Rapid repeat infection of SARS-CoV-2 by two highly distinct delta-lineage viruses

An instance of sequential infection of an individual with, firstly, the Delta variant and secondly a Delta-sub-lineage has been identified. The individual was found positive for the AY.26 lineage 22 days after being found positive for the Delta [B.1.617.2] variant. The viruses associated with the cases showed dramatic genomic difference, including 31 changes that resulted in deletions or amino acid substitutions. Seven of these differences were observed in the Spike protein. The patient in question was between 30 and 35 years old and had no underlying health conditions. Though singular, this case illustrates the possibility that infection with the Delta variant may not itself be fully protective against a population of SARS-CoV-2 variants that are becoming increasingly diverse.


Introduction
Cases of reinfection with SARS-CoV-2 are well-documented [1−3]. Efforts to determine the frequency of the phenomenon have been made and have shown that the occurrence may be rare to date [4,5]. The true incidence of reinfection is difficult to determine due to the possibility that secondary infections are harder to detect due to decreased or absent symptoms or less testing. The frequency of reinfection cases will increase over time, as more cases in the human population denotes more opportunities for the phenomenon, but also because genomic variation in the virus over the course of pandemic time can lead to variants that can evade immunities [6−9]. Variants occurring later in a pandemic's course would not be expected to be recognized with the same acuity by an immune system that has been stimulated [vaccinated] by an earlier version of a virus. The immune response to SARS-CoV-2 appears similar to that of other respiratory viruses. Within 1 to 3 weeks, antibodies including IgM, IgG and IgA class appear with specificity against spike protein, with one report showing 100% of patients seroconverting with IgG reactivity by day 15 [10,11]. Neutralizing antibodies are thought to persist but a clear decay rate of their functional titer is currently unknown and is likely to vary considerably between individuals [12−15]. Certain studies have shown that rare individuals do not develop IgG after infection [16,17].
Cases of immune evasion / vaccine breakthrough have been investigated for the "Delta" variant of SARS-CoV-2 [8,18,19]. The variant appears to possess lower sensitivity to antibody neutralization and higher transmissibility [8,18−22]. To date, the variant is highly successful and has generated multiple sub-lineages. Herein, we describe a case of infection with the Delta variant, that was followed up 22 days later by a subsequent case of infection with a sub-lineage of the Delta variant. Sequence analysis of both cases reveals substantial genetic difference between the associated viruses. Epidemiologic investigation of each case revealed contacts who were positive for SARS-CoV-2 and whose specimens were also genomically analyzed by the Nevada State Public Health Laboratory.

Materials and methods
The Nevada State Public Health Laboratory [NSPHL] performed sequencing and subsequent analysis of positive SARS-CoV-2 specimens. In each case, nasal specimens collected in viral transport medium were transported to the public health laboratory and were evaluated by real-time polymerase chain reaction [RT-PCR] to confirm reactivity and to determine cycle threshold (Ct) value. RT The workflow begins with a cDNA synthesis step [reverse transcription] followed by a target capture PCR to amplify specific regions of the SARS-CoV-2 cDNA. A modified version of the ARTIC v3 primer panel [98 tiled primers in 2 pools] is used to capture target regions of roughly 400bp in length through 2 separate PCR reactions for each sample. Following PCR, the resulting amplicons from the independent PCR reactions are pooled together and are subject to a SPRI bead cleanup, filtering out the PCR products below the expected amplicon size range and other unused PCR components. The purified amplicons of each sample are then barcoded and pooled together for library preparation. During library preparation, ONT-specific adapters are attached to each of the barcodes. An additional SPRI clean-up is performed to remove any unattached adapters from the sequencing library. The library is then mixed with other sequencing reagents, loaded onto the Flow Cell and sequenced for 8 hours. Read base calling is processed live on the GridION instrument [long-read sequencing technology]. Data is uploaded to the Clear Labs Server for bioinformatics analysis including read quality/length filtering, and consensus-based assembly. The resulting FASTQ and FASTA files are provided via the Clear Labs WGS Web App for download.
The Titan_ClearLabs workflow was utilized to generate consensus assemblies from raw Clear Labs read data and to perform SARS-CoV-2 lineage and clade designations for each sample. Briefly, human reads were removed from Clear Labs read data using the NCBI SRA-Human-Scrubber [https://github.com/ncbi/srahuman-scrubber]. De-hosted reads were then assembled as per the Artic nCoV-2019 novel coronavirus bioinformatics protocol [https://artic.network/ncov-2019/ncov2019-bioinformatics-sop. html] with a modification whereby the artic minion normalize flag  Ambiguity of the case's age is provided for deidentification purposes due to the small size of the county in which the individual resides. The patient provided consent, in writing, for the facts of this case to be published.
Identity testing was performed as previously described in Tillett et al. 2020 [23].

Results
An unvaccinated 30 to 35-year old male, resident of a rural county in Nevada, was symptomatic for COVID-19 and was tested by nasal swab on August 16, 2021, whereupon he was found reactive for SARS-CoV-2 RNA ["Case A"]. During Case A, the patient reported mild symptoms (described in Table 1) that resolved in less than a week. Three weeks later, the patient reported being exposed to additional COVID-19 cases and became symptomatic. The patient was tested again for the presence of SARS-CoV-2 by nasal swab on September 7, 2021 and was found to be reactive for SARS-CoV-2 RNA ["Case B"] [See Table 1]. The patient reported that the symptoms (Table 1) were more significant during Case B, in comparison to Case A (personal communication) No additional testing was performed on the patient between Case A and Case B.
As part of a comprehensive SARS-CoV-2 genomic surveillance program at the Nevada State Public Health Laboratory [NSPHL], samples from both instances were subjected to genomic sequencing. Successful sequencing and subsequent lineage analysis of assembled genomes revealed that the first instance of infection detected on 8/16 Fig. 1. A phylogenetic tree describing the genomic relationships of Case A and Case B relative to other cases, either from the same County as the patient, or elicited from contact tracing and investigation. C = co-community member; * = co-worker; X = direct contact to patient. was of the B.1.617.2 lineage. Sequence analysis of the specimen from the second symptomatic episode revealed virus of lineage AY.26. Both lineages were known to be circulating locally at the times of infection. As shown in Table 2 and Fig. 2A and B, the 2 genomes differed at multiple locations, resulting in 31 amino acid coding changes, including 7 in the spike protein. The genome associated with the first infection showed 4 mutations in the Spike protein relative to the reference strain (Wuhan-Hu-1). Human identity testing using short-tandem repeats was performed on both specimens and confirmed that the specimens were collected from the same individual (frequency of match: 1 in 31.62 octillion individuals); additional sequence analysis of all other reactive cases on the same plate and in the same extraction and amplification batches as the second specimen was performed and generated zero genomic matches for AY.26, ruling out contamination with a co-analyzed specimen. The genome associated with the second infection [Case B] showed 11 mutations in the spike protein relative to the reference strain. Phylogenetic analysis of the cases was performed comparing the 2 cases to those detected and sequenced from the patient's home county between July 20, 2021 and September 17, 2021 [ Fig. 1]. Interview of the patient was productive and indicated close contact with multiple known COVID-19 cases. The sole direct contact for Case A that was elicited by interview had a positive laboratory test for SARS-CoV-2 but the specimen was unavailable for sequence analysis. Interview regarding Case B elicited contacts that were all positive for SARS-CoV-2 and were assessed by genomic sequencing. Their phylogenetic relationship to the patient's cases is shown in Fig. 1. Also shown in Fig. 1 are the phylogenetic relationships to other SARS-CoV-2 positive co-workers for each case, and for 2 co-community members. These analyses reveal that plausible, genomically related sources of infection for Case A and Case B are identifiable amongst contacts and coworkers of the individual.
In lieu of intervening test results between Case A and Case B, we sought to ascertain whether infection could have been a sequence of events, or a simultaneous infection [e.g., a case of superinfection] with 2 variants. We investigated the sequence alignment file [BAM] generated by the TitanClearLabs workflow from each sequenced specimen from Case A and Case B to determine whether sequence from either case were detectable in the reads of the opposing specimen [ Fig. 2A and B]. As shown in Fig. 2C and D, mutations unique to Case A were observable at varying frequency in reads of the raw sequence data for specimen B, while the opposite was very rarely the case. The very low frequency of unique mutation associated with Case B observed in Case A reads [ Fig. 2D] is possibly the cause of the poor performance of ONT sequencing in its ability to discern low-frequency variants [24]. The observation that the frequency of Case Aspecific mutations detectable in Case B raw reads was much higher than those of the opposite condition is consistent with a sequential infection of A to B.

Discussion
Clear evidence is shown herein for an individual to have been infected with 2 distinct sub-lineages of the Delta variant within a 22day time frame. Cases A and B showed an extrapolated rate of single nucleotide variant and and multi-nucleotide variant accumulation of 501.6 substitutions per year, a rate that greatly exceeds the currently observed rate of 19.9 [25]. Reinfection with SARS-CoV-2 is welldescribed at this stage of the COVID-19 pandemic. However, the observation of reinfection within such a short timeframe, in a person devoid of known significant underlying conditions, and involving lineages of the Delta variant, is newly described. Such a finding in a relatively young person with no known underlying health conditions may indicate further challenges for controlling the pandemic. As genetic variability in the virus increases and is maintained in the population, infections with one version of the virus may not afford protection against others. It is likely that this scenario is occurring with the arrival of the Omicron variant which exhibits great antigenic (genomic) diversity relative to previous lineages [26]. A novel vaccination strategy, geared toward time-relevant variants and their sublineages may become necessary.