RESUMO
During the COVID-19 pandemic, SARS-CoV-2 surveillance efforts integrated genome sequencing of clinical samples to identify emergent viral variants and to support rapid experimental examination of genome-informed vaccine and therapeutic designs. Given the broad range of methods applied to generate new viral genomes, it is critical that consensus and variant calling tools yield consistent results across disparate pipelines. Here we examine the impact of sequencing technologies (Illumina and Oxford Nanopore) and 7 different downstream bioinformatic protocols on SARS-CoV-2 variant calling as part of the NIH Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Tracking Resistance and Coronavirus Evolution (TRACE) initiative, a public-private partnership established to address the COVID-19 outbreak. Our results indicate that bioinformatic workflows can yield consensus genomes with different single nucleotide polymorphisms, insertions, and/or deletions even when using the same raw sequence input datasets. We introduce the use of a specific suite of parameters and protocols that greatly improves the agreement among pipelines developed by diverse organizations. Such consistency among bioinformatic pipelines is fundamental to SARS-CoV-2 and future pathogen surveillance efforts. The application of analysis standards is necessary to more accurately document phylogenomic trends and support data-driven public health responses.
RESUMO
As SARS-CoV-2 variants emerge, there is a critical need to understand the effectiveness of serum elicited by different SARS-CoV-2 vaccines. A reference reagent comprised of post-vaccination sera from recipients of different vaccines allows evaluation of in vitro variant neutralization, and provides a reference for comparing assay results across laboratories. We prepared and pooled >1 L serum from donors who received the SARS-CoV-2 mRNA vaccines (BNT162b2, Pfizer and mRNA-1273, Moderna), a replication-incompetent adenovirus type 26 vaccine (Ad26.COV2.S, Johnson and Johnson), or recombinant spike protein expressed by baculovirus incorporated into a nanoparticle vaccine plus Matrix-M adjuvant (NVX-CoV2373, Novavax). Twice frozen sera were aliquoted and are available for distribution to the research community (BEI Resources). The calculated WHO titer of pooled sera to spike protein was 1,312, 1,447, 1,936, and 587 and the reciprocal RBD binding to ACE-2 IC90-titers were 60, 64, 118, and 46 for BNT162b2, mRNA1273, Ad26.CoV2373, and NVX-CoV2373 sera, respectively.
RESUMO
The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong. The genome sequence shows a 27 nt in-frame deletion ({Delta}27,264-27,290) within ORF6, predicted to result in a 9 aa deletion ({Delta}FKVSIWNLD) from the central portion of p6. This deletion is predicted to result in a dramatic alteration in the three-dimensional structure of the resultant protein (p6{Delta}22-30), possibly with significant functional implications. Analysis of the original clinical sample indicates that the deletion was not present, while sequencing of subsequent passages of the strain identifies the deletion as a majority variant. This suggests that the deletion originated ab initio during passaging and subsequently propagated into the majority, possibly due to the removal of selective pressure through the IFN-deficient Vero E6 cell line. The specific function of the SARS-CoV-2 p6 N-terminus, if any, has not yet been determined. However, this deletion is predicted to cause a shift from N-endo to N-ecto in the transmembrane localization of the SARS-CoV-2 p6{Delta}22-30 N-terminus, possibly leading to the ablation of its native function.
