key: cord-0738075-ag89fgtl
authors: Shobayo, Bode; Mishra, Mitali; Sameroff, Stephen; Petrosov, Alexandra; Ng, James; Gokden, Alper; MaCauley, Jane; Jain, Komal; Renken, Courtney; Duworko, James Tanu; Badio, Moses; Jallah, Wilhemina; Hensley, Lisa; Briese, Thomas; Lipkin, W. Ian; Mishra, Nischay
title: SARS-CoV-2 Sequence Analysis during COVID-19 Case Surge, Liberia, 2021
date: 2021-12-03
journal: Emerg Infect Dis
DOI: 10.3201/eid2712.211818
sha: 67f2061d2bef53c913dd69385651cf53cfac5fa6
doc_id: 738075
cord_uid: ag89fgtl

In June 2021, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases surged in Liberia. SARS-CoV-2 sequences from patients hospitalized during March–July 2021 revealed the Delta variant was in Liberia in early March and was dominant in June, irrespective of geography. Mutations and deletions suggest multiple SARS-CoV-2 Delta variant introductions.

B efore May 2021, Liberia reported <10 coronavirus disease (COVID-19) cases per day among its population of ≈5 million (1) . Thereafter, case numbers, hospitalizations, and deaths rapidly increased and peaked to >200 cases and 10-15 deaths per day in mid-July 2021 (Appendix Figure 1 , https://wwwnc. cdc.gov/EID/article/27/12/21-1818-App1.pdf). To determine whether the rapid case surge was associated with the introduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern or newly emerging variants, we collected nasopharyngeal swab samples from 267 hospitalized patients countrywide during March-July 2021 for high-throughput sequencing.

We collected samples in viral transport media from Bomi, Bong, Grand Cape Mount, Lofa, Margibi, Maryland, Montserrado, and Nimba Counties (Appendix Figure 2 ). We noted sample collection date and site and sex and median age of patients from whom samples were obtained (Table; Appendix   Table) . We used Buffer AVL (QIAGEN, https:// www.qiagen.com) lysis buffer to extract total nucleic acid and performed PCR by using the Triplex-CII-SARS-Cov-2 rRT PCR assay (2) . We conducted further high-throughput sequencing on 89/267 (33.3%) samples that had cycle threshold values <33 (Appendix Table) .

To prepare libraries, we used the Kapa Hyperplus Kit (Roche, https://www.roche.com) on first strand cDNA synthesized from 89 RNA samples (3), then we enriched for SARS-CoV-2 by using myBaits Custom RNA-Seq Kit (Daicel Arbor Biosciences, https://arborbiosci.com). We sequenced captured libraries on Nextseq 2000 or Nextseq 550 (Illumina, https://www.illumina.com), which yielded 5-8 million 220-bp reads per sample. We mapped reads to a SARS-CoV-2 reference sequence (GenBank accession no. NC_045512) to determine variants (Table; Appendix Table) .

Of the 89 RNA samples, 77 (86.5%) yielded complete coding sequences with a minimum depth of ≈15× (GISAID accession nos. EPI_ISL_3547663-705, EPI_ISL_3560291, and EPI_ISL_4232122-52). Using high-throughput sequencing data, we generated consensus fasta sequences of 77 SARS-CoV-2 genomic sequences and further analyzed sequences by using Geneious R10 (https://www.geneious.com), Next-Strain (4), and GISAID (5).

Among 77 genomes recovered, 4 (5.2%) were Alpha variant (B.1.1.7); 6 (7.8%) were Beta variant (B.1.351); 1 (1.3%) was Iota variant (B.1.526); 6 (7.8%) were Eta variant (B.1.525); and 56 (72.7%) were Delta variant (B.1.617.2) viruses (Table) . We identified Delta variant viruses in samples collected in early March and in April and May 2021, from Bong County. Delta variant viruses were co-circulating with Alpha, Beta, Eta, Iota, and other 20B variant viruses in Liberia. All 44 sequences recovered during June-July 2021 were from Delta variant viruses (Table) . We used complete polyprotein coding sequences from Liberia, other representative SARS-CoV-2 sequences, and variant reference sequences to create a maximum-likelihood, nucleotide-based phylogenetic tree in MEGA X (6) (Figure) .

Using reference sequence NC_045512 as a baseline, we found 3 Alpha variant-specific amino acid Recent surges in COVID-19 in many countries have been associated with the emergence of highly transmissible Delta variant viruses (7, 8) . In March 2021, the National Public Health Institute of Liberia sequenced 10 random samples from hospitalized COVID-19 patients in Monteserrado; all sequences were Alpha variant viruses (B. Shobayo, unpub. data).

A limitation of our study is the small sample sets used for analysis; nonetheless, our findings suggest that Alpha and other circulating variant viruses were replaced by Delta variant viruses countrywide in Liberia in <3 months. Mutation and phylogenetic analyses further indicate that several Delta variant strains were circulating after March 2021 and suggest multiple separate introductions.

Before June 2021, only a small percentage of the population was vaccinated in Liberia. The infections we report occurred in unvaccinated persons. The Ministry of Health, Liberia, initiated a vaccination drive in August 2021. By September, ≈130,000 persons, >2% of the population, had received a single dose of the Johnson & Johnson/Janssen vaccine (https://www.jnj.com). The COVID-19 vaccination campaign is ramping up as <30 cases/day are reported in Liberia, but the currently circulating Delta variants are a concern because they contain mutations and deletions in the surface glycoprotein that might influence vaccine efficacy (9) . Liberia should continued surveillance for SARS-CoV-2 variants of concern to determine whether additional vaccination or public health measures are needed to curb severe disease and future case surges in the country. We used the incidence of spike gene target failures identified during PCR testing to provide an early projection of the prevalence of severe acute respiratory syndrome coronavirus 2 variant B.1.1.7 in a university setting in Texas, USA, before sequencing results were available. Findings from a more recent evaluation validated those early projections.

COVID-19 coronavirs disease data

Accelerated emergency use authorization (EUA) summary the Triplex CII-SARS-CoV-2 rRT-PCR test updated 9/14/2020

Antibodies to enteroviruses in cerebrospinal fluid of patients with acute flaccid myelitis

Tracking of variants

Real-time tracking of pathogen evolution

Molecular Evolutionary Genetics Analysis across computing platforms

Effectiveness of Covid-19 vaccines against the B.1.617.2 (Delta) variant

Rapid spread of the SARS-CoV-2 Delta variant in some French regions

SARS-CoV-2 vaccines

Address for correspondence: Nischay Mishra, Center for Infection and Immunity, Columbia University, 722 W 168th St

University of Texas at Austin Office of the Vice President for Research

I dentification of the highly transmissible novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant B.1.1.7 (Alpha variant) in the United Kingdom raised concerns for renewed pandemic surges worldwide (1,2). B.1.1.7 likely arrived in the United States by

To provide local situational awareness at that pivotal moment in the coronavirus disease (COVID-19) pandemic

Mutations in the B.1.1.7 spike protein result in a failure to detect the spike gene probe in standard SARS-CoV-2 quantitative reverse transcription PCR (qRT-PCR). In estimating the prevalence of B.1.1.7 from local quantitative PCR data, we initially assumed US estimates for the proportion of spike gene target failures (SGTF) attributable to B.1.1.7 (4) and, in our retrospective analysis, update that proportion on the basis of local sequencing data. We used a Bayesian model to estimate the local growth rate of B.1.1.7 among all SARS-CoV-2 infections and applied a compartmental susceptible-exposed-infected-recovered model of SARS-CoV-2

The National Institute of Allergy and Infectious Diseases, National Institutes of Health, also participated in study design, analysis and interpretation of data, writing of the report, and decision to submit the article for publication.