key: cord-0971729-n5ujl6nm
authors: Kulkarni, Onkar; Narreddy, Suneetha; Zaveri, Lamuk; Kalal, Irawathy Goud; Tallapaka, Karthik Bharadwaj; Sowpati, Divya Tej
title: Evidence of SARS-CoV-2 reinfection without mutations in Spike protein
date: 2021-02-16
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab136
sha: 3944c805945910717a2db3a34a9ec69c4d228034
doc_id: 971729
cord_uid: n5ujl6nm

nan

M a n u s c r i p t Dear Editor -Several cases of SARS-CoV-2 reinfection have now been documented across the globe [1] [2] [3] .

Recently, Selhorst et al [4] reported a case of reinfection despite the presence of neutralizing antibodies. Their study showed the presence of S477N, an immune escape mutation [5] , in the Spike protein of the virus from the second episode. This conforms to the fact that most reported reinfections show the presence of at least one unique variation in structural proteins between episodes, particularly the Spike protein [6] . Here, we report two casesone clear case and one possible case, of SARS-CoV-2 reinfection that were detected during routine surveillance. Of note, there was no difference in the Spike protein of the virus between episodes.

To establish the genetic diversity of the virus, the samples were sequenced on Oxford Nanopore MinION following the ARTIC v3 protocol [7] , and further validated using Illumina sequencing. Genomes were assembled from raw data following a previously published method [8] , covering most of the SARS-CoV-2 genome (Table S1 ). The details of the cases and subsequent analysis are outlined below: Case 1:

A 61-year-old, apparently immunocompetent, male healthcare worker tested positive for SARS-CoV-2 using RT-PCR as part of contact tracing on 31st August 2020. After an episode of asymptomatic infection and home quarantine, he tested negative subsequently. With no travel history in between, he complained of weakness in 2nd week of November, and developed a cough 2 days later. He again tested positive for the virus on 14th November 2020. There were no other symptoms during this episode, and it was a mild disease overall. Sequencing revealed the presence of 10 unique variations between the viral genomes of both episodes (Fig 1, top, Table S2 ). No variation was observed in the Spike protein.

A 38-year-old male admitted to the hospital with symptoms of headache and fever tested positive for SARS-CoV-2 using RT-PCR on 4 th November 2020. After a day of symptoms indicated above, the patient was symptom-free. On 22nd November 2020, the patient had fever again. Sample collected on that day tested positive for SARS-CoV-2. Other than 5 days of fever following the test, there were no symptoms during the second episode. There was a history of steroid usage for a diagnosis unrelated to COVID19 (Tuberculous meningitis). Subsequent analysis revealed the presence of 3 unique variations between both the episodes, and a large number of shared variants (Fig 1, bottom, Table S3 ). One of the unique variations in episode 2 was in the Spike protein; however, it was a synonymous change.

In summary, we report two cases of SARS-CoV-2 reinfections from India, along with corresponding whole genome sequencing data, confirmed using two orthogonal sequencing M a n u s c r i p t technologies. Nextstrain analysis [9] revealed that all the 4 viral genomes belonged to the 20B clade, and carry the D614G mutation in Spike. While case 1 is a clear case of reinfection backed by a negative test between episodes and 10 unique variations between the viral strains, case 2 remains a bit unclear; 3 unique variations in 18 days is higher than expected based on current estimates, but we cannot rule out the possibility of prolonged viral shedding and accelerated viral evolution due to immunocompromised state. However, in both cases, no variation from S or E genes was identified between reinfections. Taken together, our work provides evidence for a rare but distinct possibility of reinfection without changes in the Spike protein, and highlights the need for further research to understand the genetic and molecular underpinnings of COVID19 reinfections. M a n u s c r i p t M a n u s c r i p t 

Genomic evidence for reinfection with SARS-CoV-2: a case study

Asymptomatic reinfection in two healthcare workers from India with genetically distinct SARS-CoV-2

Evidence of Severe Acute Respiratory Syndrome Coronavirus 2

Reinfection After Recovery from Mild Coronavirus Disease

Symptomatic SARS-CoV-2 reinfection of a health care worker in a Belgian nosocomial outbreak despite primary neutralizing antibody response

Landscape analysis of escape variants identifies SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization. bioRxiv

Genome Evolution Analysis Resource for COVID19

nCoV-2019 sequencing protocol v3 (LoCost) V.3. 2020

A Distinct Phylogenetic Cluster of Indian Severe Acute Respiratory Syndrome Coronavirus 2 Isolates. Open Forum Infect Dis

Nextstrain: real-time tracking of pathogen evolution

A c c e p t e d M a n u s c r i p t