Clinical Laboratories and Pathology Groups

Hosted by Robert Michel
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Researchers Use Genetic Sequencing and Wastewater Analysis to Detect SARS-CoV-2 Variants and Monkeypox within Communities

Researchers surprised that process designed to detect SARS-CoV-2 also identifies monkeypox in wastewater

Early information about an outbreak in a geographical region can inform local clinical laboratories as to which infectious agents and variants they are likely to see when testing patients who have symptoms. To that end, wastewater testing has become a rich source of early clues as to where COVID-19 outbreaks are spreading and how new variants of the coronavirus are emerging.

Now, scientists in San Diego County are adding monkeypox to its wastewater surveillance, according to an August University of California San Diego (UCSD) Health press release. The team at UCSD uses the same process for detecting SARS-CoV-2.

Ongoing advances in genetic sequencing and digital technologies are making it feasible to test wastewater for infectious agents in ways that were once too time-consuming, too expensive, or simply impossible.

Rob Knight, PhD

“Before wastewater sequencing, the only way to do this was through clinical testing, which is not feasible at large scale, especially in areas with limited resources, public participation, or the capacity to do sufficient testing and sequencing,” said Knight in a UCSD press release. “We’ve shown that wastewater sequencing can successfully track regional infection dynamics with fewer limitations and biases than clinical testing to the benefit of almost any community.” (Photo copyright: UC San Diego News.)

Same Process, Different Virus

Following August’s declaration of a state of emergency by California, San Diego County, and the federal government, UCSD researchers added monkeypox surveillance to UCSD’s existing wastewater surveillance program.

“It’s the same process as SARS-CoV-2 qPCR monitoring, except that we have been testing for a different virus. Monkeypox is a DNA virus, so it is a bit of a surprise that our process optimized for SARS-CoV-2, which is an RNA virus, works so well,” said Rob Knight, PhD, Professor of Pediatrics and Computer Science and Engineering at UCSD and one of the lead authors of the study in the press release.

Knight is also the founding director of the Center for Microbiome Innovation at UCSD.

According to the press release, RNA sequencing from wastewater has two specific benefits:

  • It avoids the potential of clinical testing biases, and
  • It can track changes in the prevalence of SARS-CoV-2 variants over time.

In 2020, at the height of the COVID-19 pandemic, scientists from the University of California San Diego and Scripps Research looked into genetic sequencing of wastewater. They wanted to see if it would provide insights into levels and variants of the SARS-CoV-2 within a specific community.

Individuals who have COVID-19 shed the virus in their stool.

The UCSD/Scripps researchers deployed commercial auto-sampling robots to collect wastewater samples at the main UCSD campus. They analyzed the samples for levels of SARS-CoV-2 RNA at the Expedited COVID-19 Identification Environment (EXCITE) lab at UCSD. After the success of the program on the campus, they extended their research to include other facilities and communities in the San Diego area.

“The coronavirus will continue to spread and evolve, which makes it imperative for public health that we detect new variants early enough to mitigate consequences,” said Knight in a July press release announcing the publication of their study in the journal Nature, titled, “Wastewater Sequencing Reveals Early Cryptic SARS-CoV-2 Variant Transmission.”

Detecting Pathogens Weeks Earlier than Traditional Clinical Laboratory Testing

In July, the scientists successfully determined the genetic mixture of SARS-CoV-2 variants present in wastewater samples by examining just two teaspoons of raw sewage. They found they could accurately identify new variants 14 days before traditional clinical laboratory testing. They detected the presence of the Omicron variant 11 days before it was first reported clinically in the community.

During the study, the team collected and analyzed 21,383 sewage samples, with most of those samples (19,944) being taken from the UCSD campus. They performed genomic sequencing on 600 of the samples and compared them to genomes obtained from clinical swabs. They also compared 31,149 genomes from clinical genomic surveillance to 837 wastewater samples taken from the community.

The scientists distinguished specific viral lineages present in the samples by sequencing the viruses’ complete set of genetic instructions. Mutational differences between the various SARS-CoV-2 variants can be minute and subtle, but also have notable biological deviations.

“Nothing like this had been done before. Sampling and detection efforts began modestly but grew steadily with increased research capacity and experience. Currently, we’re monitoring almost 350 buildings on campus,” said UCSD’s Chancellor Pradeep Khosla, PhD, in the July press release.

“The wastewater program was an essential element of UC San Diego Health’s response to the COVID pandemic,” said Robert Schooley, MD, Infectious Disease Specialist at UC San Diego Health, in the press release. Schooley is also a professor at UCSD School of Medicine, and one of the authors of the study.

“It provided us with real-time intelligence about locations on campus where virus activity was ongoing,” he added. “Wastewater sampling essentially allowed us to ‘swab the noses’ of every person upstream from the collector every day and to use that information to concentrate viral detection efforts at the individual level.”

Monkeypox Added to UCSD Wastewater Surveillance

In August, UCSD officially added the surveillance of the monkeypox virus to their ongoing wastewater surveillance program. A month earlier, the researchers had discerned 10,565.54 viral copies per liter of wastewater. They observed the levels fluctuating and increasing.

On August 2, the scientists detected 189,309.81 viral copies per liter of wastewater. However, it is not yet clear if the monitoring of monkeypox viral loads in wastewater will enable the researchers to accurately predict future infections or case rates.

“We don’t yet know if the data will anticipate case surges like with COVID,” Knight said in the August UCSD press release announcing the addition of monkeypox to the surveillance program. “It depends on when the virus is shed from the body relative to how bad the symptoms are that cause people to seek care. This is, in principle, different for each virus, although in practice wastewater seems to be predictive for multiple viruses.”

Utilization of genetic sequencing of wastewater sampling will continue to develop and improve. “It’s fairly easy to add new pathogens to the process,” said Smruthi Karthikeyan, PhD, an environmental engineer and postdoctoral researcher in Knight’s lab who has overseen wastewater monitoring at UC San Diego. “It’s doable on short notice. We can get more information in the same turnaround time.”

Thus, clinical laboratories engaged in testing programs for COVID-19 may soon see the addition of monkeypox to those processes.

-JP Schlingman

UC San Diego Researchers Add Monkeypox to Wastewater Surveillance

Wastewater Sequencing Reveals Early Cryptic SARS-CoV-2 Variant Transmission

Awash in Potential: Wastewater Provides Early Detection of SARS-CoV-2 Virus

National Wastewater Surveillance System (NWSS)

CDC National Wastewater Surveillance System Locates and Tracks SARS-CoV-2 Coronavirus in the Public’s Wastewater

CDC, HHS Create National Wastewater Surveillance System to Help Monitor and Track Spread of COVID-19

Researchers Use Machine Learning to Identify Thousands of New Marine RNA Viruses in Study of Interest to Microbiologists and Clinical Laboratory Scientists

Screening and analysis of ocean samples also identified a possible missing link in how the RNA viruses evolved

An international team of scientists has used genetic screening and machine learning techniques to identify more than 5,500 previously unknown species of marine RNA viruses and is proposing five new phyla (biological groups) of viruses. The latter would double the number of RNA virus phyla to 10, one of which may be a missing link in the early evolution of the microbes.

Though the newly-discovered viruses are not currently associated with human disease—and therefore do not drive any current medical laboratory testing—for virologists and other microbiologists, “a fuller catalog of these organisms is now available to advance scientific understanding of how viruses evolve,” said Dark Daily Editor-in-Chief Robert Michel.

“While scientists have cataloged hundreds of thousands of DNA viruses in their natural ecosystems, RNA viruses have been relatively unstudied,” wrote four microbiologists from Ohio State University (OSU) who participated in the study in an article they penned for The Conversation.

The OSU study authors included:

Zayed was lead author of the study and Sullivan led the OSU research team.

The researchers published their findings in the journal Science, titled, “Cryptic and Abundant Marine Viruses at the Evolutionary Origins of Earth’s RNA Virome.”

Matthew Sullivan, PhD
“RNA viruses are clearly important in our world, but we usually only study a tiny slice of them—the few hundred that harm humans, plants and animals,” explained Matthew Sullivan, PhD (above), Director, Center of Microbiome Science, in an OSU news story. Sullivan led the OSU research team. “We wanted to systematically study them on a very big scale and explore an environment no one had looked at deeply, and we got lucky because virtually every species was new, and many were really new,” he added. (Photo copyright: University of Ohio.)

RNA versus DNA Viruses

In contrast to the better-understood DNA virus, an RNA virus contains RNA instead of DNA as its genetic material, according to Samanthi Udayangani, PhD, in an article she penned for Difference Between. Examples of RNA viruses include:

One major difference, she explains, is that RNA viruses mutate at a higher rate than do DNA viruses.

The OSU scientists identified the new species by analyzing a database of RNA sequences from plankton collected during a series of ocean expeditions aboard a French schooner owned by the Tara Ocean Foundation.

“Plankton are any aquatic organisms that are too small to swim against the current,” the authors explained in The Conversation. “They’re a vital part of ocean food webs and are common hosts for RNA viruses.”

The team’s screening process focused on the RNA-dependent RNA polymerase (RdRp) gene, “which has evolved for billions of years in RNA viruses, and is absent from other viruses or cells,” according to the OSU news story.

“RdRp is supposed to be one of the most ancient genes—it existed before there was a need for DNA,” Zayed said.

The RdRp gene “codes for a particular protein that allows a virus to replicate its genetic material. It is the only protein that all RNA viruses share because it plays an essential role in how they propagate themselves. Each RNA virus, however, has small differences in the gene that codes for the protein that can help distinguish one type of virus from another,” the study authors explained.

The screening “ultimately identified over 44,000 genes that code for the virus protein,” they wrote.

Identifying Five New Phyla

The researchers then turned to machine learning to organize the sequences and identify their evolutionary connections based on similarities in the RdRp genes.

“The more similar two genes were, the more likely viruses with those genes were closely related,” they wrote.

The technique classified many of the sequences within the five previously known phyla of RNA viruses:

But the researchers also identified five new phyla—including two dubbed “Taraviricota” and “Arctiviricota”—that “were particularly abundant across vast oceanic regions,” they wrote. Taraviricota is named after the Tara expeditions and Arctiviricota gets its name from the Arctic Ocean.

They speculated that Taraviricota “might be the missing link in the evolution of RNA viruses that researchers have long sought, connecting two different known branches of RNA viruses that diverged in how they replicate.”

In addition to the five new phyla, the researchers are proposing at least 11 new classes of RNA viruses, according to the OSU story. The scientists plan to issue a formal proposal to the International Committee on Taxonomy of Viruses (ICTV), the body responsible for classification and naming of viruses. 

Studying RNA Viruses Outside of Disease Environments

“As the COVID-19 pandemic has shown, RNA viruses can cause deadly diseases. But RNA viruses also play a vital role in ecosystems because they can infect a wide array of organisms, including microbes that influence environments and food webs at the chemical level,” wrote the four study authors in The Conversation. “Mapping out where in the world these RNA viruses live can help clarify how they affect the organisms driving many of the ecological processes that run our planet. Our study also provides improved tools that can help researchers catalog new viruses as genetic databases grow.”

This remarkable study, which was partially funded by the US National Science Foundation, will be most intriguing to virologists and microbiologists. However, clinical laboratories also should be interested in the fact that the catalog of known viruses has just expanded by 5,500 types of RNA viruses.

Stephen Beale

Related Information:

Researchers Identified Over 5,500 New Viruses in the Ocean, Including a Missing Link in Viral Evolution

Cryptic and Abundant Marine Viruses at the Evolutionary Origins of Earth’s RNA Virome

There’s More to RNA Viruses than Diseases

Differences Between DNA and RNA Viruses

Ocean Water Samples Yield Treasure Trove of RNA Virus Data

Global Survey of Marine RNA Viruses Sheds Light on Origins and Abundance of Earth’s RNA Virome

Scientists Find Trove of over 5,000 New Viruses Hidden in Oceans

Virologists Identify More than 5,000 New Viruses in the Ocean

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