A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Dinucleotide evolutionary dynamics in influenza A virus.

Significant biases of dinucleotide composition in many RNA viruses including influenza A virus have been reported in recent years. Previous studies have showed that a codon-usage-altered influenza mutant with elevated CpG usage is attenuated in mammalian in vitro and in vivo models. However, the relationship between dinucleotide preference and codon usage bias is not entirely clear and changes in dinucleotide usage of influenza virus during evolution at segment level are yet to be investigated. In this study, a Monte Carlo type method was applied to identify under-represented or over-represented dinucleotide motifs, among different segments and different groups, in influenza viral sequences. After excluding the potential biases caused by codon usage and amino acid sequences, CpG and UpA were found under-represented in all viral segments from all groups, whereas UpG and CpA were found over-represented. We further explored the temporal changes of usage of these dinucleotides. Our analyses revealed significant decrease of CpG frequency in Segments 1, 3, 4, and 5 in seasonal H1 virus after its re-emergence in humans in 1977. Such temporal variations were mainly contributed by the dinucleotide changes at the codon positions 3-1 and 2-3 where silent mutations played a major role. The depletions of CpG and UpA through silent mutations consequently led to over-representations of UpG and CpA. We also found that dinucleotide preference directly results in significant synonymous codon usage bias. Our study helps to provide details on understanding the evolutionary history of influenza virus and selection pressures that shape the virus genome.

Mini viral RNAs act as innate immune agonists during influenza virus infection.

The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype2-4. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression5. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-ß. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias.

UNLABELLED: Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines. IMPORTANCE: Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.