Thursday, January 07, 2021

PrePrint: Genomic Evidence of a Sars-Cov-2 Reinfection Case With E484K Spike Mutation in Brazil



Barely a month ago, with long-awaited vaccines just beginning to roll out, the general consensus was how lucky we'd been that the SARS-CoV-2 virus had remained relatively stable since it acquired the D614G mutation last February.  There were mutations and variants, of course, but none were thought to change the virus's virulence of susceptibility to the vaccine. 

Vaccines were expected to be extremely effective, and for the first time there was light visible at the end of the COVID pandemic tunnel. 

Then, in mid-December the UK Health Secretary Announced A New COVID Variant had been detected in Southern England, and that it was spreading rapidly, suggesting it was more transmissible than the previous strain.  Subsequents studies have suggested this B.1.1.7 variant to be 70% more transmissible, and it has now been detected in over 40 countries. 

Its saving grace has been that it is still believed to be susceptible to the current crop of vaccines, although further testing is needed. 

Scarcely a week later, a second variant - dubbed 501Y.V2 - that had been spreading rapidly in South Africa, began to make headlines (see PrePrint: Emergence & Rapid Spread of a New SARS-CoV-2 Lineage with Multiple Spike Mutations in South Africa).

While not as widespread internationally (yet), this variant - which carries an E484K mutation which has been tentatively linked to reduced antibody recognition - is feared by some that it  could prove less susceptible to the current vaccine.

Admittedly, evidence either for or against those concerns is still slim.  Three weeks ago, this variant was barely on our radar, and proper research and analysis takes time.  

Earlier this week, yet another variant with this E484K mutation was reported in South America (see Argentina: PAIS Report On COVID E484K Variant In South America), and today we have a preprint study (not yet peer reviewed) of a reinfection in Brazil of a woman with an E484K variant virus. 

Unlike most (but not all) of the other reinfections we've seen reported, this patient experienced a more severe illness with the second infection. 

A link, and some excerpts, from the study:

Preprint Case Report Version 1 This version is not peer-reviewed
Genomic Evidence of a Sars-Cov-2 Reinfection Case With E484K Spike Mutation in Brazil
Carolina Kymie Vasques Nonaka , Marília Miranda Franco , Tiago Gräf , na Verena Almeida Mendes , Renato Santana de Aguiar , Marta Giovanetti * , Bruno Solano de Freitas Souza

Version 1 : Received: 5 January 2021 / Approved: 6 January 2021 / Online: 6 January 2021 (20:38:36 CET)

How to cite: Vasques Nonaka, C.K.; Miranda Franco, M.; Gräf, T.; Almeida Mendes, A.V.; Santana de Aguiar, R.; Giovanetti, M.; Solano de Freitas Souza, B. Genomic Evidence of a Sars-Cov-2 Reinfection Case With E484K Spike Mutation in Brazil. Preprints 2021, 2021010132 (doi: 10.20944/preprints202101.0132.v1).  

To date, uncertainty remains about how long the protective immune responses against SARS-CoV-2 persists and the first reports of suspected reinfection began to be described in recovered patients months after the first episode. 1 Viral evolution may favor reinfections, and the recently described spike mutations, particularly in the receptor binding domain (RBD) in SARS-CoV-2 lineages circulating in the UK, South Africa, and most recently in Brazil, have raised concern on their potential impact in infectivity and immune escape. 2,3,4 

We report the first case of reinfection from genetically distinct SARS-CoV-2 lineage presenting the E484K spike mutation in Brazil, a variant associated with escape from neutralizing antibodies.

A 45-year-old female healthcare executive, resident in Salvador, Bahia state, Northeast Brazil, with no comorbidities, presenting symptoms of viral infection on two occasions (May 26, 2020 and October 26, 2020). In the first episode, the patient presented diarrhea, myalgia, asthenia and odynophagia for approximately 7 days. She used 40 mg prednisone for 5 days and returned to activities 21 days later without sequelae or complaints.
In the second episode, symptomatically more severe, the patient presented headache, malaise, diarrhea, cough and sore throat that evolved to myalgia and respiratory distress, ageusia, muscle fatigue, insomnia, mild dyspnea on exertion and shortness of breath.
In both occasions, results of RT-PCR tests targeting 3 genes (N, E and RdR) were positive for SARS-CoV-2 in nasopharyngeal samples (Figure 1A). Cycle threshold values (Cts) of N, E and RdRp targets were 25, 26, and 27 in the first episode and 21, 12 and 17 in the second episode. Four weeks after testing positive by RT-PCR in the second episode, an IgG test was performed and showed a positive result (index value: 2.15 on 11/23/2020).
Sequencing was conducted on the two nasopharyngeal swabs by PGM Ion Torrent (Life Technologies, USA) according to the manufacturer’s instructions. Sequence alignment and phylogenetic analysis were performed as previously described. 3 Sequencing statistics showed 1.405.009 mapped reads for sample A and 2.570.182 reads for sample B, resulting in a sequencing mean depth >1,000X for both samples and a coverage >99%. Using the pangolin COVID-19 lineage Assigner (, sample A was identified as B.1.1.33 lineage and sample B as B.1.1.248, a lineage derived from B.1.1.28, recently identified in Brazil. 4
Phylogenetic analysis, of the two newly whole genome sequences compared with contemporaneous sequences from Brazil (supplementary table 1), clearly demonstrated that the two COVID-19 episodes, separated by a 147-day interval, were indeed caused by different SARS-CoV-2 lineages, confirming reinfection (Figure 1B).
Moreover, we identified several mutations distinguishing the two genomes (Figure 1C), three of them in the SARS-CoV-2 spike glycoprotein. In the first infection, the retrieved genome presented the S:G1219C, while in the second infection, S:E484K and S:V1176F were observed. In both samples, we found ORF1ab:P4715L, S:D614G, N:R203K, and N:G204R mutations.
Mutation analysis demonstrated, for the first time, a reinfection case with a viral variant harboring the mutation E484K, located in a key residue of the receptor binding domain, that seems to modestly enhance binding between the Spike protein and the host receptor hACE2.
The E484K mutation is part of a group of variants identified in South Africa that have been associated with increased infectivity and was also observed as occurring in the recently described B.1.1.248 lineage in Brazil. 3,4 This lineage was initially described in samples from Rio de Janeiro, but was already detected in UK, USA, Portugal and Australia.
Here we show that B.1.1.248 may be widely spread among Brazilian states as well. The RBD is also a major target of neutralizing antibodies elicited during the primary exposure to SARS-CoV-2 and the E484K mutation has been predicted to affect antibody neutralization. 5 The finding of the E484K, in an episode of SARS-CoV-2 reinfection might have major implications for public health policies, surveillance and immunization strategies.

There are limits, of course, to how much weight we should assign to a single case report. 

But this study provides us with yet another data point that suggests the E484K mutation might be more problematic when it comes to reinfections, monoclonal antibody and plasma serum treatments, and potentially (and worst case) the effectiveness of current vaccines. 

It may be weeks, or even months, before we have a solid handle on the true impact of this (and other) E484K variant(s). Not all variants that carry this mutation are likely to transmit well enough to compete with more aggressive variants (like B.1.1.7), making it too soon to say how concerned we should be. 

But this is a reminder that viruses haven't survived and thrived for millions of years on this planet without developing some pretty formidable survival skills.