COVID-19: Emergence of the D614G Mutation and the Question of Reinfection (15 July 2020)
Two commonly asked questions about SARS-CoV-2, the virus that causes the pandemic coronavirus disease (COVID-19), pertain to 1) the emergence of mutated strains of the virus and their implications, if any, for virulence, and 2) whether or not an individual can be reinfected. So, in this post I briefly discuss both the emergence of one increasingly prevalent mutation and its phenotypic effect, as well as what is known to date about immunity and reinfection with SARS-CoV-2.
By way of background, the genome of all living things is encoded along strands of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Some organisms have a genome consisting of single strands, and some have double strands; some strands are linear, and some are circular; and some organisms (e.g. bacteria) may have multiple circular strands of both chromosomal and extrachromosomal (i.e. plasmid) DNA. Nonetheless, in all cases, the strands consist of unique sequences of nucleotide bases that encode amino acids, the building blocks of proteins. A mutation occurs when bases are substituted, inserted, or deleted. Because the proofreading mechanisms are imperfect, mutations may occur during replication (i.e. when copies of the strands are synthesized for new cells). Additionally, mutations may be caused by extrinsic factors such as ultraviolet light, radiation, mutagenic chemicals, or the insertion of DNA segments (e.g. transposons) from another organism such as a virus or bacterium. The consequence of a mutation may be no discernible effect, owing to redundancy in the genetic code that enables several different three-base sequences to encode for the same amino acid (This is referred to as a “same sense mutation”). Alternatively, the mutation may encode a different amino acid (a “missense mutation”); or the mutation may prematurely terminate a reading sequence, resulting in a truncated nonfunctional protein (a “nonsense mutation”).
SARS-CoV-2 consists of a single strand of RNA encased within a 65–125 nm diameter enveloped capsid shell. Additionally, each virion contains four main structural proteins: the envelope (E) glycoprotein, the membrane (M) glycoprotein, the nucleocapsid (N) glycoprotein, and the spike (S) glycoprotein.(1) The latter is a transmembrane protein with a molecular weight of 150 kDa that facilitates the binding of the virus to angiotensin-converting enzyme 2 (ACE2) receptors expressed on the surface of cells lining the respiratory tract as well as certain cells of the gut, muscle (including the heart), kidneys, and bladder. Binding of the spike protein to the ACE2 receptor is followed by fusion of the viral membrane and host cell and subsequently, by viral entry into the host cell, replication of the viral genome, assembly of new virions, and release.
Because RNA viruses such as SARS-CoV-2 lack a proofreading mechanism, they are especially prone to misincorporation of nucleotides during genome replication. (A detailed discussion of the myriad factors contributing to the high mutation rates of RNA viruses is beyond the scope of this post, but I will refer the interested reader to the review by Sanjuán and Domingo-Calap)(2) One mutation which is increasingly prevalent in SARS-CoV-2 isolates is an adenine nucleotide to a guanine nucleotide substitution at residue 614 (position 23,403 of the Wuhan strain), which effects a change from an aspartate to glycine in the virus’s spike protein. In one study, this mutation (called “D614G”) was found in 10% of 997 global sequences before March 1st; 2020, 67% of 14,951 sequences between March 1st and March 31st; and represented 78% of 12,194 sequences between April 1st and May 18th.(3) In another similar study of isolate sequences uploaded into GenBank, D614G was not observed in February 2020, but represented 26% of sequences in March, 65% in April; and represented 70% of sequences in May.(4) The alanine to glycine substitution appears to have occurred asynchronously, appearing first in Europe, followed by North America and Oceania, and then Asia.
The increasing global prevalence of the mutated strain (now called the “D” clade) suggests that the D614G mutation confers a selective advantage over the wild type virus (i.e. the virus without the mutation). To investigate this, different groups of researchers created pseudoviruses (proxy viral constructs that express proteins of other viruses) carrying either the wild type (alanine) or mutated (glycine) version of residue 614 and then assessed infectivity. In one group, researchers using Moloney murine leukemia virus (MLV)-based pseudoviruses found that the D614G variant was up to nine-fold more efficient than the wild type version in infecting cells that express the ACE2 receptor.(5) Another group of researchers, using vesicular stomatitis virus (VSV) and lentiviral pseudoviruses, also found that the D614G variant had significantly higher infectious titers than did the wild type virus.(6) A third group of researchers using intact SARS-CoV-2 rather than pseudoviruses to infect human lung epithelial cells, also found the D614G mutation to be up to eight-fold more effective than wild-type virus at transducing cells.(7) It is important to note, however, that while the researchers in all groups showed that the D614G variant was more transmissible and reached higher viral loads than did the wild type virus, they also concluded that the variant did not appear to be associated with more severe disease. Thus, at this time, the emergence of the D614G variant is an interesting observation of unknown clinical significance.
Another pressing question for which we do not yet have a definitive answer pertains to immunity — specifically, whether an individual who was previously infected with SARS-CoV-2 is immune to reinfection, and if so, for how long? The answer to this question has wide ranging implications for herd immunity, duration of the pandemic, reemergence of the virus, and vaccine development. To date, there are no convincing documented cases of reinfection with SARS-CoV-2. In April 2020, the Korea Centers for Disease Control and Prevention (KCDC) reported on 74 patients who re-tested positive after recovering from COVID-19.8 However, the KCDC eventually concluded that these individuals had indeed recovered and were shedding inactive virus, a finding that is now widely observed (Anecdotally, I have followed patients who are clinically well but who continued to shed virus for months after recovery).
In one recent study of four common human coronaviruses (HKU1, 229E, OC43, and NL63), researchers at Columbia University followed 191 study participants between the fall of 2016 and the spring of 2018 for weekly nasal swabs and self-reported respiratory complaints. During the study period, 86 individuals tested positive for one of the coronaviruses, and 12 individuals tested positive twice, causing the researchers to conclude that “re-infections with the same endemic coronavirus are not atypical in a time window shorter than one year”.(9) Unfortunately, there is a paucity of information about whether immunity to SARS-CoV-2 will be similar to the common endemic coronaviruses such as those in this study or whether it will be longer lasting, as with the SARS-CoV-1 and MERS-CoV coronaviruses. Recall that in a prior post, I described discouraging results from two recent Chinese studies that showed that antibody titers in COVID-19 patients quickly waned, casting doubt about the longevity of immunity to SARS-CoV-2 and potentially confounding development of a vaccine.(10) In a subsequent longitudinal study of more than 90 COVID-19 patients, researchers at King’s College, London similarly found that antibody levels peaked at about three weeks after the onset of symptoms but quickly declined thereafter, with 60% of study participants mounting a robust antibody response and only 17% of them retaining a similarly “potent” antibody titer after three months.(11) However, these studies are caveated by the fact that they only looked at “neutralizing” antibodies (i.e. antibodies that directly bind to and inhibit viruses) and they did not look at antibodies that mark infected cells as targets for immune cells (a process called “antibody-dependent cellular cytotoxicity”). Moreover, we do not yet know what level of neutralizing antibodies is needed to confer protection against reinfection with SARS-CoV-2; nor do we yet know how robustly antibody-producing cells will respond to re-exposure (i.e. the “memory” or “anamnestic” response). Additionally, it is worth noting that for some other coronaviruses, while “sterilizing immunity” (i.e. protection from reinfection) may only last for several months, “protective immunity” (i.e. immunity that attenuates the severity of illness) is longer lasting. Almost certainly, SARS-CoV-2-infected individuals have some degree of short term immunity to reinfection. This is suggested by one recent study in which Rhesus macaques that were deliberately reinfected with the identical SARS-CoV-2 strain during the early recovery phase of the initial SARS-CoV-2 infection did not show detectable viral dissemination, clinical manifestations of viral disease, or histopathological changes.(12) Moreover, when the researchers compared the humoral and cellular immunity between primary infection and rechallenge, they saw enhanced neutralizing antibody and immune responses. Although these results are encouraging, it must be pointed out that the animals were re-challenged during early convalescence from the initial infection. Presumably, the researchers plan on rechallenging the animals and remeasuring their antibody titers at a later time.
Although it may seem as if the COVID-19 pandemic is interminable, it is less than seven months since the first case was reported in Wuhan, China, and the reality is that Nature does not readily give up her secrets. Hypotheses must be formulated and tested; results must be interpreted and reconciled; and conclusions must withstand the tincture of time. We know far more about this virus than we did seven months ago and presumably, we will know still more seven months hence. Nous sommes au bord des mystères et le voile s’amincit de plus en plus.(13) For now, I will endeavor to post updates that neither politicize, sensationalize, nor trivialize the pandemic, but simply provide information and perspective.
Until my next update — regards.
Michael Zapor, MD, PhD, CTropMed, FACP, FIDSA
(15 July 2020)
To read this and my other COVID-19 posts on Medium.com, please see: https://medium.com/@michaelzapor
References
1. Diabetes Metab Syndr. 2020 July-August; 14(4): 407–412
2. Cell Mol Life Sci. 2016; 73[23]: 4433–4448
3. Cell (2020), doi: https://doi.org/10.1016/j.cell.2020.06.043 (Accessed 14 July 2020)
4. https://www.scripps.edu/news-and-events/press-room/2020/20200611-choe-farzan-sars-cov-2-spike-protein.html (Accessed 14 July 2020)
5. Ibid.
6. https://www.cell.com/action/showPdf?pii=S0092-8674%2820%2930820-5 (Accessed 14 July 2020)
7. https://doi.org/10.1016/j.cell.2020.06.043 (Accessed 14 July 2020)
8. https://www.nationalreview.com/corner/how-worried-should-we-be-about-reinfection-or-reactivation-of-the-virus/ (Accessed 14 July 2020)
9. https://www.medrxiv.org/content/10.1101/2020.04.27.20082032v1 (Accessed 14 July 2020)
10. https://medium.com/@michaelzapor/covid-19-acute-and-convalescent-antibody-titers-and-their-implications-for-herd-immunity-and-dd3f891368a0 (Published 30 June 2020)
11. https://www.medrxiv.org/content/10.1101/2020.07.09.20148429v1 (Accessed 14 July 2020)
12. https://science.sciencemag.org/content/early/2020/07/01/science.abc5343 (Accessed 14 July 2020)
13. “We are on the verge of mysteries and the veil is getting thinner and thinner”; Paraphrased from the quote by Louis Pasteur: “Je suis au bord des mystères et le voile s’amincit de plus en plus.” (“I am on the verge of mysteries and the veil is getting thinner and thinner.”) From a December 1851 letter as quoted in The Great Influenza: The Epic Story of the Deadliest Plague in History by John M. Barry (Viking Press, 2004)