Limited Genetic Diversity Detected in Middle East Respiratory Syndrome-Related Coronavirus Variants Circulating in Dromedary Camels in Jordan

Viral infections by endemic, emerging, and reemerging viruses are constantly challenging public health systems and health policies all over the world [...]

M iddle East respiratory syndrome (MERS) coronavirus (MERS-CoV) represents 1 of 3 major zoonotic coronaviruses to have emerged with global impact in the past 2 decades, alongside severe acute respiratory syndrome coronavirus (SARS-CoV-1) in 2002-2003 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 2019 onward (1). The earliest known outbreak of MERS-CoV began in a hospital in Zarqa, Jordan, in April 2012 (2,3). Since that time, >2,500 cases and 880 deaths (case-fatality rate of 34%) have been reported across 27 countries worldwide (4). The fi rst detection of positive MERS-CoV by serologic testing in camels was also from Zarqa, Jordan, in 2013 (5); camels were later confi rmed as the reservoir for MERS-CoV infection in humans (6) and bats the likely ancestral host (7).Most confi rmed MERS-CoV cases have occurred within the Arabian Peninsula; Saudi Arabia, the location of ≈80% of all human cases, is the epicenter (8). Phylogenetic analyses of viral sequences isolated from camels and humans suggest that multiple camel-to-human spillover events have occurred since the initial MERS outbreaks in 2012 (9). Although humans sometimes represent a dead-end host, secondary human-to-human infection does occur, leading in some cases to large-scale outbreaks in hospital settings, such as those seen in Saudi Arabia and South Korea in recent years (10,11). Whereas infection in camels might be subclinical or cause mild upper respiratory symptoms (12,13), infection in humans can range from asymptomatic to severe acute respiratory disease or death (14).The World Health Organization has declared MERS-CoV a priority disease in its Research and Development Blueprint program as a public health risk of epidemic potential (15); vaccination of camels is a potential key component of future disease control strategies (16). Although MERS-CoV is widespread among camel populations in Africa, the Middle East,

IntroductionWhole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with ‘Ns’ inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs).MethodsIn this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell.Results and discussionUsing a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.