Sequencing COVID-19 mutations after jumping species

By November 2020, more than 200 people in Denmark were believed to have caught COVID-19 from mink. Meanwhile, the virus had been mutating within the animals, resulting in a handful of variants. Genomic analysis revealed that one variant, called Cluster 5, had unique mutations connected to the spike protein and seemed to cause moderately decreased sensitivity to neutralizing antibodies. By November 2020, Cluster 5 had been detected in 12 people in North Jutland ranging in age from 7 to 79. Danish authorities feared that Cluster 5 mutations might lead to antibody resistance and thus result in vaccines being less effective. Novel variants were already running rampant throughout the UK, Brazil and South Africa.

That’s when the Danish government made two extraordinary decisions. The first was to order the culling of every mink in Denmark – about 14 million animals. The move essentially wiped out the mink fur industry in Denmark. The animals were killed and either incinerated or coated with lime and buried. The second was to require that every positive COVID-19 case in Denmark be genetically sequenced.

Dr. Mette Christiansen, head of the Diagnostic NGS Core Facility, Molecular Medicine (MOMA) at Aarhus University hospital, as many other people never thought she would be living through a pandemic. Much less being involved in sequencing a high number of positive COVID-19. But with a calm demeanor, a steady gaze and decades of experience, the molecular biologist is not one to shy away from a challenge. She had already worked at the hospital for nearly 14 years, where she focused on the genetics of people with immune deficiencies before taking the role as NGS Core Facility lead.

Why test all positive cases?

In 2020, Denmark’s total COVID-19 caseload remained relatively low through the first half of 2020 in comparison to much of Europe. But in the summer came the mink infections, and in the fall, a variety of viral mutations. “When we had this mink mutation arise, people really didn't know what the impact would be,” says Christiansen. “The importance of sequencing all positive cases is to get a clear picture of what's going on with the pandemic here in Denmark.”

One immediate use of the test results has been to guide COVID-19 protocols in hospital environments, including guidelines for staff to better cope with infections or to reduce practices that may increase infections. “And the other really important side of it is to be able to look up those that have the more dangerous mutations that we still are not sure how will react to the vaccines,” Christiansen says. “So we are especially keen on discovering and containing those mutations.”

The data is publicly available and reveals the big picture of SARS-CoV-2 mutations in Denmark. The mink-originated Cluster 5 that had so worried health authorities seems to have died out in late 2020. The UK B.1.1.7 or alpha variant became dominant and now accounts for more than 99% of all cases in Denmark. The variants originating in Brazil (P.1) and South Africa (B.1.351) were also detected, but because health authorities knew exactly where those cases were thanks to genomic sequencing, they were able to take swift steps to contain community spread.

“Even though everybody still has to go into quarantine, we can focus our effort on those cases and make sure [the variants] don’t really gain access to the Danish community,” she says.

Keeping up with the variants

“The reason for looking into different variants is the fear of vaccine escape mutants,” says Christiansen. An escape mutant is a variant that has altered enough to avoid targeting by an antibody. Because vaccines trigger antibody production, the worry is twofold: it’s possible that vaccines may be ineffective against them, and that more mutants may evolve in vaccinated people infected with them.

“At the beginning, it was just going to be a few samples a week,” Christiansen says. But the number of positive cases spiked. “We went from having to sequence maybe 50 a week to having to sequence 1000.” The samples came from hospitals, regional clinics, mobile clinics, and especially TestCenter Denmark West, one in a national network of free testing centers established by the Statens Serum Institute (SSI), the national body for monitoring infectious disease. Samples included symptomatic patients and asymptomatic people getting tested regularly in the community.

To meet elevated demand, MOMA uses a range of QIAGEN Kits and instruments, including the QIAseq SARS-CoV-2 Primer Panel and QIAseqFX DNA Library UDI Kits for high – throughput sequencing.

“We have used them for several months now. At the moment, we're automating the setup on Hamilton robots to be able to sequence even more. This is, of course, important when you have a lot of samples,” she says. “We need to get high coverage during sequencing. One really important aspect is turnaround time – so getting the lab work finished as soon as possible,” she says. “Another is to get as many positive sample sequences as possible with high enough quality.”

What does the data show?

Of the more than 10 million PCR tests done in Denmark in 2020, about 80% of those who tested positive for COVID-19 in the spring were protected against reinfection in the fall or winter. In contrast, those who tested negative in the spring were five times more likely to test positive by the end of the year. While there were no differences between males and females, age was a factor: Protection dropped by more than 30% for people over 65.

With its population increasingly vaccinated and infection rates low, Denmark has turned the corner on COVID-19. That means Christiansen can shift focus to the lab’s other NGS responsibilities and projects, including human genome and exome sequencing.

But there is still a question of new variants arising.

“If you go back one or two months, the thought was that a lot of new mutants would arise, or at least that we would have higher numbers of infections,” Christiansen says. “It is not known yet whether there are real escape mutants,” Christiansen notes. “But all these data will be able to pinpoint whenever [a mutant] arises that might be a problem.”

“At the moment, we are quite stable. So what is going to happen within the next couple of months is going to be really interesting. Some of the people that have been vaccinated and do get infected – will they produce more mutated viruses? We don't know yet.”