Amplicon and Metagenomic Analysis of Middle East Respiratory Syndrome (MERS) Coronavirus and the Microbiome in Patients with Severe MERS.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional zoonotic spill over into humans. The severity of infection in humans is influenced by numerous factors, and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are coincident in the Middle East and thus a rapid way of sequencing MERS-CoV to derive genotype information for molecular epidemiology is needed. Additionally, complicating factors in MERS-CoV infections are coinfections that require clinical management. The ability to rapidly characterize these infections would be advantageous. To rapidly sequence MERS-CoV, an amplicon-based approach was developed and coupled to Oxford Nanopore long read length sequencing. This and a metagenomic approach were evaluated with clinical samples from patients with MERS. The data illustrated that whole-genome or near-whole-genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants, including deletion mutants. The metagenomic analysis provided information of the background microbiome. The advantage of this approach is that insertions and deletions can be identified, which are the major drivers of genotype change in coronaviruses. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in late 2012 in Saudi Arabia. The virus is a serious threat to people not only in the Middle East but also in the world and has been detected in over 27 countries. MERS-CoV is spreading in the Middle East and neighboring countries, and approximately 35% of reported patients with this virus have died. This is the most severe coronavirus infection so far described. Saudi Arabia is a destination for many millions of people in the world who visit for religious purposes (Umrah and Hajj), and so it is a very vulnerable area, which imposes unique challenges for effective control of this epidemic. The significance of our study is that clinical samples from patients with MERS were used for rapid in-depth sequencing and metagenomic analysis using long read length sequencing.

Amplicon-Based Detection and Sequencing of SARS-CoV-2 in Nasopharyngeal Swabs from Patients With COVID-19 and Identification of Deletions in the Viral Genome That Encode Proteins Involved in Interferon Antagonism.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.