Coronaviruses have encoded proofreading mechanisms, unlike other pathogenic viruses, and as a result SARS-CoV-2 has very low sequence diversity. However, natural selection can result in favorable mutations that provide an advantage to viral infectivity or transmission. Antigenic single amino acid changes are observed in seasonal influenza viruses and in previous coronaviruses. For example, a single amino acid change in spike D480A/G in the receptor-binding domain of SARS-CoV became the dominant variant in the later stages of the 2003 to 2004 pandemic.

Research partners from Los Alamos National Laboratory, Duke University, and the University of Sheffield developed a strategy for detecting phenotypic mutations in the spike protein of SARS-CoV-2 as an early warning tool by identifying variants that become increasingly prevalent in different geographic locations. They suggested that the increasing relative frequency of a particular variant observed in a distinct geographic region can become a candidate for conferring a selective advantage. Leveraging the Global Initiative for Sharing All Influenza Data (GISAID) and Nextstrain data, the bioinformatic pipeline tracts point mutations of SARS-CoV-2.

The D614G mutation

"The D614G variant first came to our attention in early April, as we had observed a strikingly repetitive pattern," said lead author Bette Korber, PhD, theoretical biologist at Los Alamos National Laboratory, in a statement. "All over the world, even when local epidemics had many cases of the original form circulating, soon after the D614G variant was introduced into a region it became the prevalent form."

The researchers found that over the course of one month, the D614G spike mutation became the globally dominant form of SARS-CoV-2. The mutation that causes the D614G amino change is transmitted as part of the conserved haplotype as part of a group of mutations that always track together.

They observed increasing frequency of the G614 variant, suggesting that it may be under positive selection. The team's early detection framework indicated that G614 consistently increased across every geographic level: country, subcountry, county, and city. Moreover, the G614 variant continued to increase in frequency, even after national stay-at-home orders were in place and traveling was significantly reduced.

The mutation was associated with higher levels of viral nucleic acid in the upper respiratory tract in human patients and showed higher infectivity in multiple pseudotyping assays. The authors suggested that the G614 variant may increase spike stability and membrane incorporation of the virus.

The G614 variant has spread faster than the D614 variant of SARS-CoV-2. The researchers suggested that this means the G614 variant is likely more infectious. However, they found no evidence of increase in the severity of illness, i.e. not significantly associated with hospitalization status.

"It is possible to track SARS-CoV-2 evolution globally because researchers worldwide are rapidly making their viral sequence data available through the GISAID viral sequence database," explained Korber.

Implications of vaccines and therapies

Determining immunologically relevant mutations is important to ensure the effectiveness of vaccines and immunotherapeutic intervention development. Most vaccines and antibody-based therapies target the trimeric spike protein, based on sequences and proteins from the Wuhan reference sequence. It is important to note that alterations from the reference sequence as the virus mutates as it spreads could potentially impact the efficacy of immune-based interventions.

It will become important to determine whether the D614 and G614 forms of SARS-CoV-2 are sensitive to neutralization by specific vaccine-elicited antibodies or antibodies produced in response to either form of the virus. The authors also noted that if the G614 variant is more infectious, than it may require higher antibody levels to confer protection by vaccines or other antibody therapies than the D614 form.

They urged that contemporary variation in the virus should be factored into vaccine and antibody-based therapy development to ensure that the interventions will be effective against circulating variants when they are eventually deployed on a global level.

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