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The lack of sequencing of SARS-CoV-2 variants is imperiling the global response to the COVID-19 pandemic

The lack of sequencing of SARS-CoV-2 variants by the U.S. and other countries is imperiling the global response to the COVID-19 pandemic, argues Dana Crawford of Case Western Reserve University in a new Viewpoint published July 15th in the journal PLOS Genetics.

Surveillance is essential to a successful and rapid response to disease outbreaks, but public health surveillance has traditionally focused on monitoring case numbers, hospitalizations and deaths. Advances in genome sequencing now allow us to track genetic variation in evolving viruses in unprecedented detail. However, despite the availability of sequencing in several countries, the adoption of genomics as a strategy for surveilling the SARS-CoV-2 virus, which causes COVID-19, has been slow, difficult and inconsistent.

Crawford points out that, as of early April 2021, the U.S. ranked 33rd in the world in SARS-CoV-2 sequencing for variant surveillance. She says that historically, the Centers for Disease Control and Prevention (CDC) has not prioritized genomic research for public health, and this bias has created a gaping hole in our understanding of the real-time evolution of SARS-CoV-2 and the impact on disease transmission and severity.

Crawford cites insufficient funding, the lack of an effective sample tracking system and strict regulations on sample and data sharing as the causes of the inadequate sequencing efforts. Still, other countries, such as China and the U.K., have successfully overcome these challenges.

The CDC recently committed more than $200 million to enhance sequencing, but Crawford points out that this late investment means that the U.S. lacks an organized database of patient information for COVID-19 studies. She cautions that investments in SARS-CoV-2 genomics need to continue and expand as new variants will likely arise due to variability in vaccination rates and adherence to COVID-19 precautions worldwide.

Given that SARS-CoV-2 is a novel zoonotic disease with no prior human infections, Crawford argues that sequencing and analysis are vital to understanding both the trajectory of the outbreak and its evolution. Provisions are also sorely needed to link genetic data to clinical and epidemiological data sources for public health research.

Finally, she concludes that ongoing international sequencing efforts are still needed to understand and respond to this continually evolving virus that knows no international boundaries.

“COVID-19 control requires near real-time host-pathogen genomic and public health data to track how SARS-CoV-2 has evolved and how this evolution affects transmission and disease severity,” Crawford adds. “US and global sequencing efforts have been too few and too disjointed, underscoring the need to engage with human genomics and integrate its technologies in emerging infectious disease public health responses.”

Serological testing can be used for diagnosis, retrospective assessment of the efficacy of control measures and testing the response to a future vaccine (Weissleder et al., 2020). Most antibody detection assays to estimate SARS-CoV-2 prevalence and incidence probe for the nucleocapsid (N) or spike (S) proteins (Weissleder et al., 2020). A recent study proposed the use of ORF8 and ORF3b antibodies as serological markers of early and late SARS-CoV-2 infection (Hachim et al., 2020).

The study is an important step towards standardization of serological assays for COVID-19 and a better understanding of SARS-CoV-2 pathogenicity. ORF8 antibodies were identified as a major marker of acute, convalescent and long-term antibody response to SARS-CoV-2. However, several lineages without a functional ORF8 exist that may affect the accuracy of serological testing (Gong et al., 2020; Pereira, 2020; Su et al., 2020; To et al., 2020). It is important to be aware of this limitation when testing for COVID-19.

The ORF8 accessory gene is specific of Betacoronavirus lineage B (subgenus Sarbecovirus) (Forni et al., 2017). The gene is poorly conserved and its function remains unknown. Strikingly, middle and late phases of the 2002/2003 SARS epidemic were characterized by the spread of viruses with either partial or complete deletions of the ORF8 gene (Consortium, 2004).

It is still a matter of debate if the truncated ORF8 may have changed the SARS-CoV replication capacity or virulence in a way that favored its adaptation to humans and/or its spread during the SARS epidemic (Forni et al., 2017; Muth et al., 2018). In the ongoing COVID-19 pandemic, a SARS-CoV-2 variant with a 382-nucleotide deletion at ORF8 emerged early in Wuhan and was exported to Singapore and Taiwan (Gong et al., 2020; Su et al., 2020).

The deleted variant resulted in a less severe infection and lower concentrations of proinflammatory cytokines, chemokines and growth factors that are strongly associated with severe COVID-19 (Young et al., 2020).

I recently identified several nonsense mutations and additional deletions in the ORF8 gene that either remove or significantly change the ORF8 protein (Pereira, 2020). Currently, 17 nonsense mutations and eight deletions resulting in truncated ORF8 proteins, or even with the complete removal of the gene, are described in public databases (Table 1 ).

In total, mutations were observed in 660 genomes from viruses sampled in patients from different regions of the world (Table 1). Strikingly, the first reported case of a re-infection with SARS-CoV-2 was in a patient whose first infection resulted from a variant with a nonsense mutation at ORF8 (To et al., 2020). The available genomic data suggests that deleted or truncated ORF8 proteins are not a sporadic event and occurred in different times and places.

Moreover, the lower severity of infections resulting from variants without a functional ORF8 (Young et al., 2020) suggests that these lineages may escape detection as carriers are not always tested. It is therefore probable that the current list is an underrepresentation of the real number of circulating variants without a functional ORF8. Furthermore, the ORF8 stands out as being one of the most variable SARS-CoV-2 genes (Pereira, 2020).

Currently, 194 ORF8 positions were found to have missense variants (; accessed on 26 October 2020). Such non-synonymous variants may also be a problem for serological testing by potentially leading to epitope loss and a null serological response.

Table 1

List of nonsense mutations and deletions detected in SARS-CoV-2 ORF8. Data from the China National Center for Bioinformation ( and CoV-GLUE database ( accessed on 26 October 2020.

Genome positionN° of casesGenomic changeSampling location
27,9131T > ABangladesh
27,91538G > TUganda, USA, UAE, Wales, Japan, India
27,945152C > TUSA, India, Canada, England, Scotland, Portugal, Belgium, Italy, South Africa
27,94816G > TEngland, USA
27,9605C > TEngland, USA
27,97264C > TEngland, Scotland, Wales, Sweden, USA, Canada, Peru, India
27,9782C > TEngland
27,9864T > A; T > GEngland, Georgia, Sweden
28,0274G > AUSA
28,0414G > TChina, USA
28.0501A > TEngland
28,06818G > TUSA, Australia, Singapore, Slovakia, England, Switzerland
28,0722T > AUSA
28,0762C > AEngland
28,083195G > TIndia, Bangladesh, Hong Kong, England, Northern Ireland, Scotland, Latvia, Italy, Canada, Gambia, South Africa
28,20929G > TEngland, Wales, Spain, Iceland, Croatia, Japan, Hong Kong, South Korea, Bangladesh, USA, Australia
28,221102G > TUSA, India, England, Belgium, Netherlands, Poland
27,847–28,2309DeletionSingapore, Taiwan
Total cases660

The utility of serological tests depends on the sensitivity and specificity of the assay. Nevertheless, caution is necessary when detecting antibody responses directed against ORF8 antigens, as the absence of a functional gene may render the test inefficient. In case of patients who test negative for ORF8 antibody, it is recommended to always consider other targets (e.g., N or S proteins), particularly for those in which the suspicion for a past infection is high.

It is therefore highly recommended to test the antibody responses in patients with COVID-19 infected with the deficient ORF8 variants reported here (Table 1). If that is not possible, the serological test should be accompanied by a note of caution for its use and a disclaimer for patients who tested negative for ORF8 antibodies.

referene link:

More information: Crawford DC, Williams SM (2021) Global variation in sequencing impedes SARS-CoV-2 surveillance. PLoS Genet 17(7): e1009620.



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