Isolation and characterization of SARS-CoV-2 in Kenya

The emergence of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) from Wuhan, China, in December 2019 raised a global health concern that eventually became a pandemic affecting almost all countries worldwide. The respiratory disease has infected over 530 million people worldwide, with over 950,000 deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region. Author Summary The Coronavirus disease 2019 (COVID-19) pandemic is caused by a type of coronavirus that emerged in Wuhan, China in December 2019 and later spread to almost all countries. Many countries are still finding ways to contain it. The virus has been studied in many ways to investigate its origin, infectivity, and evolution. Different variants of the virus have emerged and spread, causing a lot of concern as to whether the pandemic will end soon. Significant studies have proven the ability of the virus to grow in the laboratory using cell lines that offer the necessary conditions. Therefore, this study sought to find out the growth of the virus in specific monkey cell line and the variant circulating within the Kenyan population. We found that the selected cell lines supported viral growth outside a human host system. In addition, the circulating virus was found to have evolved to enhance its survival mechanism. This is the first study in Kenya to report this virus’s isolation, culture, and identification in monkey kidney cells. These cells supported the growth of the virus in the laboratory and analysing the genome of the growth products showed the virus was related to previously reported strains with multiple changes in its whole DNA sequence.


Introduction
The coronaviruses (coronaviridae) were first recognized as a new family of viruses in 1968. They are enveloped, positive-sense-stranded ribonucleic acid (RNA) viruses with a genome of 26-30kb, with the largest genome among all the known RNA viruses (1). The name of these viruses is derived from their morphology with spike proteins on their surface that appear like a crown shape (2). Phylogenetically, coronaviruses are classified into four genus: Alphacoronaviruses, However, recently, a new coronavirus strain emerged in Wuhan, China, that has caused a worldwide pandemic that was later named SARS-CoV-2 by the International Committee on Taxonomy of Viruses (5). Sequence and phylogenetic analysis of various published strains show close similarity (99% homology) to the wild type SARS-CoV strain (6). Research is still underway to understand the aetiology and pathophysiology of the virus.
As of 15 th July 2022, COVID-19 had infected over 560 million individuals with over 6 million deaths globally, of which 9.1 million cases and over 173,000 mortalities occurred in Africa (7).
Almost all the regions across the world have recorded COVID-19 cases. However, some countries such as Turkmenistan have not yet registered any cases (8). The previous outbreaks of coronaviruses, including SARS and MERS, affected over 26 and 27 nations, (9) with morbidity of 8,000 and 2,519 for SARS and MERS, respectively (10). Compared to other pandemics, COVID-19 has caused higher morbidities and an overall lower case fatality ratio (CFR) as of 14 th July 2022 (11). Comorbidities such as diabetes, cancer, and hypertension have been linked to high mortalities due to COVID-19. On the other hand, Africa is known to be high in morbidities of infectious diseases such as HIV/AIDS and Malaria (12,13,14). Recent studies have been able to link such morbidities with COVID-19 co-infection and severe disease (15,16).
SARS-CoV-2 has been conducted in countries such as South Korea, where in February 2020, nasopharyngeal (NP) and oropharyngeal (OP) samples were isolated from confirmed cases of COVID-19 patients where isolation and replication were confirmed through viral culture and gene 6 sequencing (17). In June 2020, in the same country, serum, urine, and stool specimens were used to isolate the SARS-CoV-2, where the presence of the virus was evaluated using real-time RT-PCR (18). As of 15 th July 2022, the Kenya Ministry of Health had recorded a cumulative COVID-19 caseload of 336,445 and 5,668 related deaths (19). This paper describes the isolation, characterization, and viral dynamic of SARS-CoV2 Kenyan strain.

Isolation of SARS-CoV-2 in culture
Three of the four clinical samples indicated cytopathic effect (CPE) in Vero CCL-81 cells (001, 003, and 004) three days post infection (Figure 1 and 2) following the initial infection and infectious culture fluids (ICFs) were frozen for further analysis. Sample 002 did not show CPE and ICF was kept for blind passaging. Sample 004 ICF was selected for sequencing.

Quantification of SARS-CoV-2 by plaque assay
The isolated SARS-CoV-2 formed distinctly visible plaques, and the viral titer was determined as 1·65 X 10 5 pfu/ml.
Gel results were as shown below (Figures 3 and 4).
Out of the 14 samples used in this study, 13 appeared positive for SARS-CoV-2 RdRp and S genes (92.9%).

Whole-genome sequencing and phylogenetic analysis
We successfully assembled the whole genomes and confirmed the identity of SARS-CoV-2 virus isolates from our cultured isolates. The length of the assembled genomes from isolates 001 and 002 was 29,829 bp and 29,903bp, respectively. This represents 100% coverage of the reference genome. Comparisons of the sequences from this study to previous isolates from Kenya and the rest of the world in databases show apparent similarity, particularly to samples from Africa. We

Nucleotide substitutions and amino acid changes
To confirm substitutions at the protein level and follow up on the Nextclade classification of the isolates to the clade 20C, we aligned the sequences and profile mutations as shown in Table 1.
While variations across the entire genome, pronounced differences in nucleotide sequences are evident in the S protein coding sequences (CDS) (Supplementary Figure1). However, most of these are silent mutations, as we could identify only seven mutations in each sequence (Table 1 and Supplementary figure 2).

Conclusion
This study showed that Vero CCL-81 cells could support SARS-CoV-2 virus isolation and characterization through the formation of viral plaques. Detection of SARS-CoV-2 targeting the S and RdRp genes was effective. However, a limitation of this study was that the isolates cultured were from samples collected in July, 2020, when the pandemic emerged, with very few strains reported. As we are aware, the virus has dynamically evolved; hence future studies should focus on recent samples to sequence viral genomes from different isolates collected in different regions to increase the probability of identifying multiple strains circulating in Kenya and even possible novel mutations, which will also offer a road map in the development of diagnostics and vaccines to control the spread of the disease. Another significant area of study could be on co-infections of SARS-CoV-2 with other endemic diseases such as HIV/AIDS and malaria that have continued to affect the Kenyan population for a long time so as to depict disease severity due to co-infection within a human host.

Specimen collection
NP and OP swabs were collected as described (37)

Isolation of SARS-CoV-2 from clinical samples
Isolation and propagation of high titers of infectious SARS-CoV-2 were done in a biosafety level 3 laboratory by qualified staff using a modified method by Tastan and colleagues (39). Following plates and flasks were maintained in a humidified 5% CO 2 atmosphere in an incubator at 37 o C.
Cells were monitored daily for CPE and at 85% of CPE; the ICF from the 75cm 2 flask was harvested by centrifuging for 10 minutes at 4 o C. The supernatant was aliquoted and kept at -80 o C as seed virus stock.

SARS-CoV-2 viral titer determination
Plaque assays were done based on SARS-CoV-2 and MERS-CoV protocols (40) with a few modifications. Vero CCL-81 cells were seeded in 6-well culture plates to attain a 90% cell confluence before inoculation with 200µl of seed virus stock. After overlaying the infected cells with agarose gel, the plates were incubated at 37°C in a 5% carbon dioxide gas atmosphere for 3-4 days until plaques were evident. Staining was done with neutral red dye to visualize the plaques, and after overnight incubation, the plaques were counted and determined using the formula, Titer (pfu/ml) = number of plaques X dilution factor X 1/volume of virus added to cells in a well (ml).

Viral RNA Extraction
Viral RNA was extracted from the supernatants using the viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and temporarily kept at -20 o C awaiting reverse transcription.

Reverse-transcription Polymerase Chain Reaction (RT-PCR)
Reverse transcription polymerase chain reaction was done as described (19

Whole genome sequencing of the RT-PCR Product
A total of 50ng cleaned-up PCR product was repaired using NEBNext Ultra II End repair/dA- PRJNA825709. The quality threshold was set at 20, and a minimum length of 150 was allowed.
Trimmomatic tool (42) was used to remove adapter sequences, followed by a post-trimming quality assessment based on the FastQC report. We used SPADES (43) to assemble raw sequences with reference genome retrieved from the SARS-CoV-2 RefSeq database: https://ftp.ncbi.nlm.nih.gov/refseq/release/viral/; March 10 th version. Raw sequences were deposited in the NCBI's Sequence Retrieval Archive (SRA) under accessions SRS12585595 and SRS12585594. The sequences were identified using nucleotide Basic Local Alignment Search Tool_BLAST (44). Upon confirming the sequence, we performed cluster analysis of the isolates using Nextstrain/Nextclade v1.14.0 (45) server. Equally, mutations in amino acid residues were conducted using Nextclade. Sequence alignment and phylogenetic comparisons were achieved using MUSCLE (46) and MEGAX (47).