BACKGROUND: As a result of antigenic drift, current influenza vaccines provide limited protection against circulating influenza viruses, and vaccines with broad cross protection are urgently needed. Hemagglutinin stalk domain and ectodomain of matrix protein 2 are highly conserved among influenza viruses and have great potential for use as a universal vaccine. METHODS: In this study, we co-expressed the stalk domain and M2e on the surface of cell membranes and generated chimeric and standard virus-like particles of influenza to improve antigen immunogenicity. We subsequently immunized BALB/c mice through intranasal and intramuscular routes. RESULTS: Data obtained demonstrated that vaccination with VLPs elicited high levels of serum-specific IgG (approximately 30-fold higher than that obtained with soluble protein), induced increased ADCC activity to the influenza virus, and enhanced T cell as well as mucosal immune responses. Furthermore, mice immunized by VLP had elevated level of mucosal HA and 4M2e specific IgA titers and cytokine production as compared to mice immunized with soluble protein. Additionally, the VLP-immunized group exhibited long-lasting humoral antibody responses and effectively reduced lung viral titers after the challenge. Compared to the 4M2e-VLP and mHA-VLP groups, the chimeric VLP group experienced cross-protection against the lethal challenge with homologous and heterologous viruses. The stalk domain specific antibody conferred better protection than the 4M2e specific antibody. CONCLUSION: Our findings demonstrated that the chimeric VLPs anchored with the stalk domain and M2e showed efficacy in reducing viral loads after the influenza virus challenge in the mice model. This antibody can be used in humans to broadly protect against a variety of influenza virus subtypes. The chimeric VLPs represent a novel approach to increase antigen immunogenicity and are promising candidates for a universal influenza vaccine.

Since December 2019, various types of strategies have been applied due to the emergent need to investigate the biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to discover a functional treatment. Different disease modeling systems, such as mini-organ technology, have been used to improve our understanding of SARS-CoV-2 physiology and pathology. During the past 2 years, regenerative medicine research has shown the supportive role of organoid modeling in controlling coronavirus disease 2019 (COVID-19) through optimal drug and therapeutic approach improvement. Here, we overview some efforts that have been made to study SARS-CoV-2 by mimicking COVID-19 using stem cells. In addition, we summarize a perspective of drug development in COVID-19 treatment via organoid-based studies.

BACKGROUND: Variation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses that was first documented in an infectious isolate of the betacoronavirus SARS-CoV-2, obtained from 3 patients in Hong Kong that had a 5'-untranslated region segment at the end of the ORF6 gene that in its new location translated into an ORF6 protein with a predicted modified carboxyl terminus. While comparing the amino acid sequences of translated ORF8 genes in the GenBank database, we found a subsegment of the same 5'-UTR-derived amino acid sequence modifying the distal end of ORF8 of an isolate from the United States and decided to carry out a systematic search. METHODS: Using the nucleotide and in the case of SARS-CoV-2 also the translated amino acid sequence in three reading frames of the genomic termini of coronaviruses as query sequences, we searched for 5'-UTR sequences in regions other than the 5'-UTR in SARS-CoV-2 and reference strains of alpha-, beta-, gamma-, and delta-coronaviruses. RESULTS: We here report numerous genomic insertions of 5'-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, but not delta- or gammacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions would change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain inserted 5'-UTR sequences. CONCLUSION: The intragenomic rearrangements involving 5'-untranslated region sequences described here, which in several cases affect highly conserved genes with a low propensity for recombination, may underlie the generation of variants homotypic with those of concern or interest and with potentially differing pathogenic profiles. Intragenomic rearrangements thus add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

BACKGROUND: The COVID-19 pandemic highlighted the importance of diagnostic testing against curbing the spread of SARS-CoV-2. The urgent need and scale for diagnostic tools resulted in manufacturers of SARS-CoV-2 assays receiving emergency authorization that lacked robust analytical or clinical evaluation. As it is highly likely that testing for SARS-CoV-2 will continue to play a central role in public health, the performance characteristics of assays should be evaluated to ensure reliable diagnostic outcomes are achieved. METHODS: VALCOR or "VALidation of SARS-CORona Virus-2 assays" is a study protocol designed to set up a framework for test validation of SARS-CoV-2 virus assays. Using clinical samples collated from VALCOR, the performance of Aptima SARS-CoV-2 assay was assessed against a standard comparator assay. Diagnostic test parameters such as sensitivity, specificity and overall per cent agreement were calculated for the clinical performance of Aptima SARS-CoV-2 assay. RESULTS: A total of 180 clinical samples were tested with an addition of 40 diluted clinical specimens to determine the limit of detection. When compared to the standard comparator assay Aptima had a sensitivity of 100.0% [95% CI 95.9-100.0] and specificity of 96.7% [95% CI 90.8-99.3]. The overall percent agreement was 98.3% with an excellent Cohen's coefficient of κ = 0.967 [95% CI 0.929-1.000]. For the limit of detection, Aptima was able to detect all of the diluted clinical samples. CONCLUSION: In conclusion. validation of Aptima SARS-CoV-2 assay using clinical samples collated through the VALCOR protocol showed excellent test performance. Additionally, Aptima demonstrated high analytical sensitivity by detecting all diluted clinical samples corresponding to a low limit of detection.

BACKGROUND: Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is an important type of brain inflammation caused by autoantibody. As one of the primary agents responsible for respiratory tract infection, the human respiratory syncytial virus (hRSV) has also been reported to be capable of causing extrapulmonary diseases. Here, we first describe a case of anti-NMDAR encephalitis when hRSV was shown to be present in the cerebrospinal fluid. CASE PRESENTATION: The child was noted to have ataxia and positive anti-NMDA receptors in the cerebrospinal fluid, diagnosed as anti-NMDA receptor encephalitis in combination with cranial MRI images. After high-dose hormone pulse therapy and medication, the disease improved, and he was discharged. However, a relapse occurred almost a year later, and the cranial MRI imaging showed progressive cerebellar atrophy. An hRSV strain from group B was detected in his cerebrospinal fluid, and the whole genome sequence was recovered using transcriptome sequencing. CONCLUSIONS: To our knowledge, this is the first report of hRSV being found in the cerebrospinal fluid of a patient with anti-NMDAR encephalitis. Even though more clinical records and experimental evidence are needed for validation, this work expands the types of diseases linked to hRSV and the likely cause of anti-NMDAR encephalitis.

BACKGROUND: Neutralizing anti-interferon (IFN)-γ autoantibodies are linked to adult-onset immunodeficiency and opportunistic infections. METHODS: To explore whether anti-IFN-γ autoantibodies are associated with disease severity of coronavirus disease 2019 (COVID-19), we examined the titers and functional neutralization of anti-IFN-γ autoantibodies in COVID-19 patients. In 127 COVID-19 patients and 22 healthy controls, serum titers of anti-IFN-γ autoantibodies were quantified using enzyme-linked immunosorbent assay, and the presence of autoantibodies was verified with immunoblotting assay. The neutralizing capacity against IFN-γ was evaluated with flow cytometry analysis and immunoblotting, and serum cytokines levels were determined using the MULTIPLEX platform. RESULTS: A higher proportion of severe/critical COVID-19 patients had positivity for anti-IFN-γ autoantibodies (18.0%) compared with non-severe patients (3.4%, p < 0.01) or healthy control (HC) (0.0%, p < 0.05). Severe/critical COVID-19 patients also had higher median titers of anti-IFN-γ autoantibodies (5.01) compared with non-severe patients (1.33) or HC (0.44). The immunoblotting assay could verify the detectable anti-IFN-γ autoantibodies and revealed more effective inhibition of signal transducer and activator of transcription (STAT1) phosphorylation on THP-1 cells treated with serum samples from anti-IFN-γ autoantibodies-positive patients compared with those from HC (2.21 ± 0.33 versus 4.47 ± 1.64, p < 0.05). In flow-cytometry analysis, sera from autoantibodies-positive patients could also significantly more effectively suppress the STAT1 phosphorylation (median,67.28%, interquartile range [IQR] 55.2-78.0%) compared with serum from HC (median,106.7%, IQR 100.0-117.8%, p < 0.05) or autoantibodies-negative patients (median,105.9%, IQR 85.5-116.3%, p < 0.05). Multivariate analysis revealed that the positivity and titers of anti-IFN-γ autoantibodies were significant predictors of severe/critical COVID-19. Compared with non-severe COVID-19 patients, we reveal that a significantly higher proportion of severe/critical COVID-19 patients are positive for anti-IFN-γ autoantibodies with neutralizing capacity. CONCLUSION: Our results would add COVID-19 to the list of diseases with the presence of neutralizing anti-IFN-γ autoAbs. Anti-IFN-γ autoantibodies positivity is a potential predictor of severe/critical COVID-19.

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

BACKGROUND: IL-17A is a pleiotropic cytokine and intimately associated with asthma, but its role in respiratory syncytial virus (RSV) infection is conflicting in the literature. METHODS: Children hospitalized in the respiratory department with RSV infection during RSV pandemic season of 2018-2020 were included. Nasopharyngeal aspirates were collected for pathogen and cytokines determination. In the murine model, RSV intranasal administrations were performed in wild-type and IL-17A-/- mice. Leukocytes and cytokines in bronchoalveolar lavage fluid (BALF), lung histopathology, and airway hyperresponsiveness (AHR) were measured. RORγt mRNA and IL-23R mRNA were semi-quantified by qPCR. RESULTS: IL-17A increased significantly in RSV-infected children and was positively associated with pneumonia severity. In the murine model, IL-17A significantly increased in BALF of mice with RSV infection. Airway inflammation, lung tissue damage and AHR were significantly alleviated in wild-type mice following IL-17A neutralization and in the IL-17A-/- mice. IL-17A decreased by removing CD4+ T cells but increased by depleting CD8+ T cells. IL-6, IL-21, RORγt mRNA and IL-23R mRNA dramatically increased in parallel with the rise of IL-17A. CONCLUSIONS: IL-17A contributes to the airway dysfunctions induced by RSV in children and murine. CD3+CD4+T cells are its major cellular sources and the IL-6/IL-21-IL-23R-RORγt signaling pathway might participate in its regulation.