Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as “long COVID”, post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. Methods We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. Results Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. Conclusions Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene–environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity–environment interactions are likely to be under strong positive selection.

The β-1, 3 (d)-glucan (β-glucan) present in the cell wall of Candida albicans induces epigenetic changes in human monocytes resulting in primed macrophages exhibiting increased cytokine responsiveness to reinfection. This phenomenon is referred to as trained immunity or innate immune memory. However, whether β-glucan can reprogramme murine monocytes in vitro or induce lasting effects in vivo has yet to be elucidated. Thus, purified murine spleen-derived monocytes were primed with β-glucan in vitro and assessed for markers of differentiation and survival. Important macrophage cell markers during monocyte-to-macrophage differentiation were downregulated and survival enhanced due to partial inhibition of apoptosis. Increased survival and not the β-glucan training effect explained the elevated production of tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) induced by subsequent lipopolysaccharide (LPS) challenge. In vivo, 4 days after systemic administration of β-glucan, mice were more responsive to LPS challenge as shown by the increased serum levels of TNFα, IL-6 and IL-10, an effect shown to be short lived as enhanced cytokine production was lost by day 20. Here, we have characterised murine macrophages derived from β-glucan-primed monocytes based on their surface marker expression and for the first time provide evidence that the training effect of β-glucan in vivo declines within a 3-week period.

Spring viremia carp virus (SVCV) is a rhabdovirus seasonally affecting warm-water cyprinid fish farming causing high impacts in worldwide economy. Because of the lack of effective preventive treatments, the identification of multipath genes involved in SVCV infection might be an alternative to explore the possibilities of using drugs for seasonal prevention of this fish disease. Because the zebrafish (Danio rerio) is a cyprinid susceptible to SVCV and their genetics and genome sequence are well advanced, it has been chosen as a model for SVCV infections. We have used newly designed pathway-targeted microarrays 3-4-fold enriched for immune/infection functional-relevant probes by using zebrafish orthologous to human genes from selected pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG). The comparative analysis of differential expression of genes through 20 pathways in 2-day exposed or 30-day survivors of SVCV infection allowed the identification of 16 multipath genes common to more than 6 pathways. In addition, receptors (Toll-like, B-cell, T-cell, RIG1-like) as well as viral RNA infection pathways were identified as the most important human-like pathways targeted by SVCV infection. Furthermore, by using bioinformatic tools to compare the promoter sequences corresponding to up and downregulated multipath gene groups, we identified putative common transcription factors which might be controlling such responses in a coordinated manner. Possible drug candidates to be tested in fish, can be identified now through search of data bases among those associated with the human orthologous to the zebrafish multipath genes. With the use of pathway-targeted microarrays, we identified some of the most important genes and transcription factors which might be implicated in viral shutoff and/or host survival responses after SVCV infection. These results could contribute to develop novel drug-based prevention methods and consolidate the zebrafish/SVCV as a model for vertebrate viral diseases.

The dengue virus (DENV) nonstructural protein 5 (NS5) contains both an N-terminal methyltransferase domain and a C-terminal RNA-dependent RNA polymerase domain. Polymerase activity is responsible for viral RNA synthesis by a de novo initiation mechanism and represents an attractive target for antiviral therapy. The incidence of DENV has grown rapidly and it is now estimated that half of the human population is at risk of becoming infected with this virus. Despite this, there are no effective drugs to treat DENV infections. The present in silico study aimed at finding new inhibitors of the NS5 RNA-dependent RNA polymerase of the four serotypes of DENV. We used a chemical library comprising 372,792 nonnucleotide compounds (around 325,319 natural compounds) to perform molecular docking experiments against a binding site of the RNA template tunnel of the virus polymerase. Compounds with high negative free energy variation (ΔG <−10.5 kcal/mol) were selected as putative inhibitors. Additional filters for favorable druggability and good absorption, distribution, metabolism, excretion, and toxicity were applied. Finally, after the screening process was completed, we identified 39 compounds as lead DENV polymerase inhibitor candidates. Potentially, these compounds could act as efficient DENV polymerase inhibitors in vitro and in vivo.

It has not been elucidated whether or not autophagy is induced by rhabdoviral G glycoproteins (G) in vertebrate organisms for which rhabdovirus infection is lethal. Our work provides the first evidence that both mammalian (vesicular stomatitis virus, VSV) and fish (viral hemorrhagic septicemia virus, VHSV, and spring viremia carp virus, SVCV) rhabdoviral Gs induce an autophagic antiviral program in vertebrate cell lines. The transcriptomic profiles obtained from zebrafish genetically immunized with either Gsvcv or Gvhsv suggest that autophagy is induced shortly after immunization and therefore, it may be an important component of the strong antiviral immune responses elicited by these viral proteins. Pepscan mapping of autophagy-inducing linear determinants of Gvhsv and Gvsv showed that peptides located in their fusion domains induce autophagy. Altogether these results suggest that strategies aimed at modulating autophagy could be used for the prevention and treatment of rhabdoviral infections such as rabies, which causes thousands of human deaths every year.

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To investigate fish innate immunity, we have conducted organ and cell immune-related transcriptomic as well as immunohistologic analysis in mutant zebra fish (Danio rerio) lacking adaptive immunity (rag1−/−) at different developmental stages (egg, larvae, and adult), before and after infection with spring viremia carp virus (SVCV). The results revealed that, compared to immunocompetent zebra fish (rag1+/+), rag1−/− acquired increased resistance to SVCV with age, correlating with elevated transcript levels of immune genes in skin/fins and lymphoid organs (head kidney and spleen). Gene sets corresponding to apoptotic functions, immune-related multigene families, and interferon-related genes were constitutively upregulated in uninfected adult rag1−/− zebra fish. Overexpression of activated CASPASE-3 in different tissues before and after infection with SVCV further confirmed increased apoptotic function in rag1−/− zebra fish. Concurrently, staining of different tissue samples with a pan-leukocyte antibody marker showed abundant leukocyte infiltrations in SVCV-infected rag1−/− fish, coinciding with increased transcript expression of genes related to NK-cells and macrophages, suggesting that these genes played a key role in the enhanced immune response of rag1−/− zebra fish to SVCV lethal infection. Overall, we present evidence that indicates that rag1−/− zebra fish acquire an antiviral alert state while they reach adulthood in the absence of adaptive immunity. This antiviral state was characterized by (i) a more rapid response to viral infection, which resulted in increased survival, (ii) the involvement of NK-cell- and macrophage-mediated transcript responses rather than B- and/or T-cell dependent cells, and (iii) enhanced apoptosis, described here for the first time, as well as the similar modulation of multigene family/interferon-related genes previously associated to fish that survived lethal viral infections. From this and other studies, it might be concluded that some of the characteristics of mammalian trained immunity are present in lower vertebrates.