Circular RNAs (circRNAs) are a newly recognized component of the transcriptome with critical roles in autoimmune diseases and viral pathogenesis. To address the importance of circRNA in RNA viral transcriptome, we systematically identified and characterized circRNAs encoded by the RNA genomes of betacoronaviruses using both bioinformatical and experimental approaches. We predicted 351, 224, and 2764 circRNAs derived from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), SARS‐CoV, and Middle East respiratory syndrome coronavirus, respectively. We experimentally identified 75 potential SARS‐CoV‐2 circRNAs from RNA samples extracted from SARS‐CoV‐2‐infected Vero E6 cells. A systematic comparison of viral and host circRNA features, including abundance, strand preference, length distribution, circular exon numbers, and breakpoint sequences, demonstrated that coronavirus‐derived circRNAs had a spliceosome‐independent origin. We further showed that back‐splice junctions (BSJs) captured by inverse reverse‐transcription polymerase chain reaction have different level of resistance to RNase R. Through northern blotting with a BSJ‐spanning probe targeting N gene, we identified three RNase R‐resistant bands that represent SARS‐CoV‐2 circRNAs that are detected cytoplasmic by single‐molecule and amplified fluorescence in situ hybridization assays. Lastly, analyses of 169 sequenced BSJs showed that both back‐splice and forward‐splice junctions were flanked by homologous and reverse complementary sequences, including but not limited to the canonical transcriptional regulatory sequences. Our findings highlight circRNAs as an important component of the coronavirus transcriptome, offer important evaluation of bioinformatic tools in the analysis of circRNAs from an RNA genome, and shed light on the mechanism of discontinuous RNA synthesis.

Influenza D virus (IDV), the cattle flu virus, is a novel multi-host RNA virus, circulating silently worldwide, with widespread seropositivity among US cattle, reaching up to 80% in some areas raising a potential threat of cattle-to-human transmission. Currently, five genetic lineages of IDV have been described, but their evolutionary dynamics have not been studied. Although IDV was first identified in 2011, our comprehensive analysis of all known IDV genomes suggests that the earliest ancestors of IDV likely to have evolved towards the end of the 20^th century and D/OK lineage appears to have emerged in 2005. We confirmed a significantly higher substitution rate in IDV than in Influenza C virus, which is consistent with their global distribution and multi-host tropism. We identified multiple sub-populations within the D/OK lineage, highlighting extensive diversification and dissemination. Other findings are evidence for potential reassortment among IDV strains in the USA and transboundary circulation of IDV in Europe with introductions into Danish cattle, some of which potentially originated from France. IDV, an emerging virus with a higher rate of evolution and uncontrolled circulation, could facilitate its adaptation to humans. Our findings underscore the importance of targeted surveillance for IDV in humans and at-risk animal populations.

The Coronavirus disease 2019 (COVID-19) pandemic disproportionately affects immunocompetent and immunocompromised individuals, with the latter group being more vulnerable to severe disease and death. However, the differential pathogenesis of SARS-CoV-2 in the context of a specific immunological niche remains unknown. Similarly, systematic analysis of disease pathology in various extrapulmonary organs in immunocompetent and immunocompromised hosts during SARS-CoV-2 infection is not fully understood. We used a hamster model of SARS-CoV-2 infection, which recapitulates the pathophysiology of patients with mild-to-moderate COVID-19, to determine the dynamics of SARS-CoV-2 replication and histopathology at organ-level niches and map how COVID-19 symptoms vary in different immune contexts. Hamsters were intranasally infected with low (LD) or high (HD) inoculums of SARS-CoV-2, and the kinetics of disease pathology and viral load in multiple organs, antibody response, inflammatory cytokine expression, and genome-wide lung transcriptome by RNAseq analysis were determined and compared against corresponding responses from chemically induced immunocompromised hamsters. We observed transient body weight loss proportional to the SARS-CoV-2 infectious dose in immunocompetent hamsters. The kinetics of viral replication and peak viral loads were similar between LD and HD groups, although the latter developed more severe disease pathology in organs. Both groups generated a robust serum antibody response. In contrast, infected immunocompromised animals showed more prolonged body weight loss and mounted an inadequate SARS-CoV-2-neutralizing antibody response. The live virus was detected in the pulmonary and extrapulmonary organs for extended periods. These hamsters also had persistent inflammation with severe bronchiolar-alveolar hyperplasia/metaplasia. Consistent with the differential disease presentation, distinct changes in inflammation and immune cell response pathways and network gene expression were seen in the lungs of SARS-CoV-2-infected immunocompetent and immunocompromised animals.

Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses have shown unprecedented host range and pathogenicity, including infections in cattle, previously not susceptible to H5N1. We investigated whether selection pressures on clade 2.3.4.4b viral genes could shed light on their unique epidemiological features. Our analysis revealed that while the gene products of clade 2.3.4.4b H5N1 primarily undergo purifying selection, there are notable instances of episodic diversifying selection. Specifically, the polymerase genes PB2, PB1, and PA exhibit significantly greater selection pressures in clade 2.3.4.4b than all earlier H5N1 virus clades. Polymerases play critical roles in influenza virus adaptation, including viral fitness, interspecies transmission, and virulence. Our findings provide evidence that significant selection pressures have shaped the evolution of the H5N1 clade 2.3.4.4b viruses, facilitating their expanded host tropism and the potential for further adaptation to mammalian hosts. We discuss how exogenous factors, such as altered bird migration patterns and increased host susceptibility, may have contributed to the expanded host range. As H5N1 viruses continue to infect new hosts, there is a greater risk of emergent novel variants with increased pathogenicity in humans and animals. Thus, comprehensive One Health surveillance is critical to monitor transmission among avian and mammalian hosts.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1–4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

The pathologic consequences of Coronavirus Disease-2019 (COVID-19) include elevated inflammation and dysregulated vascular functions associated with thrombosis. In general, disruption of vascular homeostasis and ensuing prothrombotic events are driven by activated platelets, monocytes, and macrophages, which form aggregates (thrombi) attached to the endothelium lining of vessel walls. However, molecular pathways underpinning the pathological interactions between myeloid cells and endothelium during COVID-19 remain undefined. Here, we tested the hypothesis that modulations in the expression of cellular receptors angiotensin-converting enzyme 2 (ACE2), CD147, and glucose-regulated protein 78 (GRP78), which are involved in homeostasis and endothelial performance, are the hallmark responses induced by SARS-CoV-2 infection. Cultured macrophages and lungs of hamster model systems were used to test this hypothesis. The results indicate that while macrophages and endothelial cells are less likely to support SARS-CoV-2 proliferation, these cells may readily respond to inflammatory stimuli generated by the infected lung epithelium. SARS-CoV-2 induced modulations of tested cellular receptors correlated with corresponding changes in the mRNA expression of coagulation cascade regulators and endothelial integrity components in infected hamster lungs. Among these markers, tissue factor (TF) had the best correlation for prothrombotic events during SARS-CoV-2 infection. Furthermore, the single-molecule fluorescence in situ hybridization (smFISH) method alone was sufficient to determine the peak and resolution phases of SARS-CoV-2 infection and enabled screening for cellular markers co-expressed with the virus. These findings suggest possible molecular pathways for exploration of novel drugs capable of blocking the prothrombotic shift events that exacerbate COVID-19 pathophysiology and control the disease.