RNA structural elements occur in numerous single stranded (+)-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' UTR in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including SARS-CoV and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts implicates a key role of s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodelled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as anti-viral agents.IMPORTANCEThe highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The anti-viral potential of this conserved element represents a promising start for further research into targeting conserved elements in RNA viruses.

Intrinsically disordered regions/proteins (IDRs) are abundant across all the domains of life, where they perform important regulatory roles and supplement the biological functions of structured proteins/regions (SRs). Despite the multifunctionality features of IDRs, several interrogations on the evolution of viral genomic regions encoding IDRs in diverse viral proteins remain unreciprocated. To fill this gap, we benchmarked the findings of two most widely used and reliable intrinsic disorder prediction algorithms (IUPred2A and ESpritz) to a dataset of 6108 reference viral proteomes to unravel the multifaceted evolutionary forces that shape the codon usage in the viral genomic regions encoding for IDRs and SRs. We found persuasive evidence that the natural selection predominantly governs the evolution of codon usage in regions encoding IDRs by most of the viruses. In addition, we confirm not only that codon usage in regions encoding IDRs is less optimized for the protein synthesis machinery (transfer RNAs pool) of their host than for those encoding SRs, but also that the selective constraints imposed by codon bias sustain this reduced optimization in IDRs. Our analysis also establishes that IDRs in viruses are likely to tolerate more translational errors than SRs. All these findings hold true, irrespective of the disorder prediction algorithms used to classify IDRs. In conclusion, our study offers a novel perspective on the evolution of viral IDRs and the evolutionary adaptability to multiple taxonomically divergent hosts.

The presence of foot-and-mouth disease virus (FMDV) of the O/ME-SA/Ind-2001e sublineage within Pakistan was initially detected in two samples collected during 2019. Analysis of further serotype O FMDVs responsible for disease outbreaks in 2019-2020 in the country has now identified the spread of this sublineage to 10 districts within two separate provinces in North-Eastern and North-Western Pakistan. Phylogenetic analysis indicates that these viruses are closely related to those circulating in Bhutan, Nepal and India. The VP1 coding sequences of these viruses from Pakistan belong to three distinct clusters, which may indicate multiple introductions of this virus sublineage, although the routes of introduction are unknown. Vaccine matching studies against O1 Manisa, O 3039 and O TUR/5/2009 support the suitability of existing vaccine strains to control current field outbreaks, but further studies are warranted to monitor the spread and evolution of the O/ME-SA/Ind-2001e sublineage in the region.

As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes

Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-β and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-β were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1β and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-β, and reporting some peculiarity of swine, which should be considered in translational studies.