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Gene sequencing could lead to the discovery of a vaccine or ways to prevent ferret coronaviruses.
In June 2015, Michigan State University launched a crowdfunding campaign to raise money for ferret virus sequencing research. The goal is to sequence the entire genome of both ferret enteric (related to the intestines) and ferret systemic coronavirus, plus the genome of mink systemic coronavirus. Researchers hope that this information will lead to the development of accurate diagnostic tests for these coronaviruses and ways to prevent them.
The crowdfunding project is in its final week, but how did the research get to this point, what’s being done with the funds raised? For answers, we interviewed Dr. Matti Kiupel, professor in the department of Pathobiology and Diagnostic Investigation and section chief of anatomic pathology at the Diagnostic Center for Population and Animal Health (DCPAH), who is leading the project.
The History Behind Kiupel’s Coronavirus Search
Kiupel has been on the trail of coronavirus infection in ferrets since his graduate student days at Purdue University in 1998 when he did necropsies on several ferrets that had suffered from what was then called green slime disease. He found coronavirus particles in the feces and then an antibody that has cross reactivity to group 1 coronaviruses.
“Dr. Bruce Williams had heard about my discovery,” Kiupel said. “He had been working on this syndrome for a while and had accumulated a large series of formalin fixed tissues from more than 100 affected ferrets with the same clinical disease. We tested a series of these animals with immunohistochemistry using the same antibody and confirmed a consistent association of the coronavirus with the intestinal lesions, thereby suggesting that this coronavirus was the most likely cause.”
They named the disease epizootic catarrhal enteritis and published their findings in the “Journal of the American Veterinary Medical Association.”
In 2001, Kiupel became assistant professor at Michigan State University. Among his many projects he continued working on ferret coronaviruses.
“I had teamed up with Roger Maes and Annabel Wise, virologists at DCPAH,” Kiupel said, “and together we further characterized the coronavirus associated with ECE as a novel group 1 coronavirus and named it ferret enteric coronavirus (FRECV). The findings were published in 2006.”
This study provided genetic evidence that a previously unknown coronavirus caused the disease ECE. While this helped to further identify the cause of ECE, it did nothing to advance diagnosis or prevention. Meanwhile, a few reports of ferrets suffering from something similar to feline infectious peritonitis (FIP) were surfacing in Europe. Kiupel hoped to do genetic tests on these, but could not get any frozen tissue samples because importation of frozen tissues wasn’t possible.
“Through collaboration with Michael Garner we finally received frozen tissues from ferrets with FIP-like disease and could identify a coronavirus similar to FRECV within lesions,” Kiupel said. “The findings were published in 2008.”
“Additional research into samples from ferrets with ECE and FIP-like disease allowed us to further characterize the coronaviruses identified in both disease processes as FRECV and ferret systemic coronavirus (FRSCV).”
Kiupel added that they recently discovered an FIP-like disease in mink.
“Since mink also have an enteric coronavirus, a similar disease spectrum as in cats and ferrets is expected,” Kiupel said. “The findings of this discovery have been presented at national meetings, but have yet to be published.”
This expected similarity between the cat, ferret and mink diseases is why sequencing the genome of the mink systemic coronavirus is included in the current research goals.
Via Michigan State University Ferret Health Advancement
The core team working on ferret research at MSU is Matti Kiupel, DVM, MS, PhD, DACVP (left), Roger Maes, DVM, PhD (middle) and Annabel Wise, DVM, MS, PhD.
Current Ferret Coronavirus Research At MSU
The researchers are using whole genome sequencing using the Illumina platform, Kiupel said. He added that two thirds of the genome is still to be sequenced, and he expects the process to take several months.
“It involves not only the sequencing itself, which is automated, but also the sequence assembly,” he said.
The funds raised allow the researchers to contract with the Genomics facility.
“The contract involves both computer and researcher time,” Kiupel said. “The cost for sequencing each virus will be around $4,000 to 5,000. We had planned to use funds previously raised through the MSU web page to allow us to sequence additional samples. This web page has been essential to allow a small but steady stream of support, which together with the current campaign will finally allow us to get more complete sequence data.”
Although the crowdfunding campaign has surpassed the $5,000 goal, every dollar over this helps further the research. Kiupel said that the funds raised are being combined with past funds raised to sequence a single strain.
“Any additional funds can be used for sequencing of additional strains which allow us to focus on the identification of virulence markers and their genomic location,” Kiupel said. “When we say strains we mean different isolates from different ferrets. Since we can’t isolate the virus and don’t know how much genetic variations will exist between viruses causing disease in different animals, we refer to them as strains. If we identify some particular regions of interest in the genome, we would want to screen a larger number of such strains to confirm that the genetic change is of relevance.”
After sequencing is complete, the next step is polymerase chain reaction (PCR).
“There are two genotypes of ferret coronavirus. Both have been associated with the enteric and the systemic form,” Kiupel said. “Since it has become clear that these genotypes are not strictly associated with a specific pathotype (enteric or systemic), we will run the PCR for both genotypes on all diagnostic cases. The initial format will be two individual genotype-specific assays. The next format will be a duplex real-time PCR.”
Kiupel has no estimates on when a vaccine might be developed, but that’s not the only option for prevention.
“This is analogous to FIP in cats, where effective vaccines are still elusive,” Kiupel said. “We are also contemplating investigating other approaches such as the use of broad-spectrum inhibitors against 3C-like proteases of coronaviruses as potential antiviral drugs.”
Why Coronavirus Research Is Needed
Kiupel said that coronaviruses are widespread and cause illness or disease in a variety of species. That’s why it’s important to study them. He believes the research into ferret coronaviruses also will benefit other animals.
“Identification of virulence markers that are conserved or semi-conserved will be very important for comparative molecular pathogenesis studies and form the rationale for the logical design of immunization strategies,” he said.
But you might be wondering why genetic study is required.
“As a rule, cell culture propagation of members of this family is challenging,” Kiupel said. “A genomics-based approach is therefore the most logical one towards the discovery of viral genes that are mediators of virulence and host immune responses.”
It Takes A Team To Tackle Coronavirus
Kiupel said the core team at the College of Veterinary Medicine at MSU doing the research are Roger Maes, Annabel Wise and himself. Maes is a professor and section head of the virology laboratory at DCPAH. Wise is an academic specialist in virology. But the team also includes many others.
“We have always collaborated closely with clinicians and researchers from diverse fields to further our understanding of different ferret diseases,” Kiupel said. “Some of the clinicians we closely collaborated with include Jerry Murray, Katrina Ramsell, Angela Lennox, Natalie Antinoff, Ruth Heller, Sandra Mitchell among many others. We also collaborate with other pathologists such as Michael Garner or Bruce Williams. We collaborate with James Fox at MIT, and I had the opportunity to help author the virology and neoplasia chapter of the most recent edition of “Biology and Diseases of the Ferret.” Currently we collaborate with Bob Wagner and Kevin Carr on different ferret coronavirus projects. Kevin runs the Genomics Core Facility at MSU and plays an essential part in the project that we raised money for through the web campaign. We have also collaborated with a pediatric oncologist, David Wilson, to shed light on the mechanisms of ferret adrenal disease.”
In addition to the people doing the work, people to fund the research are also part of the team. Although the crowdfunding campaign ends shortly, the need for funds is ongoing, and donations can always be made through the Ferret Health Advancement website.