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.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Introduction: Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. Methods: This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. Results: The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/µL, and both assays were completed in 30 min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. Discussion: The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

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.

2A2 protein of DHAV-1 induces duck embryo fibroblasts gasdermin E-mediated pyroptosis.

The duck hepatitis A virus type 1 (DHAV-1) causes rapid death in ducklings by triggering a severe cytokine storm. Pyroptosis is an inflammatory form of programmed cell death that is directly related to an increase in pro-inflammatory cytokine levels. Only a few studies have explored the mechanisms underlying pyroptosis in virus-infected avian cells. In this study, we established an avian infection model in vitro by infecting duck embryo fibroblasts (DEFs) with the virulent DHAV-1 LY0801 strain. DHAV-1 infection induced pyroptosis in the DEFs by activating gasdermin E (GSDME) protein via caspase-3-mediated cleavage. The genes encoding the different structural and non-structural DHAV-1 proteins were cloned into eukaryotic expression plasmids, and the 2A2 protein was identified as the key protein involved in pyroptosis. The HPLC-tandem mass spectrometry (HPLC-MS/MS) and co-immunoprecipitation (Co-IP) analysis established that DHAV-1 2A2 directly interacted with the mitochondrial anti-viral signaling protein (MAVS) both intracellularly and in vitro. Furthermore, we got the results that N-terminal 1-130 aa of 2A2 was involved in the interaction with MAVS and the C-terminal TM domain of MAVS is necessary for the interaction with 2A2 by Co-IP analysis. To our knowledge, this is the first study to reveal that DHAV-1 protein interacts with host proteins to induce pyroptosis. Our findings provide new insights into the molecular pathogenesis of DHAV-1 infection, and a scientific basis for the prevention and treatment of duck viral hepatitis.

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).

The role of duck LGP2 in innate immune response of host anti-tembusu virus.

Laboratory of Genetics and Physiology 2 (LGP2), along with Retinoic Acid Induced Gene-I (RIG-I) and Melanoma Differentiation Associated Gene 5, are members of the retinoic acid-inducible gene-I-like receptors (RLRs) in pattern recognition receptors, playing an important role in the host's innate immunity. Due to lacking a caspase activation and recruitment domain, LGP2 is controversially regarded as a positive or negative regulator in the antiviral response. This study aimed to explore how duck LGP2 (duLGP2) participates in duck innate immunity and its role in countering the duck Tembusu virus (DTMUV). In duck embryo fibroblast cells, the overexpression of duLGP2 significantly reduced the cell's antiviral capacity by inhibiting type I interferon (IFN) production and the expression of downstream IFN-stimulated genes. Conversely, duLGP2 knockdown had the opposite effect. For the first time, we introduced the LGP2 gene fragment into duck embryos using a lentiviral vector to ensure persistent expression and generated gene-edited ducks with LGP2 overexpression. We demonstrated that duLGP2 facilitates DTMUV replication in both in vitro and in vivo experiments, leading to robust inflammatory and antiviral responses. Interestingly, the repressive effects of duLGP2 on type I IFN production were only observed in the early stage of DTMUV infection, with type I IFN responses becoming enhanced as the viral load increased. These results indicate that duLGP2 acts as a negative regulator during the resting state and early stages of DTMUV infection. This study provides a theoretical basis for further research on duck RLRs and developing new anti-DTMUV drugs or vaccine adjuvants.

Alternative splicing of histone demethylase Kdm6bb mediates temperature-induced sex reversal in the Nile tilapia.

Sex determination in many fish species is remarkably plastic and temperature sensitive. Nile tilapia display a genetic sex-determination system (XX/XY). However, high-temperature treatment during critical thermosensitive periods can induce XX females into XXm pseudo-males, and this phenomenon is termed temperature-induced sex reversal (TISR). To investigate the molecular mechanism of TISR in Nile tilapia, we performed Iso-seq analysis and found a dramatic effect of high temperature on gene alternative splicing (AS). Kdm6bb histone demethylase showed a novel AS at intron 5 that generates Kdm6bb_tv1 transcripts without intron 5 and Kdm6bb_tv2 with intron 5. Kdm6bb_tv1 encodes a full-length protein while Kdm6bb_tv2 encodes a truncated protein. Expression analysis revealed that intron 5 splicing of Kdm6bb is male and gonad biased at larval stage, and only gonad biased at adult stage. High-temperature treatment induced intron 5 splicing in the gonads of XX and XY fish, resulting in increased Kdm6bb_tv1 expression. To directly test the role of Kdm6bb_tv1 in Nile tilapia TISR, we knocked out expression of Kdm6bb_tv1. However, Kdm6bb_tv1-/- homozygous mutants showed embryonic lethality. Overexpression of Kdm6bb_tv1, but not Kdm6bb_tv2, induced sex reversal of XX females into pseudo-males. Overexpression of Kdm6bb_tv1, as with high-temperature treatment, modified the promotor region of Gsdf and Dmrt1 by demethylating the trimethylated lysine 27 of histone 3 (H3K27me3), thereby increasing expression. Collectively, these studies demonstrate that AS of Kdm6bb intron 5 increases the expression of Kdm6bb_tv1, which acts as a direct link between high temperature and activation of Gsdf and Dmrt1 expression, leading to male sex determination.

Research Note: Genetic characterization of novel duck reoviruses from Shandong Province, China in 2022.

Since 2005, novel duck reoviruses have been outbreaks in duck breeding areas such as central China and South China. In recent years, the incidence rate of this disease is still increasing, bringing serious economic losses to waterfowl breeding industry. This study isolated 3 novel duck reoviruses (NDRV-SDLS, NDRV-SDWF, and NDRV-SDYC) from sick ducks in 3 local duck farms in Shandong Province. The study aimed to investigate the characteristics of these viruses. The virus is inoculated into duck embryo fibroblasts, where the virus replicates to produce syncytium and dies within 3 to 5 d. The viruses were also isolated from infected ducks, and RT-PCR amplified the whole genomes after passage purification in duck embryos. The resulting whole genome was analyzed for genetic evolution. The total length of the gene sequencing was 23,418 bp, divided into 10 fragments. Gene sequence comparison showed that the 3 strains had high similarity with novel duck reoviruses (NDRV) but low similarity with chicken-origin reovirus (chicken ARV) and Muscovy duck reovirus (MDRV), especially in the σC segment. Phylogenetic analysis of the 10 fragments showed that the 3 isolates constituted the same evolutionary clade as other DRV reference strains and were far related to ARV and MDRV in different evolutionary clades. The results of all 10 segments indicate that the isolates are in the evolutionary branch of NDRV, suggesting that the novel waterfowl reovirus is the dominant circulating strain in Shandong. This study complements the gene bank information of NDRV and provides references for vaccine research and disease prediction of NDRV in Shandong.

Stimulation of lipopolysaccharide from Pseudomonas aeruginosa following H9N2 IAV infection exacerbates inflammatory responses of alveolar macrophages and decreases virus replication.

H9N2 IAV infection contributed to P. aeruginosa coinfection, causing severe hemorrhagic pneumonia in mink. In this study, the in vitro alveolar macrophage models were developed to investigate the innate immune responses to P. aeruginosa LPS stimulation following H9N2 IAV infection, using MH-S cells. The cytokine levels, apoptosis levels and the viral nucleic acid levels were detected and analyzed. As a result, the levels of IFN-α, IL-1ß, TNF-α, and IL-10 in MH-S cells with P. aeruginosa LPS stimulation following H9N2 IAV infection were significantly higher than those in MH-S cells with single H9N2 IAV infection and single LPS stimulation (P < 0.05), exacerbating inflammatory responses. LPS stimulation aggravated the apoptosis of MH-S cells with H9N2 IAV infection. Interestingly, LPS stimulation influences H9N2 IAV replication and indirectly reduced H9N2 IAV replications in in vitro AMs. It implied that LPS should play an important role in the pathogenesis of H9N2 IAV and P. aeruginosa coinfection.

Research Note: Complete genome cloning and genetic evolution analysis of four Cherry Valley duck circovirus strains in China in 2022.

In recent years, with the expansion of duck breeding industry in China, the infection rate of duck circovirus (DuCV) in duck and the mixed infection rate of DuCV with other diseases increased significantly, which seriously endanger the development of duck breeding industry. To study the epidemic status of duck circovirus in China, analyze the virus's genetics and evolution, and establish a foundation for scientific prevention and control of duck circovirus, our laboratory collected 4 disease materials preliminarily diagnosed as duck circovirus infections. Conventional PCR was used to amplify 4 strains of duck circovirus with a full length of 1993bp, and their sequences were compared and analyzed. The analysis showed that the 4 DuCVs had typical circovirus characteristics, including 3 major ORFs: ORFV1 (Rep protein), ORFC1 (Cap protein), ORFC2 (apoptosis-related protein), and a stem ring structure. The 4 strains were compared with 22 other reference strains, and the results revealed that all 4 strains belonged to the DuCV-I type represented by the German strain AY228555. Furthermore, the homology between the 4 DuCVs and the reference strains was up to 98.6%, which help us to understand the genotype and genetic variation of DuCV in these regions and provide a reference for the prevention and control of DuCV.

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.

miR-155-1 as a positive factor for novel duck reovirus replication by regulating SOCS5-mediated interferons.

Diseases caused by novel duck reovirus (NDRV) have brought considerable economic losses to the poultry industry. MicroRNAs (miRNAs) have an impact on virus replication and antiviral immunity. However, the miRNA profile upon NDRV infection in duck embryo fibroblasts (DEFs) remains to be discovered. In this study, small RNA (sRNA) sequencing was performed to decipher the cellular miRNA response to NDRV infection. Based on 26 differentially expressed miRNAs (19 upregulated and 7 downregulated miRNAs) obtained from sequencing data and their target genes predicted by software, GO and KEGG analyses were performed to elucidate the functions of miRNAs in NDRV invasion, replication, and virus spread. "FoxO signaling pathway", "autophagy", and "Toll-like receptor signaling pathway" might participate in NDRV replication as revealed by KEGG enrichment analysis. The miR-155-1 sequence was found to be identical to rno-miR-155-5p and was sharply increased with the progression of NDRV infection. Moreover, NDRV-induced miR-155-1 could act as a positive factor for virus replication in DEFs, which inhibited type I interferon (IFN-I) production. Luciferase assay confirmed that miR-155-1 disturbed the abundance of suppressor of cytokine signaling (SOCS) 5 by targeting 3'-UTR. SOCS5, which is linked to increased IRF7 expression, restricts IFN expression and promotes NDRV replication in DEFs. Therefore, this study proposed that miR-155-1 was used by NDRV to restrict SOCS5 expression, attenuating the production of IFN-I and creating a favorable environment for virus replication.

Establishment of indirect ELISA method for Salmonella antibody detection from ducks based on PagN protein.

BACKGROUND: Salmonella as an important food-borne zoonotic bacterial pathogen, infection in ducks is a recessive infection, however, it can also cause high mortality and threat to food safety. Preventing and controlling the infection and transmission of Salmonella in ducks critically require rapid and sensitive detection method. Full-length Salmonella-specific protein PagN was induced and expressed in E.coil BL21 and was purified as an antigen to establish an indirect enzyme-linked immunosorbent assays (iELSA) detection kit. RESULTS: The recombinant PagN protein has a molecular weight of 43 kDa containing a His-tag, was recognized by an anti-Salmonella positive serum by Western blot assay. The optimal concentration of PagN as a coating antigen in the iELISA was 1 µg/mL, and the optimal dilution of enzyme-labeled secondary antibody was 1:4000 (0.025 µg/mL). The cutoff OD450 value was established at 0.268. The iELISA kit showed high selectivity since no cross-reaction with E. coli, Staphylococcus aureus and Streptococcus was observed. iELISA method and Dot-blot test were performed on 100 clinical sera samples collected from duck farms, and the actual coincidence rate was 89% (89/100). 613 duck serum samples from 3 different farms were tested using established method and commercial ELISA kit. The concordance between the two methods was 94.1%. CONCLUSION: Anti-PagN based iELISA can serve as a useful tool for diagnosis of Salmonella infection.

Epidemiological investigation on drug resistance of Salmonella isolates from duck breeding farms in Shandong Province and surrounding areas, China.

Duck salmonellosis is a common acute septic infectious disease that spreads rapidly, with serious harm to the duck breeding industry and public health. To date, there are few reports about the epidemiological characteristics of drug resistance in Salmonella from ducks. In this study, an epidemiological investigation was conducted on drug resistance of 110 Salmonella strains isolated from multiple duck farms in Shandong Province and surrounding areas, China. The multidrug-resistant (MDR) rate for 110 Salmonella strains was up to 71.82% (79/110), and 12 types of drug resistance genes were detected in all isolates, including ß-lactams, aminoglycosides, tetracycline, macrolides, and quinolones resistance genes. Using the multilocus sequence typing (MLST) based on 7 housekeeping genes, 13 various ST types were identified among all strains, and ST19 (32/110, 29.09%) was the primary type. As the dominant serotypes, S. Kottbus and S. Typhimurium were divided into multiple ST types. A total of 6 kinds of plasmid incompatibility groups were carried in the Salmonella strains, of which IncFIIs (29/110, 26.36%) was most prevalent, and the class I integrons were detected in 78.18% (86/110) of strains. Furthermore, we found that some drug resistance genes, plasmid incompatibility groups, and class I integrons coexist in the same strain. This phenomenon indicates that class I integrons and plasmids are important ways for the spread of drug resistance genes. Therefore, the spread of antibiotic resistance in Salmonella had been facilitated, especially erythromycin (108/110, 98.18%), streptomycin (93/110, 84.54%), and tetracycline (53/110, 48.18%). The above research results broadened ideas and provided directions for the transmission mechanism of Salmonella resistance.

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.

Occurrence and molecular epidemiology of fosA3-bearing Escherichia coli from ducks in Shandong province of China.

The plasmid-borne fosfomycin resistance gene fosA3 has been identified in Escherichia coli (E. coli) from various animals but has rarely been reported in ducks. In this study, we investigated the fosA3 prevalence and molecular characteristics of fosA3-harboring E. coli strains from ducks in Shandong province of China. In 416 E. coli isolates, 91 (21.88%) were identified as fosA3-bearing strains, and the fosfomycin-resistant phenotype of 88 of the 91 fosA3-harboring strains was successfully transferred to the recipient strains. Seven different genetic structures surrounding the fosA3 gene were detected and 2 new contexts were discovered among the fosA3-carrying E. coli. Twenty fosA3-harboring isolates and their trans-conjugants were randomly selected for pulsed-field gel electrophoresis (PFGE) typing and S1-nuclease PFGE, respectively. The PFGE patterns revealed that the 20 randomly selected fosA3-bearing isolates were not a result of clonal dissemination. S1-PFGE showed that 15 of the 20 randomly selected trans-conjugants carried a single plasmid, and these 15 plasmids that harbored fosA3 (55-190 kb) were distributed into the following replicon types: IncF (n = 11), IncI1 (n = 1), IncN (n = 1), untypable (n = 1), and W-FIC (n = 1). Additionally, as vectors for fosA3 in E. coli, F-:A1:B6, N/ST1, IncI1/ST2, W-FIC, and one untypable plasmid had never been reported before. These observations highlighted the importance of ducks as a reservoir for multidrug-resistant fosA3-carrying E. coli.

Klebsiella pneumoniae infection following H9N2 influenza A virus infection contributes to the development of pneumonia in mice.

In this study, whether H9N2 influenza A virus (IAV) infection contributed to secondary Klebsiella pneumoniae infection was investigated. From post-infection onwards, clinical symptoms were monitored, examined and recorded daily for 11 days. As a result, no clinical signs were observed in the mice infected with single H9N2 IAV, implying that H9N2 IAV was less pathogenic to mice. Compared to single K. pneumonia infection, K. pneumoniae infection following H9N2 IAV infection exacerbates lung histopathological lesions and apoptosis, resulting in more severe diseases. Lung index of the mice with H9N2 IAV and K. pneumoniae co-infection was significantly higher than those in the other groups. Bacterial loads in the tissues in H9N2 IAV and K. pneumoniae co-infection group were significantly higher than those in the single K. pneumoniae infection group at 7 dpi. It demonstrated that prior H9N2 IAV infection contributed to K. pneumonia proliferation and delayed bacterial clearance in mice. Secondary K. pneumoniae infection influences seroconversion of anti-H9N2 antibody titers and the cytokine profiles. The findings demonstrated that H9N2 IAV infection facilitated secondary K. pneumonia infection, causing severe the diseases in mice.

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.

Nucleotide sequences of the infectious DNA clones of two mink enteritis virus isolates exhibit the diversity of the terminal palindromic sequences and predicted secondary structures.

In this study, the infectious RF-DNA clones of two mink enteritis viruses, MEV-SD1 and MEV-SD7, were constructed, which generated progeny virions and seemed to contain an almost or completely full-length genome. The genomes of MEV-SD1 and MEV-SD7 were 5162 bp and 5113 bp in length, respectively. The genomic organizations of MEV-SD1 and MEV-SD7 were similar to that of the other carnivore parvoviruses. The 3'-UTR of the virion strand of MEV-SD1 and MEV-SD7 were 311 bp and 313 bp in length, respectively, containing a 208 bp palindromic sequence assuming Y-shaped configurations. Interestingly, the difference of the 3' palindromic sequences between MEV-SD1 and MEV-SD7 resulted in the orientation inversion of the hairpin ears. And the 5'-UTRs of MEV-SD1 and MEV-SD7 were 582 bp and 531 bp, respectively, containing a 198 bp palindromic sequence assuming U-shaped configurations, a triplet mismatch (5'-TAC-3') in the center of the duplex stem and a triplet mismatch (5'-AGA-3') forming a small asymmetric bubble. The findings demonstrated that the genomic termini of the carnivore parvoviruses showed the diversity in length, base composition, and predicted secondary structure.