Evidence for cross-species transmission of human coronavirus OC43 through bioinformatics and modeling infections in porcine intestinal organoids.

Cross-species transmission of coronaviruses has been continuously posing a major challenge to public health. Pigs, as the major animal reservoirs for many zoonotic viruses, frequently mediate viral transmission to humans. This study comprehensively mapped the relationship between human and porcine coronaviruses through in-depth bioinformatics analysis. We found that human coronavirus OC43 and porcine coronavirus PHEV share a close phylogenetic relationship, evidenced by high genomic homology, similar codon usage patterns and comparable tertiary structure in spike proteins. Inoculation of infectious OC43 viruses in organoids derived from porcine small and large intestine demonstrated that porcine intestinal organoids (pIOs) are highly susceptible to human coronavirus OC43 infection and support infectious virus production. Using transmission electron microscopy, we visualized OC43 viral particles in both intracellular and extracellular compartments, and observed abnormalities of multiple organelles in infected organoid cells. Robust OC43 infections in pIOs result in a significant reduction of organoids viability and widespread cell death. This study bears essential implications for better understanding the evolutionary origin of human coronavirus OC43, and provides a proof-of-concept for using pIOs as a model to investigate cross-species transmission of human coronavirus.

Coagulase-negative staphylococci are the main causes of bacterial meningitis in duck.

Since September 2018, serious meningitis has been found on some breeding-duck farms in Shandong Province, China. A large number of ducks exhibit severe neurological symptoms. The ducks were randomly selected for laboratory testing. Duck brain samples were collected using standard sterile techniques, and the staphylococci isolates were detected in 404 (70.14%) out of 576 brain samples. A total of 525 coagulase-negative staphylococci (CoNS) strains were isolated, including 6 species: Staphylococcus sciuri (S. sciuri) (67.24%, 353/525), Staphylococcus epidermidis (S. epidermidis) (9.71%, 51/525), Staphylococcus saprophyticus (S. saprophyticus) (8.38%, 44/525), Staphylococcus lentus (S. lentus) (7.62%, 40/525), Staphylococcus haemolyticus (S. haemolyticus) (2.48%, 13/525), and Staphylococcus xylosus (S. xylosus) (4.57%, 24/525). Mixed strain infections were detected in 121 (29.95%) infected presentations. The antimicrobial susceptibility testing indicated that 40.38% of the isolates exhibited multi-drug resistance, and 53.90% of the strains were methicillin-resistant strains by amplification of the methicillin resistance gene (mecA) gene. Through experimental reproduction of the disease, we determined that the CoNS strains were the leading pathogens causing bacterial meningitis in ducks. Although these CoNS strains does not directly cause the death of sick ducks, they still cause large economic losses due to the retarded growth and development of the sick ducks, lower feed returns, and lower grades of processed duck products. The results of this study will contribute to our understanding of the epidemiology and pathogenesis of CoNS and be helpful in the prevention and treatment of the infection.

Duck hepatitis A virus type 1 infection induces hepatic metabolite and gut microbiota changes in ducklings.

Duck hepatitis A virus type 1 (DHAV-1) can cause severe liver damage in infected ducklings and is a fatal and contagious pathogen that endangers the Chinese duck industry. The objective of this study was to explore the correlation mechanism of liver metabolism-gut microbiota in DHAV-1 infection. Briefly, liquid chromatography-mass spectrometry and 16S rDNA sequencing combined with multivariate statistical analysis were used to evaluate the effects of DHAV-1 infection on liver metabolism, gut microbiota regulation, and other potential mechanisms in ducklings. In DHAV-1-infected ducklings at 72 h postinfection, changes were found in metabolites associated with key metabolic pathways such as lipid metabolism, sugar metabolism, and nucleotide metabolism, which participated in signaling networks and ultimately affecting the function of the liver. The abundance and composition of gut microbiota were also changed, and gut microbiota is significantly involved in lipid metabolism in the liver. The evident correlation between gut microbiota and liver metabolites indicates that DHAV-host gut microbiome interactions play important roles in the development of duck viral hepatitis (DVH).

Mpox virus infection and drug treatment modelled in human skin organoids.

Mpox virus (MPXV) primarily infects human skin to cause lesions. Currently, robust models that recapitulate skin infection by MPXV are lacking. Here we demonstrate that human induced pluripotent stem cell-derived skin organoids are susceptible to MPXV infection and support infectious virus production. Keratinocytes, the predominant cell type of the skin epithelium, effectively support MPXV infection. Using transmission electron microscopy, we visualized the four stages of intracellular virus particle assembly: crescent formation, immature virions, mature virions and wrapped virions. Transcriptional analysis showed that MPXV infection rewires the host transcriptome and triggers abundant expression of viral transcripts. Early treatment with the antiviral drug tecovirimat effectively inhibits infectious virus production and prevents host transcriptome rewiring. Delayed treatment with tecovirimat also inhibits infectious MPXV particle production, albeit to a lesser extent. This study establishes human skin organoids as a robust experimental model for studying MPXV infection, mapping virus-host interactions and testing therapeutics.

Nonstructural protein 2A2 from Duck hepatitis A virus type 1 inhibits interferon beta production by interaction with mitochondrial antiviral signaling protein and TANK-binding kinase 1.

Type I interferon (IFN-I) is essential for the regulation of host-virus interactions, and viruses have evolved strategies to escape the host immune response. Duck hepatitis A virus type 1 (DHAV-1) causes severe liver necrosis and hemorrhage, neurological symptoms, and high mortality in ducklings. However, how DHAV-1 interacts with the duck innate immune system remains unclear. In this study, DHAV-1-encoded proteins were cloned, and DHAV-1 2A2 was shown to strongly suppress IFN-ß-luciferase activity, triggered by Sendai virus and polyriboinosinic polyribocytidylic acid [poly(I:C)], along with the transcription of IFN-ß and downstream antiviral genes, including OASL, PKR, and TNF-a. In addition, 2A2 interacts with the central adaptor proteins mitochondrial antiviral signaling (MAVS) and TANK-binding kinase 1 (TBK1) by its N-terminal 1-100 amino acids (aa), thus leading to the inhibition of IFN-ß production. Importantly, the deletion of the N-terminal 1-100 aa region of 2A2 abolished inhibition of IFN-I production. Moreover, the transmembrane domain of the MAVS protein and the ubiquitin domain of TBK1 were demonstrated to be required for interaction with DHAV-1 2A2. These findings revealed a novel strategy by which DHAV-1 hijacks cellular immunosurveillance and provided new insights into controlling the disease.

Duck hepatitis A virus type 1 transmission by exosomes establishes a productive infection in vivo and in vitro.

Duck hepatitis A virus type 1 (DHAV-1) infection causes an acute and highly fatal disease in young ducklings. Exosomes are nano-sized small extracellular vesicles secreted by various cells, which participate in intercellular communication and play a key role in the physiological and pathological processes. However, the role of exosomes in DHAV-1 transmission remains unknown. In this study, through RT-PCR, WB analysis and TEM observation, the complete DHAV-1 genomic RNA, partial viral proteins, and virions were respectively identified in the exosomes derived from DHAV-1-infected duck embryo fibroblasts (DEFs). The productive DHAV-1 infection was transmitted by exosomes in DEFs, duck embryos, and ducklings, and high titers of neutralizing antibodies completely blocked DHAV-1 infection but did not significantly neutralize exosome-mediated DHAV-1 infection. To the best of our knowledge, this is the first report that exosome-mediated DHAV-1 infection was resistant to antibody neutralization in vivo and in vitro, which might be an immune evasion mechanism of DHAV-1.

Long Noncoding RNA Expression Rofiles Elucidate the Potential Roles of lncRNA- XR_003496198 in Duck Hepatitis A Virus Type 1 Infection.

Duck hepatitis A virus type 1 (DHAV-1) is a highly lethal virus that severely affects the duck industry worldwide. Long noncoding RNAs (lncRNAs) exert crucial roles in pathogen attacks. Here, we conducted deep transcriptome analysis to investigate the dynamic changes of host lncRNAs profiles in DHAV-1-infected duck embryo fibroblasts. We identified 16,589 lncRNAs in total and characterized their genomic features. Moreover, 772 and 616 differentially expressed lncRNAs (DELs) were screened at 12 and 24 h post-infection. Additionally, we predicted the DELs' cis- and trans-target genes and constructed lncRNA-target genes regulatory networks. Functional annotation analyses indicated that the putative target genes of DELs participated in diverse vital biological processed, including immune responses, cellular metabolism, and autophagy. For example, we confirmed the dysregulation of pattern recognition receptors (TLR3, RIG-I, MDA5, LGP2, cGAS), signal transducers (STAT1), transcription factors (IRF7), immune response mediators (IL6, IL10, TRIM25, TRIM35, TRIM60, IFITM1, IFITM3, IFITM5), and autophagy-related genes (ULK1, ULK2, EIF4EBP2) using RT-qPCR. Finally, we confirmed that one DHAV-1 induced lncRNA-XR_003496198 is likely to inhibit DHAV-1 replication in DEFs. Our study comprehensively analyzed the lncRNA profiles upon DHAV-1 infection and screened the target genes involved in the innate immune response and autophagy signaling pathway, thereby revealing the essential roles of duck lncRNAs and broadening our understanding of host-virus interactions.

Construction of Recombinant Lactococcus lactis Strain Expressing VP1 Fusion Protein of Duck Hepatitis A Virus Type 1 and Evaluation of Its Immune Effect.

With the continuous development of duck farming and the increasing breeding density, the incidence of duck hepatitis A virus type 1 (DHAV-1) has been on the rise, seriously endangering the development of duck farming. To reduce the use of antibiotics in duck breeding, susceptibility risks and mortality, and avoid virulence recovery and immune failure risk, this study aims to develop a new type of mucosal immune probiotics and make full use of molecular biology techniques, on the level of genetic engineering, to modify Lactococcus lactis (L. lactis). In this study, a secretory recombinant L. lactis named MG1363-VP1 with an enhanced Green Fluorescent Protein (eGFP) and translation enhancer T7g10L was constructed, which could express the VP1-eGFP fusion protein of DHAV-1. The animal experiment in ducklings was performed to detect the immune response and protection effect of oral microecologics by recombinant L. lactis. The results showed that oral L. lactis MG1363-VP1 significantly induced the body's humoral immune system and mucosal immune system to produce specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) for DHAV-1 in ducklings, and cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon gamma (IFN-γ). The mortality rate was monitored simultaneously by the natural infestation in the process of production and breeding; notably, the ducklings vaccinated with L. lactis MG1363-VP1 were effectively protected against the nature infection of DHAV-1. The recombinant L. lactis MG1363-VP1 constructed in this study provides a new means of preventing and controlling DHAV-1 infection in the future.

Isolation and characterization of a naturally attenuated novel duck reovirus strain as a live vaccine candidate.

Novel duck reovirus (NDRV) causes high morbidity in ducklings, and recovered ducklings are often remarkably stunted in growth. In this study, four NDRV strains were isolated from the NDRV outbreaks that occurred in different regions of Shandong province, China. The biological characteristics and pathogenicity of the four NDRV strains were elucidated, and the N20 was identified as a naturally attenuated strain. Three-day-old ducklings were immunized with live N20 strain (100 ELD50/duck), and challenged with 104.52 ELD50 of virulent N19 strain at 7 days post immunization. The vaccinated ducklings showed no evidence of clinical signs, gross and histopathological lesions, or loss of body weight, and 100 % protection against the virulent NDRV N19 infection. The NDRV-specific antibodies were generated in the immunized ducklings and could neutralize different NDRV strains. These results indicated that the N20 strain was a promising live attenuated vaccine candidate against highly pathogenic NDRV infection.

Integrated miRNA and mRNA Expression Profiles Reveal Differentially Expressed miR-222a as an Antiviral Factor Against Duck Hepatitis A Virus Type 1 Infection.

Duck hepatitis A virus 1 (DHAV-1) is a highly contagious etiological agent that causes acute hepatitis in young ducklings. MicroRNAs (miRNAs) play important regulatory roles in response to pathogens. However, the interplay between DHAV-1 infection and miRNAs remains ambiguous. We characterized and compared miRNA and mRNA expression profiles in duck embryo fibroblasts cells (DEFs) infected with DHAV-1. In total, 36 and 96 differentially expressed (DE) miRNAs, and 4110 and 2595 DE mRNAs, were identified at 12 and 24 h after infection. In particular, 126 and 275 miRNA-mRNA pairs with a negative correlation were chosen to construct an interaction network. Subsequently, we identified the functional annotation of DE mRNAs and target genes of DE miRNAs enriched in diverse Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may be important for virus resistance, cell proliferation, and metabolism. Moreover, upregulated miR-222a could negatively regulate DHAV-1 replication in DEFs and downregulate integrin subunit beta 3 (ITGB3) expression by targeting the 3' untranslated region (3'UTR), indicating that miR-222a may modulate DHAV-1 replication via interaction with ITGB3. In conclusion, the results reveal changes of mRNAs and miRNAs during DHAV-1 infection and suggest miR-222a as an antiviral factor against DHAV-1.