Editors: Donna E. Hansel, MD, PhD, chair of pathology, Oregon Health and Science University, Portland; Richard D. Press, MD, PhD, professor and director of molecular pathology, OHSU; James Solomon, MD, PhD, assistant professor, Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York; Sounak Gupta, MBBS, PhD, senior associate consultant, Mayo Clinic, Rochester, Minn.; Tauangtham Anekpuritanang, MD, molecular pathology fellow, Department of Pathology, OHSU; Hassan Ghani, MD, molecular genetic pathology fellow, Department of Pathology, OHSU; and Fei Yang, MD, assistant professor, Department of Pathology, OHSU.
Assessment of how the SARS-CoV-2 virus infects target cells
May 2020—Each year a growing number of novel viruses are discovered in the wild animal kingdom with the use of next-generation sequencing technology. However, the current method of functionally assessing the zoonotic potential—that is, the potential to be transmitted to humans—of novel viruses involves synthesis of viral genomes (tens of thousands of bases) and reverse genetic engineering to produce a recombinant virus. This is expensive and time-consuming, and it is not practical due to the scale of new viral strains being discovered. To overcome this hurdle, Letko, et al., developed a rapid and cost-effective method that could functionally assess a number of related viruses for zoonotic potential. The essential component of viral cross-species transmission is cell entry, which is a multi-step process involving attachment of the virus to the host cell surface, receptor engagement, processing of host proteases, and downstream membrane fusion. Previous studies have shown that for betacoronaviruses, such as SARS-CoV-2, the receptor-binding domain (RBD) within the viral spike protein is the single critical region mediating host cell receptor recognition and engagement. Therefore, the roughly 200-base RBD was synthesized and engineered to generate a chimeric spike expression construct for the subsequent receptor cell entry assay. Using this same approach, Letko, et al., constructed the chimeric RBD spike of the SARS-CoV-2 virus and observed that this virus used the human angiotensin-converting enzyme 2 (ACE2) receptor but no other known coronavirus receptors for host cell entry. This result was confirmed by independent virus infectivity studies using the full virus isolated from patients (Zhou, et al.). To further demonstrate the utility of this new approach, Letko, et al., studied other strains of lineage B betacoronaviruses. RBD sequences of these strains are clustered into three major clades based on phylogenetic analysis. Viruses with clade one RBD, which includes the SARS-CoV that emerged in humans in 2003, but not clade two or three RBD, can infect host cells expressing human ACE2. The authors found that none of the viruses with any of the three clades entered an array of cell lines expressing other human receptors known to interact with coronaviruses. The RBD of SARS-CoV-2 has sequence characteristics found in all three clades but was not clustered into any of the clades. The authors hypothesized that these clades can rearrange genetic material to convey zoonotic potential since coronaviruses often undergo genetic recombination. They developed a scalable, rapid, and cost-effective method for testing a large number of novel viruses for their ability to infect human cells, without the need for viral isolation. Through this study of lineage B coronaviruses, the authors postulated that viruses can gain virulence through genetic recombination and that several other lineage B coronaviruses can enter human cells through unknown receptors. This new platform could potentially serve as a means of swiftly surveilling emerging zoonotic viruses to better prepare for future outbreaks.
Letko M, Marzi A, Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol. 2020;5:562–569.
Correspondence: Dr. Michael Letko at michael.letko@nih.gov, or Dr. Vincent Munster at vincent.munster@nih.gov
Zhou P, Yang X, Wang X, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–273.
Correspondence: Dr. Zheng-Li Shi at zlshi@wh.iov.cn
Rapid NGS-based diagnosis of bacteremia in septic patients
Timely administration of antimicrobial therapy to patients with bacteremia, especially those with septic shock, is associated with improved clinical outcome. The standard of care for identifying the culprit pathogen, however, still relies on traditional blood culture, which takes one to two days, and, therefore, prevents early administration of appropriate antibiotics. Polymerase chain reaction-based methods, in comparison, can improve turnaround time but can assess only a small number of targeted pathogens. A higher throughput next-generation sequencing (NGS) approach can assess a broader range of microorganisms. Moreover, cell-free DNA (cfDNA) in plasma has recently been validated as an effective biomarker for identifying causative pathogens. Rigorous studies have shown that cfDNA-based NGS methods have a sensitivity and specificity comparable to culture-based methods. However, the traditional Illumina NGS sequencers, known for their high sequence quality, generally take 24 to 30 hours to produce data for analysis (Blauwkamp, et al.). The MinIon portable third-generation Nanopore sequencer, on the other hand, can generate sequence results in real time for immediate data analysis. To take advantage of the speed of MinIon, Grumaz, et al., developed a rapid workflow on MinIon that involved library preparation protocols for short cfDNA fragments and bioinformatics adaptations. Eight samples from four sepsis patients and three healthy control subjects were tested in this study, and the total time from blood draw to pathogen identification was no more than six hours. In one sample with high microbial load, the diagnostic result was obtained one minute after starting sequencing. Because Nanopore sequencers are known to have higher sequencing error rates than Illumina sequencers, the authors applied several measures to MinIon to mitigate this limitation, including applying more stringent thresholds to the sequence read coverages for calling a positive result, assembling the reference database with only sepsis-relevant pathogenic species, and subtracting artifacts identified in a set of noninfected control subjects. A retrospective extrapolation of real-time sequencing performance on a cohort of 239 septic patient samples sequenced with a validated Illumina platform showed that 90 percent of pathogens identified by the Illumina workflow would have been identified by the MinIon workflow. These findings support the conclusion that an optimized workflow using a rapid NGS methodology to diagnose sepsis within a critically short time frame may be an attractive future point-of-care tool.
Blauwkamp TA, Thair S, Rosen MJ, et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat Microbiol. 2019;4:663–674.
Correspondence: Dr. Timothy A. Blauwkamp at tim.blauwkamp@kariusdx.com
Grumaz C, Hoffmann A, Vainshtein Y, et al. Rapid next-generation sequencing–based diagnostics of bacteremia in septic patients. J Mol Diagn. 2020;22(3):405–418.
Correspondence: Dr. Kai Sohn at kai.sohn@igb.fraunhofer.de