Host-Virus Chimeric Events in SARS-CoV-2-Infected Cells Are Infrequent and Artifactual

Abstract

Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a potential pathogenic mechanism. Since SARS-CoV2 is a positive-sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle. Thus, it is biologically unlikely to be in a location where splicing events could result in genome integration. Therefore, we investigated the biological authenticity of HVC events. In contrast to true biological events such as mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and infected cell lines were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ∼1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events was, in fact, not greater than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes. Importance The pathogenic mechanisms underlying SARS-CoV2, the virus responsible for COVID-19, are not fully understood. In particular, relatively little is known why some individuals develop life-threatening or persistent COVID-19. Recent studies identified host-virus chimeric (HVC) reads in RNA-sequencing data from SARS-CoV2 infected cells and suggested that HVC events support potential “human genome invasion” and “integration” by SARS-CoV2. This suggestion has fueled concerns about the long-term effects of current mRNA vaccines that incorporate elements of the viral genome. SARS-CoV2 is a positive-sense, single-stranded RNA virus that does not encode a reverse transcriptase and does not include a nuclear phase in its life cycle, so some doubts have rightfully been expressed regarding the authenticity of HVCs and the role played by endogenous retrotransposons in this phenomenon. Thus, it is important to independently authenticate these HVC events. Here we provide several evidences suggesting that the observed HVC events are likely artifactual.

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

“…More importantly, chimeric reads were also identified in uninfected zebrafish embryos when mixing their RNAs with the RNAs from infected human cells at library construction step, therefore, providing the solid evidence that chimeric reads in viral infected samples are artificially introduced mainly through random ligations during library construction. In support, another team also observed the similar results based on their RNA-seq analysis ( 27 ). Although Zhang et al found that SARS-CoV-2 could be reverse-transcribed and integrated into the genome of cultured cells overexpressing LINE-1 ( 20 ), it might be mainly due to the activation of retrotransposon but not natural characters of SARS-CoV-2.…”

Comprehensive analysis of RNA-seq and whole genome sequencing data reveals no evidence for SARS-CoV-2 integrating into host genome

“…More importantly, chimeric reads were also identified in uninfected zebrafish embryos when mixing their RNAs with the RNAs from infected human cells at library construction step, therefore, providing the solid evidence that chimeric reads in viral infected samples are artificially introduced mainly through random ligations during library construction. In support, another team also observed the similar results based on their RNA-seq analysis (Yan et al, 2021). Although Zhang et al found that SARS-CoV-2 could be reverse-transcribed and integrated into the genome of cultured cells overexpressing LINE1 (Zhang et al, 2021), it might be mainly due to the activation of retrotransposon but not natural characters of SARS-CoV-2.…”

No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing

“…In a recent study, Zhang et al overexpressed L1 in HEK293T cells, infected these with SARS-CoV-2, and identified DNA fragments of the virus through PCR amplification 29 . These results, alongside other less direct 30,31 analyses, were interpreted as evidence of SARS-CoV-2 genomic integration 29 . Crucially, Zhang et al then detected 63 putative SARS-CoV-2 integrants by Oxford Nanopore Technologies (ONT) long-read sequencing.…”