Outbreak of Natural Severe Fever with Thrombocytopenia Syndrome Virus Infection in Farmed Minks, China.

We isolated severe fever with thrombocytopenia syndrome virus (SFTSV) from farmed minks in China, providing evidence of natural SFTSV infection in farmed minks. Our findings support the potential role of farmed minks in maintaining SFTSV and are helpful for the development of public health interventions to reduce human infection.

Enhancing dendrobine production in Dendrobium nobile through mono-culturing of endophytic fungi, Trichoderma longibrachiatum (MD33) in a temporary immersion bioreactor system.

Introduction: Dendrobine, a valuable alkaloid found in Dendrobium nobile, possesses significant pharmaceutical potential. Methods: In this study, we explored innovative approaches to enhance dendrobine production by utilizing endophytic fungi in a Temporary Immersion Bioreactor System (TIBS, Nanjing BioFunction Co. Ltd., China) and traditional test bottles. Dendrobine was unequivocally identified and characterised in D. nobile co-culture seedlings through UHPLC analysis and LC-MS qTOF analysis, supported by reference standards. Results: The CGTB (control group) and EGTB (experimental group) 12-month-old D. nobile seedlings exhibited similar peak retention times at 7.6±0.1 minutes, with dendrobine identified as C16H25NO2 (molecular weight 264.195). The EGTB, co-cultured with Trichoderma longibrachiatum (MD33), displayed a 2.6-fold dendrobine increase (1804.23 ng/ml) compared to the CGTB (685.95 ng/ml). Furthermore, a bioanalytical approach was applied to investigate the mono-culture of T. longibrachiatum MD33 with or without D. nobile seedlings in test bottles. The newly developed UHPLC-MS method allowed for dendrobine identification at a retention time of 7.6±0.1 minutes for control and 7.6±0.1 minutes for co-culture. Additionally, we explored TIBS to enhance dendrobine production. Co-culturing D. nobile seedlings with Trichoderma longibrachiatum (MD33) in the TIBS system led to a substantial 9.7-fold dendrobine increase (4415.77 ng/ml) compared to the control (454.01 ng/ml) after just 7 days. The comparative analysis of dendrobine concentration between EGTB and EGTIBS highlighted the remarkable potential of TIBS for optimizing dendrobine production. Future research may focus on scaling up the TIBS approach for commercial dendrobine production and investigating the underlying mechanisms for enhanced dendrobine biosynthesis in D. nobile. The structural elucidation of dendrobine was achieved through 1H and 13C NMR spectroscopy, revealing a complex array of proton environments and distinct carbon environments, providing essential insights for the comprehensive characterization of the compound. Discussion: These findings hold promise for pharmaceutical and industrial applications of dendrobine and underline the role of endophytic fungi in enhancing secondary metabolite production in medicinal plants.

Classification of genotypes based on the VP1 gene of feline calicivirus and study of cross-protection between different genotypes.

Feline calicivirus (FCV) causes upper respiratory tract diseases and even death in cats, thereby acting as a great threat to feline animals. Currently, FCV prevention is mainly achieved through vaccination, but the effectiveness of vaccination is limited. In this study, 105 FCV strain VP1 sequences with clear backgrounds were downloaded from the NCBI and subjected to a maximum likelihood method for systematic evolutionary analysis. Based on the genetic analysis results, FCV-positive sera were prepared using SPF mice and Chinese field cats as target animals, followed by a cross-neutralization assay conducted on the different genotype strains and in vivo challenge tests were carried out to further verify with the strain with best cross-protection effect. The results revealed that FCV was mainly divided into two genotypes: GI and GII. The GI genotype strains are prevalent worldwide, but all GII genotype strains were isolated from Asia, indicating a clear geographical feature. This may form resistance to FCV prevention in Asia. The in vitro neutralization assay conducted using murine serum demonstrated that the cross-protection effect varied among strains. A strain with broad-spectrum neutralization properties, DL39, was screened. This strain could produce neutralizing titers (10 × 23.08-10 × 20.25) against all strains used in this study. The antibody titers against the GI strains were 10 × 23.08-10 × 20.5 and those against the GII strains were 10 × 20.75-10 × 20.25. Preliminary evidence suggested that the antibody titer of the DL39 strain against GI was higher than that against GII. Subsequent cross-neutralization assays with cat serum prepared with the DL39 strain and each strain simultaneously yielded results similar to those described above. In vivo challenge tests revealed that the DL39 strain-immunized cats outperformed the positive controls in all measures. The results of several trials demonstrated that strain DL39 can potentially be used as a vaccine strain. The study attempted to combine the genetic diversity and phylogenetic analysis of FCV with the discovery of potential vaccines, which is crucial for developing highly effective FCV vaccines.

Pmorf0222, a Virulence Factor in Pasteurella multocida, Activates Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase via Toll-Like Receptor 1/2.

Pasteurella multocida primarily causes hemorrhagic septicemia and pneumonia in poultry and livestock. Identification of the relevant virulence factors is therefore essential for understanding its pathogenicity. Pmorf0222, encoding the PM0222 protein, is located on a specific prophage island of the pathogenic strain C48-1 of P. multocida. Its role in the pathogenesis of P. multocida infection is still unknown. The proinflammatory cytokine plays an important role in P. multocida infection; therefore, murine peritoneal exudate macrophages were treated with the purified recombinant PM0222, which induced the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) via the Toll-like receptor 1/2 (TLR1/2)-nuclear factor kappa B (NF-κB)/mitogen-activated protein kinase (MAPK) signaling and inflammasome activation. Additionally, the mutant strain and complemented strain were evaluated in the mouse model with P. multocida infection, and PM0222 was identified as a virulence factor, which was secreted by outer membrane vesicles of P. multocida. Further results revealed that Pmorf0222 affected the synthesis of the capsule, adhesion, serum sensitivity, and biofilm formation. Thus, we identified Pmorf0222 as a novel virulence factor in the C48-1 strain of P. multocida, explaining the high pathogenicity of this pathogenic strain.

Isolation, identification and pathogenic characteristics of tick-derived parainfluenza virus 5 in northeast China.

The number of parainfluenza virus 5 (PIV5) infection cases has increased worldwide over the past six decades; however, factors underlying this increase remain unclear. PIV5 has been emerging or re-emerging in humans and animal species. To date, no information is yet available regarding PIV5 infection in arthropod ticks. Here, we successfully isolated tick-derived PIV5 from the Ixodes persulcatus species designated as HLJ/Tick/2019 in Heilongjiang, China. Phylogenetic analysis revealed that the tick-derived PIV5 is closely related to subclade 2.2.6, which has become the dominant subtype prevalent in dogs, pigs and wildlife across China. Further experiments to understand the importance of this virus as an infectious vector revealed that a ferret animal model experimentally infected with Tick/HLJ/2019 via the oronasal and ocular inoculation routes developed moderate respiratory distress with pneumonia and neurologic tissue damage from inflammation for the first time. Further surveillance of PIV5 in vectors of viral transmission is necessary to enhance our knowledge of its ecology in reservoirs and facilitate the control of re-emerging diseases.

Development of a method for the isolation and culture of astrocytes from the canine cerebral cortex.

BACKGROUND: Astrocytes are considered key players in neuroimmunopathological processes, and they play a certain role in neuroinflammation. Rodent primary astrocyte cultures are commonly used in the study of human neuroinflammation. However, gene sequence homologies are closer between humans and dogs than between humans and rodents. NEW METHOD: We established protocols to isolate astrocytes from the canine forebrain. Cerebral hemispheres of 3-4-week-old dogs were used. The isolation procedure included the use of the Neural Tissue Dissociation Kit P, demyelination by the magnetic bead method, and separation and preparation by differential adhesion. RESULTS: We found a 96% astrocyte purification rate after isolation by differential adhesion. Purified canine astrocytes increased the secretion of interleukin-1ß, interleukin-6, and tumor necrosis factor-alpha, and increased the expression of glial fibrillary acidic protein after lipopolysaccharide stimulation. We sequenced the transcriptome of the purified canine astrocytes and analyzed the differentially expressed genes among the rodent, human, and canine astrocytes. Transcriptome profiling and gene ontology analysis of the genes co-expressed in humans and canines indicate that human and canine astrocytes may be different from their rodent counterparts in terms of mediated interactions with metals. COMPARED WITH THE EXISTING METHODS: The cells prepared by our method allow for the rapid separation of astrocytes with a relatively small resource scheme. The method also retains the cell phenotype and has an in vitro culture lifetime of approximately 2-3 months. CONCLUSION: We established a method for preparing canine astrocytes with high purity, which can be used to study the biological function of astrocytes in vitro.

Establishment and application of ERA-LFD method for rapid detection of feline calicivirus.

Feline calicivirus (FCV) has a single-stranded, positive-sense RNA genome, and it is responsible for many infectious respiratory diseases in cats. In addition, more worryingly, highly virulent strains of FCV can cause high mortality in felines. Therefore, a rapid and reliable diagnosis tool plays an important role in controlling the outbreak of FCV. In this study, enzymatic recombinase amplification (ERA) assay combined with lateral flow dipstick (LFD) was developed for the detection of FCV, targeting a relatively conversed position of FCV-ORF1. The results showed that the optimal reaction condition was at 40 °C for 30 min. ERA-LFD method was highly sensitive with the detection limit as low as 3.2 TCID50 of FCV RNA per reaction. The specificity analysis demonstrated no cross-reactivity with feline parvovirus (FPV), feline herpesvirus (FHV) and feline infectious peritonitis virus (FIPV). ERA-LFD was highly repeatable and reproducible, with the intra-assay and inter-assay coefficients of variation for this method both less than 7%. The general test showed that all the recombinant plasmids with known mutant sites and FCV strains with different mutant sites stored in our laboratory were all detected by this method. Of the 23 samples, 14 samples were tested positive for FCV by ERA-LFD and RT-qPCR, respectively. In summary, ERA-LFD assay was a fast, accurate and convenient diagnosis tool for the detection of FCV. KEY POINTS: • The detection principle of ERA-LFD was introduced. • Almost all the currently known FCV strains can be detected. • ERA-LFD is easy to operate and can be used for field detection.

Identification and biochemical characterisation of a novel methionine aminopeptidase from the taiga tick Ixodes persulcatus.

Methionine aminopeptidases (MetAPs), which remove the initiator methionine from nascent peptides, are essential in all organisms and considered to be a valuable targets for the treatment of various diseases, including cancer, malaria, and bacterial infections. However, MetAPs have not been reported in hard ticks (family Ixodidae), and their bioinformatics characterisation in tick's genome sequences is limited. In this study, we cloned, identified, and characterised a novel MetAP from Ixodes persulcatus, a vector for pathogens causing Lyme borreliosis and tick-borne encephalitis. The sequence analysis showed that I. persulcatus MetAP was a type 1 enzyme carrying C-terminal motifs conserved in the M24A family of metallopeptidases. Protein-protein docking simulations using human MetAP revealed conservation of substrate and metal-binding residues in the catalytic site cleft of the novel enzyme, which was designated IpMetAP. Recombinant IpMetAP expressed in Escherichia coli revealed its significant enzymatic activity with the synthetic substrate H-Met-4-methyl-coumaryl-7-amide at pH 7.5 with Km of 0.014 mM, kcat of 0.25 s-1, and overall catalytic efficiency (kcat/Km) of 18.36 mM-1 s-1. The activity of IpMetAP was enhanced by the addition of divalent cations Mn2+ and Co2+ and significantly inhibited by EDTA and bestatin. Site-directed mutagenesis of conserved amino acids indicated that the substitution of metal-binding residues D226 and H288 completely abolished the IpMetAP enzymatic activity, whereas that of the other sites had only moderate effects on substrate hydrolysis. The catalytic properties of IpMetAP suggest that the enzyme behaves similar to other MetAPs and such characterization expands our knowledge of aminopeptidases and protein metabolism of tick.

Enhancing the production of light olefins and aromatics from catalytic fast pyrolysis of cellulose in a dual-catalyst fixed bed reactor.

In this study, the effects of a macroporous catalyst (CaO), mesoporous catalyst (MCM-41), and microporous catalysts (ZSM-5 and SAPO-34) on the production of light olefins and aromatics from cellulose catalytic fast pyrolysis were investigated in a dual-catalyst fixed bed reactor. Further the fractional catalytic pyrolysis of MCM-41 or CaO with ZSM-5 or SAPO-34 was explored. The results showed that ZSM-5 was the most efficient catalyst for the formation of light olefins and aromatics followed by MCM-41, CaO and SAPO-34, and no aromatics were found with SAPO-34 only. Moreover, 15% CaO combined 85% ZSM-5 produced the highest yield of light olefins (5.59%) and aromatic (13.42%). The addition of CaO and MCM-41 promoted the selectivity of C2H4 and decreased the production of naphthalene.

The conversion of biomass to light olefins on Fe-modified ZSM-5 catalyst: Effect of pyrolysis parameters.

Light olefins are the key building blocks for the petrochemical industry. In this study, the effects of in-situ and ex-situ process, temperature, Fe loading, catalyst to feed ratio and gas flow rate on the olefins carbon yield and selectivity were explored. The results showed that Fe-modified ZSM-5 catalyst increased the olefins yield significantly, and the ex-situ process was much better than in-situ. With the increasing of temperature, Fe-loading amount, catalyst to feed ratio, and gas flow rate, the carbon yields of light olefins were firstly increased and further decreased. The maximum carbon yield of light olefins (6.98% C-mol) was obtained at the pyrolysis temperature of 600°C, catalyst to feed ratio of 2, gas flow rate of 100ml/min, and 3wt% Fe/ZSM-5 for cellulose. The selectivity of C2H4 was more than 60% for all feedstock, and the total light olefins followed the decreasing order of cellulose, corn stalk, hemicelluloses and lignin.

Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii.

In the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a K m value of 0·255 µ m and a k cat value of 35·6 s-1. The overall catalytic efficiency (k cat/K m) of the rTgAPP was 139·6 × 105 M-1 s-1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP.