Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Yan, Bingyu; Chakravorty, Srishti; Mirabelli, Carmen; Wang, Luopin; Trujillo-Ochoa, Jorge L; Chauss, Daniel; Kumar, Dhaneshwar; Lionakis, Michail S; Olson, Matthew R.; Wobus, Christiane E.; Afzali, Behdad; Kazemian, Majid

doi:10.1101/2021.02.17.431704

Cited by 8 publications

(11 citation statements)

References 22 publications

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“…Instead, our study examined specifically the extent to which such integration events can be supported by the detection of chimeric reads between SARS-CoV-2 RNA and human RNA. At least at the level that can be determined by RNA-seq data analysis, our findings do not indicate frequent genomic integration and subsequent expression of SARS-CoV-2 RNA, and similar conclusions were reached by independent analysis ( Yan et al, 2021 ).…”

Section: Discussionsupporting

confidence: 86%

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

Kazachenka

Kassiotis

2021

Front. Microbiol.

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The human genome bears evidence of extensive invasion by retroviruses and other retroelements, as well as by diverse RNA and DNA viruses. High frequency of somatic integration of the RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the DNA of infected cells was recently suggested, based on a number of observations. One key observation was the presence of chimeric RNA-sequencing (RNA-seq) reads between SARS-CoV-2 RNA and RNA transcribed from human host DNA. Here, we examined the possible origin specifically of human-SARS-CoV-2 chimeric reads in RNA-seq libraries and provide alternative explanations for their origin. Chimeric reads were frequently detected also between SARS-CoV-2 RNA and RNA transcribed from mitochondrial DNA or episomal adenoviral DNA present in transfected cell lines, which was unlikely the result of SARS-CoV-2 integration. Furthermore, chimeric reads between SARS-CoV-2 RNA and RNA transcribed from nuclear DNA were highly enriched for host exonic, rather than intronic or intergenic sequences and often involved the same, highly expressed host genes. Although these findings do not rule out SARS-CoV-2 somatic integration, they nevertheless suggest that human-SARS-CoV-2 chimeric reads found in RNA-seq data may arise during library preparation and do not necessarily signify SARS-CoV-2 reverse transcription, integration in to host DNA and further transcription.

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Section: Discussionsupporting

confidence: 86%

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

Kazachenka

Kassiotis

2021

Front. Microbiol.

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“…This is consistent with the finding that NC RNA is the most abundant SARS-CoV-2 subgenomic RNA ( 56 ), making it the most likely target for reverse transcription and integration. However, recent data showed that up to 1% of RNA-seq reads from SARS-CoV-2–infected cells can be artifactually chimeric as a result of RT switching between RNA templates, which can occur during the cDNA synthesis step in the preparation of a RNA-seq library ( 57 ). Thus, because there is a mixture of host mRNAs and positive-strand viral mRNAs in infected cells, the identification of genuine chimeric viral–cellular RNA transcripts is compromised by the generation of artifactual chimeras in the assays.…”

Section: Resultsmentioning

confidence: 99%

Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues

Zhang

Richards

Barrasa

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

223

238

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Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral–host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3′ end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.

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“…Other studies suggested different interpretations for the data identifying virus/host chimeric reads after next generation sequencing (NGS) of SARS-CoV-2 infected cells. These works pointed out the possibility of fusion events happening during library preparation [22,27] that could explain the nature of the identified chimeric DNA fragments containing both human and viral sequences. Specifically, Yan et al [22] found that the observed chimeric events [7] fell below background and were likely due to template switching of the reverse transcriptase enzyme used during RNAseq library preparation.…”

Section: Introductionmentioning

confidence: 99%

“…These works pointed out the possibility of fusion events happening during library preparation [22,27] that could explain the nature of the identified chimeric DNA fragments containing both human and viral sequences. Specifically, Yan et al [22] found that the observed chimeric events [7] fell below background and were likely due to template switching of the reverse transcriptase enzyme used during RNAseq library preparation. They concluded that the chimeric events observed were rare, unreproducible, and unlikely to have occurred biologically [22].…”

Section: Introductionmentioning

confidence: 99%

ASSESSMENT OF POTENTIAL SARS-CoV-2 VIRUS N GENE INTEGRATION INTO HUMAN GENOME REVEALS NO SIGNIFICANT IMPACT ON RT-qPCR COVID-19 DIAGNOSTIC TESTING

Briggs¹,

Ward²,

Rey³

et al. 2021

Preprint

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The SARS Coronavirus 2 (SARS-CoV-2) pandemic presents new scientific and scale-up challenges for diagnostic capabilities worldwide. The gold standard diagnostic for SARS-CoV-2 infection is a reverse transcription/quantitative PCR (RT-qPCR) which targets the viral genome, an assay that has now been performed on millions of patient specimens worldwide regardless of symptomatic status. Recently Zhang et al. suggested the possibility that the SARS-CoV-2 N gene could integrate into host cell DNA through the action of the LINE-1 retrotransposon, a mobile element that is potentially active in human somatic cells, thereby calling into question the veracity of N-gene based RT-qPCR for detection of SARS-CoV-2 infection. Accordingly, we assessed the potential impact of these purported integration events on nasal swab specimens tested at our clinical laboratory. Using an N-gene based RT-qPCR assay, we tested 768 arbitrarily selected specimens and identified 2 samples which resulted in a positive detection of viral sequence in the absence of reverse transcriptase, a necessary but not sufficient signal consistent with possible integration of the SARS-CoV-2 N gene into the host genome. Regardless of possible viral N gene integration into the genome, in this small subset of samples, all patients were still positive for SARS-CoV-2 infection, as indicated by a much lower Ct value for reactions performed in the presence of reverse transcriptase (RT) versus reactions performed without RT. Moreover, one of the two positives observed in the absence of RT also tested positive when using primers targeting ORF1ab, a gene closer to the 5 prime end of the genome. These data are inconsistent with the N gene integration hypothesis suggested by the studies by Zhang et al., and importantly, our results suggest little to no practical impact of possible SARS-CoV-2 genome integration events on RT-qPCR testing.

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Host-virus chimeric events in SARS-CoV2 infected cells are infrequent and artifactual

Cited by 8 publications

References 22 publications

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA

Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues

ASSESSMENT OF POTENTIAL SARS-CoV-2 VIRUS N GENE INTEGRATION INTO HUMAN GENOME REVEALS NO SIGNIFICANT IMPACT ON RT-qPCR COVID-19 DIAGNOSTIC TESTING

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