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A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus., Graphical abstract, In response to a recent claim that SARS-CoV-2 can be incorporated into human DNA by LINE-1 retrotransposon proteins, Smits et al. apply long-read DNA sequencing to cultured HEK293T cells infected with SARS-CoV-2. They find no evidence for genomic integration of the virus, despite demonstrated availability of the LINE-1 protein machinery.

SUMMARYA recent study proposed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we applied deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2, and did not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolved 78 L1 insertions arisingin vitroin the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV) positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions via ONT sequencing. That we found no evidence of SARS-CoV-2 integration suggests such events are, at most, extremely rarein vivo, and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.

The gastrointestinal tract represents the largest immune interface with the environment. Exposure to large numbers of dietary and microbial antigens requires complex and highly regulated intestinal immune responses by different immune cell types for the maintenance of oral tolerance. Defective immune homeostasis can cause gut barrier dysfunction and breakdown of tolerance, leading to chronic inflammation and autoimmunity. In this review, we summarize the key immune cell populations involved in oral tolerance. We also describe diet-modifiable aspects of gut immunity that alter the intricate balance between inflammatory and tolerogenic immune responses in the gut and contribute to disease development.

Complementation of movement and coat proteins of the orchid-infecting potexvirus Cymbidium mosaic virus (CymMV) and tobamovirus Odontoglossum ringspot virus (ORSV) was investigated. Nicotiana benthamiana, which is susceptible to both CymMV and ORSV, was used as a model system. Four transgenic lines, each harbouring one of the movement protein (MP) or coat protein (CP) genes of CymMV or ORSV, were constructed. The MP of CymMV consists of three overlapping open reading frames, together called the triple-gene block (TGB). CymMV and ORSV mutants, each carrying an inactivated MP or CP, were generated from the respective biologically active full-length cDNA clones. Complementation was studied by infecting transgenic plants with in vitro transcripts generated from these mutants. The cell-to-cell movement of a movement-deficient CymMV was restored in transgenic plants carrying the ORSV MP transgene. Similarly, CymMV TGB1 transgenic plants were able to rescue the cell-to-cell movement of a movement-deficient ORSV mutant. ORSV CP transgenic plants supported systemic movement of a CymMV CP-deficient mutant. However, in these plants, neither encapsidation of CymMV RNA with ORSV CP nor CymMV CP expression was detected. Long-distance movement of an ORSV CP-deficient mutant was not supported by CymMV CP. The complementation of MPs and CPs of CymMV and ORSV facilitates movement of these viruses in plants, except for long-distance movement of ORSV RNA by CymMV CP.

The variability in coat protein gene sequences of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) that naturally infect orchids worldwide was investigated. Samples were collected from Korea, Singapore and Taiwan. The sequence data were compared with available published coat protein gene sequences of CymMV and ORSV, including those from Japan and Thailand. Among CymMV isolates, the homology was 89.1%-99.7% and 93.2%-100% at the nucleotide and amino acid levels, respectively. Among the ORSV isolates, the homology was 95.5%-100% and 93%-100% at the nucleotide and amino acid levels, respectively. No particular region of variability could be defined in either of the viruses. In deduced amino acid sequence, the N-terminal was more conserved than the C-terminal in both CymMV and ORSV. By comparing all sequences determined in this study and those that are published in the GenBank databases, we did not find clustering based on geographical distribution or sequence identity. Such high sequence conservation suggests that both CymMV and ORSV coat protein genes are suitable candidates to provide resistance to orchids cultivated in different geographical locations.