A new study has identified small mutations in genes of early COVID-19 viruses that appeared to have substantially increased the risk of severe disease in patients who contracted them.

The researchers, who carried out extensive, whole-genome sequencing of the viruses taken from patients primarily in the first year of the pandemic, spotted in particular a group of four genetic mutations that were linked to a 5.46-fold increase in hospitalization.

“5.46 is a large effect,” said Dr. Lue Ping Zhao, a biostatistician at Fred Hutchinson Cancer Research Center, who is the lead author of a paper on the study published online by the journal Scientific Reports on Jan. 24.

While much of coronavirus research focuses on ways to block transmission, this study sought to identify virulence factors — traits that would make the virus cause more serious illness. To do so, it sequenced every viral genome from virus samples taken from 683 patients from Washington state. Roughly half the patients were so sick they were hospitalized, while the rest were treated in a medical office setting.

When the scientists analyzed the genomic data, one particular set of mutations stood out among the hospitalized patients — just four letters in the genetic code that differed from that of the most common reference strains of the virus. It was identified by the four changed letters: caac.

Zhao believes the work, which was funded by grants from the National Institutes of Health, could be helpful in identifying targets for new drugs that could disrupt processes these genes are involved with that might cause the virus to make people sicker.

The research was carried out on “a mixed bag of COVID-19 viruses,” Zhao said, but did not involve more recent, highly transmissible variants-of-concern such as delta or omicron. The four-letter caac mutation that was linked to increased virulence showed up in nearly 17% of patients whose viral genomes were sequenced in Washington state.

In one cohort of patients used in the analysis, 22% of those hospitalized had viruses with the caac mutation, compared to only 7% of patients who did not need hospitalization.

“If you carry that mutation, the chances that you would get serious disease is high,” Zhao said.

Much of the research on vaccines, treatments and COVID-19 transmission focuses on the distinctive spike proteins that dot the surface of SARS-CoV-2, the virus that causes the disease. Because it uses the spike to land on human cells and pry its way into them, the spike has been the primary target of vaccines designed to block SARS-CoV-2 and eventually break the chain of transmission.

The spike is also the site of the majority of mutations in the highly transmissible omicron variant, and scientists believe that allows it to partially escape immunity.

Curiously, the Fred Hutch study did not find an association between severity of disease and any of the frequently found mutations in the spike protein. Instead, the suspected virulence factors are genes involved in controlling the internal machinery of the virus.

“The mutations we identified that correlate with development of more severe disease are in viral proteins important for the reproduction of the virus after it infects host cells,” said study co-author Dr. Terry Lybrand, a professor of chemistry at Vanderbilt University, in Nashville, Tennessee. “In the past, less attention has been paid to mutations in these proteins.”

The caac mutations appear across just two genes that each encode a different protein involved in how the virus operates once inside an infected cell. Like those of many viruses, COVID-19 genes are made from ribonucleic acid, or RNA, a cousin of DNA. Three of the mutations are single nucleotides — single-letter code changes — in a gene that carries the blueprints for making a nucleocapsid. That is a large protein involved in packaging and protecting SARS-CoV-2’s entire complement of genes.

The other mutation in caac is a single-letter change in a gene coding for endoRNAse, a compact enzyme that cuts RNA and is thought to be involved in viral replication.

The study involved patients who were treated for COVID-19 in hospitals serving the Seattle metropolitan area and from hospitals in Washington state’s rural Yakima county. The caac mutations first appeared in April of 2020, peaked in June, and then faded to low levels in subsequent months’ samples.

However, the number of specimens containing caac mutations rose in January 2021, just as the study period was wrapping up. That was months before the sudden emergence of the delta variant, which was more transmissible than most variants in 2020 and the ancestral strain that emerged in Wuhan, China, in late 2019. In late November 2021 came the explosive appearance of omicron, a variant more than twice as transmissible as delta — but deemed less likely to cause severe disease.

Zhao said that, fortunately, sequences of the omicron variant show it does not carry the full caac package of mutations previously linked to higher risk of hospitalization. Nevertheless, the researchers are continuing to sequence omicron samples, watching out for changes that might increase or decrease its ability to cause severe disease.

Fred Hutch geneticist Dr. Daniel Geraghty, who is senior author of the paper, stressed that if the connections between sequence mutations and patient outcomes could be actively determined in real time, public health resources related to the pandemic could be managed more effectively.

“This is critical going forward, if and when new variants arise,” he said.